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Cisco leads a $14 million funding round for Embrane to enhance its go-to-market strategy, Arista expands its cloud platform with two new 100GbE modules, and Dell launches new networking solutions, featuring the Z9500 Fabric Switch. Read More
Author Archives: John Rath
Zettaset Adds Data-in-Motion Encryption for Hadoop
Big data management company Zettaset has added a new data-in-motion encryption capability to its Orchestrator management and security add-on application for Hadoop. Read More
NVIDIA Targets Need for Speed With Ultra-Fast GPU Interconnect
At its annual GPU Technology Conference (GTC) in San Jose this week NVIDIA (NVDA) laid the foundation for its Pascal GPU architecture with NVLink high-speed integration, launched a GPU rendering appliance, and introduced a new Tegra K1 powered development kit for the embedded market. Read More
Dell Adds to Software Portfolio With StatSoft Acquisition
Dell announced the acquisition of StatSoft, a provider of advanced analytics solutions that deliver a wide range of data mining, predictive analytics and data visualization capabilities. Read More
Hortonworks Nets $100 Million to Accelerate Enterprise Hadoop
Enterprise Hadoop provider Hortonworks announced an oversubscribed $100 million funding round. The investment will help the company continue to grow its ecosystem and global operations. Read More
Titanfall Taps Windows Azure Cloud for Low-Latency Gaming
Cloud infrastructure has been well tested for enterprise workloads. But can it handle the demands of gamers, who are ultra-sensitive to network lag? Behold Titanfall, running on the Microsoft Windows Azure cloud. Read More
Actifio Secures $100 Million, Funding Values Company at $1.1 Billion
With 182 percent annual growth rate and over 1 Exabyte of data under management, copy data virtualization company Actifio announced it has secured an oversubscribed $100 million funding round led by Tiger Global Management, with participation from current investors. Read More
Iowa Data Center Boom Continues with $255 Million Project Alluvion
Another mystery data center project may land in Iowa, with West Des Moines set to review an application for Project Alluvion – a $255 million data center project from an unnamed company. Read More
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Network virtualization company Midokura announced a partnership with Cumulus Networks to offer a joint technology solution that will enable customers to manage workloads on virtual and non-virtualized infrastructure. Read More


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9 reviews | 3.9 out of 5
Great product and the new tube allows you to get all of the cream out. It works just like other benzoyl peroxide products but is less than half the price.
Been using this for years and it always seems to be sold out at my local Walmart, so when I do spot it on the shelf, I stock up. This works much better than the name brand comparatives, and is obviously way cheaper. Just noticed that it is now FINALLY available online - YEA!!!
The only acne medicine I use. It must be good because I had to order it online because I can rarely find it stores.
Best acne fighter I've tried
This is the old stand-by: benzoiyle peroxide, so it seems to work fine
This cream is very runny and comes pouring out of the tube as soon as cap is off. Much product is wasted because of this. Too runny to put on face..supposed to be a spot treatment but cream is too thin and won't stay on the spot! Hard to say if it actually works.
We use this instead of the name brand and it works just as good if not better
This is just as effective as Proactiv Advanced Blemish treatment, but cost a whole heck of a lot less! I can get 4 tubes of this for the price of just one tiny tube of Proactiv.
Thought it was a good product for the price.

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The Other Civil War: Slavery and Struggle in Civil War America (Paperback)
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The Other Civil War offers historian and activist Howard Zinn's view of the social and civil background of the American Civil War a view that is rarely provided in standard historical texts. Drawn from his New York Times bestseller A People's History of the United States, this set of essays recounts the history of American labor, free and not free, in the years leading up to and during the Civil War. He offers an alternative yet necessary account of that terrible nation-defining epoch.
About the Author
Howard Zinn (1922-2010) was a historian, playwright, and social activist. His many books include A People's History of the United States, which has sold more than two million copies.


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Lions assistant Kris Kocurek: "I'm coaching them just like they're my own this week. That's the mentality. I told them the first night, 'We're going to be the hardest-working group on the field.' "
- Filed Under
MOBILE, Ala. -- The defensive linemen had dominated a drill in practice Wednesday morning, and they were feeling pretty good about themselves. They came over to the sideline all smiles, hooting and hollering, laughing and joking.
Then Kris Kocurek stepped in. He got in a few faces.
"Do not be content!" he barked at them. "Get better!" ...


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Raina scored 84 before getting out while Dhoni bludgeoned his way to a 19-ball 63 as he hammered eight sixes in his astonishing unbeaten innings to power CSK to 202 for four. (Getty Images)
Ranchi: Unbeaten so far in the tournament, in-form team Chennai Super Kings will eye a hat-trick of wins and seal the semi-final berth when they take on the struggling Brisbane Heat of Australia in Ranchi on Saturday in their Group B match. Chennai are on top of the point table while Brisbane are at the bottom.
Batting firepower will give CSK a definitive edge over Brisbane. With the in-form limited-overs specialists MS Dhoni and Suresh Raina, Chennai can take any opposition line-up to task. Their match against Sunrisers Hyderabad on Thursday once again showed the T20 ability of Raina and skipper MSD. Raina smashed a quickfire half-century while skipper Dhoni produced a belligerent unbeaten knock of 63 off just 19 balls for CSK to post a stunning 202 on the board against Hyderabad.
So far Mike Hussey and Murali Vijay have failed to give the home team a good start and the Chennai franchise will be hoping that the duo get some runs under their belt ahead of the semis. Star allrounder Ravindra Jadeja too hasn't quite dazzled so far either with the bat or ball.
Chennai have some bowling issues to deal with given that they ended up conceding 190 runs to Hyderabad. The likes of Mohit Sharma and Albie Morkel would have to watch their economy rate or else they could end up helping Brisbane batsmen regain lost form.
Brisbane, on the other hand, are all but eliminated from the competition. Handed defeats in the two matches they have played so far, Brisbane need to win on Saturday to stay alive in the event. The depleted Australian outfit, which failed miserably with the bat in the games against Trinidad & Tobago and the Titans, will be expecting its batsmen to show more responsibility.
Brisbane would hope that captain James Hopes, who has plenty of international experience and has also captained the IPL side Delhi Daredevils, gives them a solid start along with Burns. Besides, allrounder Dan Christian will also have to carry the batting burden. Chris Lynn, the big-hitting middle-order batsman, is another player who can make an impact.
The bowling unit has done well so far but have found little or no support from the team's batsmen.
Chennai Super Kings: 1 Michael Hussey, 2 Murali Vijay, 3 Suresh Raina, 4 S Badrinath, 5 MS Dhoni (capt/wk), 6 Dwayne Bravo, 7 Ravindra Jadeja, 8 Albie Morkel, 9 Ravichandran Ashwin, 10 Jason Holder, 11 Mohit Sharma.
Brisbane Heat: 1 James Hopes (capt), 2 Joe Burns, 3 Peter Forrest, 4 Daniel Christian, 5 Chris Lynn, 6 Chris Hartley (wk), 7 Ben Cutting, 8 Chris Sabburg, 9 Nathan Hauritz, 10 Matthew Gale, 11 Alister McDermott


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Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea. While previously characterized arginases have an alkaline pH optimum and require activation with manganese, arginase from Helicobacter pylori is optimally active with cobalt at pH 6. The arginase from Bacillus anthracis is not well characterized; therefore, this arginase was investigated by a variety of strategies and the enzyme was purified.
The rocF gene from B. anthracis was cloned and expressed in E. coli and compared with E. coli expressing H. pylori rocF. In the native organisms B. anthracis arginase was up to 1,000 times more active than H. pylori arginase and displayed remarkable activity in the absence of exogenous metals, although manganese, cobalt, and nickel all improved activity. Optimal B. anthracis arginase activity occurred with nickel at an alkaline pH. Either B. anthracis arginase expressed in E. coli or purified B. anthracis RocF showed similar findings. The B. anthracis arginase expressed in E. coli shifted its metal preference from Ni > Co > Mn when assayed at pH 6 to Ni > Mn > Co at pH 9. Using a viable cell arginase assay, B. anthracis arginase increased dramatically when the cells were grown with manganese, even at final concentrations of <1 μM, whereas B. anthracis grown with cobalt or nickel (≥500 μM) showed no such increase, suggesting existence of a high affinity and specificity manganese transporter.
Unlike other eubacterial arginases, B. anthracis arginase displays unusual metal promiscuity. The unique properties of B. anthracis arginase may allow utilization of a specific metal, depending on the in vivo niches occupied by this organism.
Bacillus anthracis is a highly invasive, exceptionally virulent pathogen of mammals, with humans as accidental hosts [1]. B. anthracis possesses two virulence plasmids, pXO1 and pXO2, that encode for a tripartite secreted toxin and a poly-D-glutamic acid capsule, respectively [2-4]. The toxins can cause death or produce serious edema in infected individuals [5,6]. Despite many years of research, chromosomal loci have received little attention; however, a chromosomally-encoded cytotoxin, anthrolysin O, has been recently reported [7].
Arginase hydrolyzes L-arginine to L-ornithine and urea. This enzyme is found in both eubacteria and eucaryotes. However, relatively few eubacterial arginases have been characterized; most characterized arginases are from animals and yeasts [8-10]. We recently characterized an arginase (RocF) from the gastric pathogen, Helicobacter pylori [11], discovering a number of interesting and unique properties among the arginase superfamily. When we began this current study, the closest homolog of the H. pylori arginase (NCBI Accession Number: NP_208190) was that from B. anthracis (NCBI Accession number: YP_016761 [12]. Yet, the two RocF proteins share only 20% amino acid identity over the entire protein (27% when comparing the first 283 amino acids of H. pylori with the first 271 amino acids of B. anthracis). Arginase activity from H. pylori has recently been measured using a highly sensitive and quantitative assay that can determine enzyme activity from extracts, viable cells or the purified enzyme [11]. The rocF gene from H. pylori has been cloned into E. coli DH5α and confers arginase activity to E. coli (which does not possess a native arginase), suggesting that the rocF gene alone is sufficient to confer arginase activity [11].
L-arginine is used by macrophages to produce nitric oxide and other downstream reactive nitrogen species [13]. The production of nitric oxide by activated host macrophages is an effective antimicrobial agent and serves as an initial innate defense mechanism against pathogens [13]. Interestingly, the H. pylori arginase inhibits host nitric oxide production, allowing for survival of the organism when co-cultured with activated macrophages [14]. A similar situation likely occurs with the B. anthracis enzyme [15]. Additionally, H. pylori arginase decreases T lymphocyte proliferation and CD3ζ expression [16], arguing for the importance of this enzyme in multiple facets of host-pathogen interactions.
In addition to roles in pathogenesis, it is speculated that arginases have evolved to regulate the levels of arginine and ornithine within the cell [17]. Regulation of these amino acids is pivotal in protein synthesis, polyamine and nitric oxide production, and other cellular processes [17]. For example in H. pylori, expression of a second copy of the arginase gene, rocF, does not double the arginase activity, suggesting the bacterium has mechanisms to limit hydrolysis of the essential amino acid, arginine [18].
In organisms having a complete urea cycle, arginase is the final enzyme of the cycle; however, arginase is not found in most eubacteria [19]. In H. pylori, the urea is further hydrolyzed into carbon dioxide and ammonia via the enzyme urease [20]. Most eubacteria that contain an arginase also have the entire urea cycle. Examples include H. pylori and B. subtilis [20,21]. However, several arginase-containing bacteria apparently lack a complete urea cycle. Such organisms include B. licheniformis [22] and B. anthracis. This suggests that arginase in these latter bacteria has evolved a unique physiologic role in the cell. Several biochemical properties of the B. anthracis arginase had been preliminarily described [23]. Purified B. anthracis arginase had to be preactivated with 1 μM Mn2+ (MnSO4) and heat (37°C for 3 hours) (designated as heat-activation) for activity to be measured using a urea detection method [23]. Indeed, nearly all arginases require some type of heat activation step in the presence of manganese for catalytic activity [24]. For B. anthracis arginase, manganese was previously considered the most efficient cation activator and stabilizer, allowing the Mn2+-preactivated enzyme to maintain stability and activity even after exposure to 50–60°C for one hour [23]. Additionally, the enzyme showed optimal catalytic activity between pH 9.8 and 10.0 [23], a finding consistent will all arginases [25], except the H. pylori enzyme (see results) [11].
Activity of the purified B. anthracis arginase decreased significantly after dialysis and lyophilization; if the manganese-preactivated enzyme was treated with other divalent cations, a large decrease in arginase catalytic activity occurred [23]. Collectively, these earlier data suggested that the metal cofactor involved in B. anthracis arginase activity was manganese [23], with nickel and cobalt having no role or an inhibitory role. The earlier data also indicated that heat-activation was absolutely required for catalytic activity. Identification of the gene responsible for B. anthracis arginase was not previously reported.
While the toxin components produced by B. anthracis have been the primary focus of research, there is limited research on the organism's other genes and gene products. In this study, we sought to characterize in depth the chromosomally-encoded B. anthracis arginase using a variety of conditions not previously examined, using a sensitive enzyme assay. We also established an E. coli model for the B. anthracis arginase and determined whether arginase could be detected and characterized from viable organisms. We provide evidence that the enzyme has novel features not previously recognized. For example, the B. anthracis arginase displayed optimal activity with nickel in extracts and manganese in viable cells, but surprisingly did not absolutely require either heat activation or the addition of exogenous metal to detect arginase activity. The B. anthracis arginase gene could complement an H. pylori arginase mutant, conferring B. anthracis arginase-like properties to H. pylori.
Characterization of arginase-containing extracts of B. anthracis 7702
Many holes remain in our knowledge of the chromosomally-encoded arginase from B. anthracis. Also, no two eubacterial arginases have ever been directly compared for their properties under identical conditions. We therefore sought to investigate the B. anthracis arginase in much more detail and compared it to the H. pylori arginase. Extracts from B. anthracis 7702 assayed with manganese (Mn2+) or cobalt (Co2+) displayed arginase activity at both pH 6.3 and 9.0; however, when assayed in the presence of nickel (Ni2+) at pH 9.0, the activity was up to 500-fold higher than with Mn2+ or Co2+ (Fig. 1A &1B). This is in striking contrast to what was reported previously for this enzyme, in which Ni2+ inhibited activity [23].
Figure 1. B. anthracis 7702 extracts show optimal catalytic activity with nickel. Cells were grown on CBA plates at 37°C for 24 h in 5% CO2 in air, harvested, and extracts prepared as described in Methods. The extracts were assayed with 5 mM (final concentration) of the appropriate metal after heat activation at pH 6.3 (A) or 9.0 (B) (Bis Tris Propane [BTP]-buffered arginine).
B. anthracis arginase activity was determined at various concentrations of nickel, manganese, or cobalt. Far lower concentrations of nickel were needed to obtain abundant arginase activity, than when cobalt or manganese were used (Fig. 2). Micromolar nickel activated arginase >2-fold more than the optimal level of manganese (400 U versus 125 U) and >100-fold higher than the optimal level of cobalt (400 U versus 12 U). Optimal concentrations for the three metals were as follows: nickel, ≥ 500 μM; manganese, ≥ 12.5 mM; and cobalt 5 mM (Fig. 2A–D). Since all three metals caused near optimal arginase activity at 5 mM, this concentration was used for most subsequent experiments.
Figure 2. Effect of metal concentration on arginase activity in B. anthracis 7702 extracts. Cells were grown on CBA plates at 37°C for 24 h in 5% CO2 in air, harvested, and extracts prepared as described in Methods. The extracts were heat-activated (50–55°C for 30 minutes) with increasing concentrations of NiCl2 (A, high concentrations; B, low concentrations), MnSO4 (C), or CoCl2(D) at pH 9.0 (in BTP-buffered arginine).
Heat activation is required for all previously characterized arginases [24,26], presumably to allow partial unfolding of the enzyme and incorporation of the metal cofactor. In a previous study, B. anthracis arginase activity decreased in samples exposed to heat, if the enzyme had not been already pre-activated with manganese [23]. In contrast with this earlier finding, we observed that B. anthracis arginase activity surprisingly increased about 10-fold following treatment with heat (50–55°C, 30 min) at pH 9.0 in the absence of metal versus no heat (Fig. 3). However, in the presence of various metals, heat treatment did not affect arginase activity at either pH 6.0 or 9.0, when compared to samples that were not heat-treated (Fig. 3). Under all conditions, nickel conferred that highest arginase catalytic activity.
Figure 3. Effect of heat activation on the arginase activity of B. anthracis 7702 extracts. Cells were grown on CBA plates at 37°C for 24 h in 5% CO2 in air, harvested, and extracts prepared as described in Methods. The extracts were assayed with or without heat activation (50–55°C or on ice for 30 minutes, respectively) in the absence (no metal) or presence of 5 mM metal (Co, Mn, or Ni) at pH 6.0 (in MES-buffered arginine) or 9.0 (Tris-buffered arginine). There was no evidence of a buffer effect on activity (data not shown).
Comparative characteristics of the H. pylori and B. anthracis arginases
Since the H. pylori and B. anthracis arginases share low amino acid similarity (20% identity), we questioned whether the properties of these two enzymes were similar or distinct, when assayed under identical conditions. H. pylori and B. anthracis arginase activities expressed in E. coli DH5α were first compared (Fig. 4A). E. coli (pBS-barocF) had optimal arginase activity at both pH 6.0 and 9.0 with nickel, with lower activity in the presence of cobalt or manganese (Fig. 4A). At pH 6.0, E. coli (pBS-barocF) showed better activity with cobalt than with manganese (p < 0.05, Co versus Mn) (Fig. 4A). At pH 9.0, the optimal activating metal was nickel for E. coli (pBS-barocF), but higher arginase activity occurred when assayed with manganese rather than cobalt. With E. coli (pBS-rocF), optimal arginase activity was with cobalt (p < 0.05, Co versus Mn) (Fig. 4A) at either pH 6.0 or 9.0.
Figure 4. Characteristics of arginase extracts from E. coli DH5α expressing the H. pylori or B. anthracis enzymes or from native organisms. Extracts were prepared from E. coli carrying H. pylori (pBS-rocF) or B. anthracis rocF (pBS-barocF) and grown in L broth at 37°C with aeration (A) or from 24 hour cultures of B. anthracis and H. pylori grown on CBA at 37°C in 10% CO2, 5% O2 (B). Extracts were heat-activated for 30 minutes at 50–55°C in the presence of ddH2O (no metal control), 5 mM CoCl2, MnSO4, or NiCl2 and assayed at pH 6.0 or 9.0 in MES-buffered arginine or Tris-buffered arginine, respectively (A) or at pH 6.3 or pH 9.0 in BTP-buffered arginine (B). Arginase activity data are the mean arginase activity ± standard deviation of one representative of at least two experiments (independently prepared extracts) conducted in triplicate. Under these conditions, E. coli DH5α(pBS) showed a background of ~1000 pmol L-orn/min/mg protein [U]. Lack of error bars on some bars indicates that the standard deviation was minimal and did not appear on the graph. NM, no metal.
Arginase activity of E. coli (pBS-barocF) was approximately 10-fold higher at pH 9.0 than at 6.0 in the presence of manganese (Fig. 4A, p < 0.005). While E. coli (pBS-rocF) displayed optimal activity with cobalt when assayed at different pHs, E. coli (pBS-barocF) had optimal activity with nickel at different pHs. As previously reported [11], the H. pylori arginase in the E. coli model (pBS-rocF) displayed optimal catalysis with cobalt at pH 6.0.
In addition, enzyme activities from native organisms were compared after growth under identical conditions (Campylobacter blood agar, 24 h, 37°C, 5% O2/10% CO2). While the B. anthracis enzyme had greater activity with nickel than without metal (>50-fold difference irrespective of pH), the enzyme still surprisingly retained activity even in the absence of metal (Fig. 4B, NM). At pH 9.0 arginase activity in B. anthracis extracts was up to 1000-fold higher than in H. pylori extracts depending on the metal present and the pH of assay (Fig. 4B). Even when assayed under sub-optimal conditions (e. g., pH 6 with manganese), the B. anthracis arginase was still more activity than the H. pylori enzyme (Fig. 4B). The only condition in which the H. pylori arginase was as active as the B. anthracis enzyme was when the enzymes were assayed at pH 6.3 in cobalt (Fig. 4B). The results suggest that the E. coli models nearly completely mimic the data obtained from native organisms.
Comparison of arginase activity between E. coli DH5α pBS-barocF and B. anthracis 7702
To strengthen the validity of the E. coli arginase model, we directly compared the arginase activities between E. coli (pBS-barocF) versus B. anthracis under identical conditions. Under most assay conditions, the arginase expressed in E. coli versus B. anthracis showed similar trends in that highest arginase activity occurred at pH 9 with nickel (data not shown). However, arginase activity in E. coli (pBS-barocF) was 5–15-fold higher than in B. anthracis under most conditions, probably due to increased gene dosage from the high-copy-number plasmid. The only case in which B. anthracis arginase activity was not significantly lower than arginase expressed in the E. coli model was when the extracts were assayed without metal. The second most optimal metal shifted from manganese at pH 9.0 to cobalt at pH 6.3, especially in the E. coli model (data not shown). In addition, there was substantial activity without heat treatment or without addition of exogenous metal (data not shown).
Metal dose-dependency of B. anthracis arginase activity using viable cells
The B. anthracis arginase characteristics in live cells was examined to better ascertain whether the in vitro biochemical results would hold true in viable cells. Previously, we showed that E. coli (pBS-rocF) increases its arginase activity when grown with increasing concentrations of metal [11]. Here, we determined whether arginase could be detected in viable cells of B. anthracis or in E. coli (pBS-barocF), and if so, what metal was ideal for catalytic activity. In both viable cell models, arginase activity was detectable (Fig. 5 and 6). At pH 6.0 with E. coli (pBS-barocF), growing the cells in the presence of manganese (≥ 100 μM) increased arginase activity of viable cells (Fig. 5A); cobalt and nickel had no significant effect. At pH 9.0, there was evidence of a slight dose-dependent increase with cobalt, nickel, or manganese (Fig. 5B), with manganese showing the greatest effect.
Figure 5. Effect of metal concentration on arginase activity in E. coli (pBS-barocF) whole cells. Cells were grown in the absence or presence of various concentrations of metal (CoCl2, MnSO4, or NiCl2). No effect on growth was observed at the concentrations used. Whole cells were harvested and assayed directly without heat treatment (ice, 30 min) at pH 6.0 (A) or 9.0 (B) using MES- or Tris-arginine buffer, respectively. Data shown is represented as the fold increase over the no metal control. Under these conditions, E. coli DH5α(pBS) showed a background of ~1000 pmol L-orn/min/mg protein [U]. Before fold-increases were calculated, 1000 U was subtracted from each E. coli DH5α (pBS-barocF) data point.
Figure 6. Evidence that B. anthracis 7702 has a high affinity and specificity manganese transporter involved in arginase activity. B. anthracis 7702 were grown in BHI broth in the absence or presence of metal (CoCl2, MnSO4, NiCl2) at various concentrations. No effect on growth was observed at the concentrations used. Cells were harvested and assayed directly (viable whole cells) without heat treatment (ice, 30 min) at pH 9.0 using BTP-buffered arginine. Inset. B. anthracis grown with 0.2 to 1.0 μM MnSO4.
These experiments were also conducted in B. anthracis. Remarkably, arginase activity increased when the bacteria were grown with exceptionally low manganese concentrations (200 nM; Fig. 6 inset). Even at the highest level of cobalt or nickel that can be used without adversely affecting growth (500 μM for cobalt; 1 mM for nickel), cobalt or nickel had no effect on arginase activity (Fig. 6).
Disruption of the B. anthracis rocF in pBS-barocF abolishes arginase activity in E. coli
Above, it was demonstrated that B. anthracis rocF confers arginase activity to E. coli, suggesting that rocF is necessary and sufficient to confer arginase activity to E. coli. Further proof was obtained by disrupting the rocF gene in pBS-barocF and transforming the resultant plasmid, pBS-barocF::kan, into E. coli to yield two transformants. One transformant had the kanamycin cassette in the forward (F) orientation with respect to rocF, while the other had the cassette in the reverse (R) orientation. E. coli carrying either clone had no detectable arginase activity (0.84 ± 0.06 nmol L-ornithine/min/mg prot [U] for pBS-barocF::kan F and 0.73 ± 0.2 U for pBS-barocF::kan R), in contrast with pBS-barocF (3,320 ± 360 U) (heat-activated with manganese, BTP-arginine buffer, pH 9.0). E. coli carrying the insert-free vector control, pBS, had 0.57 ± 0.03 U of background activity under these conditions.
Characteristics of purified B. anthracis RocF
To determine whether the experimental results with B. anthracis and the E. coli model also hold true for the purified protein, we constructed an E. coli strain carrying the B. anthracis rocF gene translationally-fused to a his6 tag at the N-terminus (designated His6-BaRocF). Previous results indicated that the six histidine tag did not interfere with arginase activity [11]. The B. anthracis His6-BaRocF protein was purified to >95% homogeneity (Fig. 7A). Purified His6-BaRocF had a Km for arginine of 10 mM and a Vmax of 500 pmol/sec at pH 9.0 in the presence of 5 mM nickel.
Figure 7. Characteristics of purified B. anthracis His6-BaRocF. A. Purification of His6-BaRocF. His6-BaRocF was purified as described in Methods and the flow-through (FT), washes (W) and elutions (E) analyzed by Coomassie blue-stained SDS-PAGE. Intervening lanes between W7 and E1 have been cropped out. B and C. Purified His6-BaRocF displays optimal catalytic activity with nickel. The purified enzyme was assayed with 5 mM of the appropriate metal after heat activation at pH 6.3 (B) or pH 9.0 (C) in Bis Tris Propane [BTP]-buffered arginine. At least two experiments were conducted for each sample in triplicate. Data are presented as mean arginase activity (nmol L-orn/min/mg protein) ± standard deviation. No Metal, assayed without metal. D. Effect of heat activation on the activity of His6-BaRocF. The purified arginase samples were assayed with or without heat activation (50–55°C or on ice for 30 minutes, respectively) in the absence (No Metal) or presence of 5 mM metal (CoCl2, MnSO4, or NiCl2) at pH 6.0 (in MES-buffered arginine) or 9.0 (Tris-buffered arginine). There was no evidence of a buffer effect on activity (data not shown). At least two experiments were conducted in triplicate for each sample. Data are presented as mean arginase activity (nmol L-orn/min/mg protein) ± standard deviation.
When purified His6-BaRocF samples were assayed in the presence of nickel, the activity was up to 100-fold higher than with manganese or cobalt (Fig. 7B, 7C), similar to the findings in B. anthracis extracts (Fig. 1). Little to no arginase activity occurred in the presence of other divalent cations. Additionally, the metal preference shifted from Ni > Co > Mn at pH 6.3 to Ni > Mn > Co at pH 9.0, as was observed in the E. coli model (Fig. 4A).
In a previous study, B. anthracis arginase activity decreased in samples exposed to heat (50–60°C), if the enzyme had not been pre-activated with manganese [23]. We determined that in the presence of cobalt, heat activation did not adversely affect arginase activity at pH 6.0 or 9.0, when compared to samples that were not heat-treated (Fig. 7D). Remarkably, heat treatment dramatically increased arginase activity in the presence of nickel at either pH 6.0 or 9.0. As observed in B. anthracis extracts and in extracts using the E. coli model, optimal catalytic activity was observed when the purified enzyme was assayed in the presence of nickel (Fig. 7D), rather than manganese or cobalt.
Catalytic activity of purified B. anthracis arginase was determined at various concentrations of nickel, manganese, or cobalt (Fig. 8). Nickel showed an increase in activity at a much lower concentration when compared to manganese or cobalt, confirming the results found in extract samples from the native organism and in E. coli expressing the B. anthracis arginase. Micromolar nickel concentrations activated arginase >2-fold more than the optimal level of manganese (544 nmol L-orn/min/mg prot [U] versus 251 U), similar to evidence from extract samples. Additionally, optimal concentrations for the three metals were: nickel, ≥ 12.5 mM; manganese, ≥ 5 mM; and cobalt, 25 mM (Fig. 8). At these optimal concentrations of metal, nickel conferred eight times more arginase activity than either manganese or cobalt.
Figure 8. His6-BaRocF assayed in the presence of various concentrations of different metals. The purified arginase samples were heat activated (50–55°C for 30 minutes) with increasing concentrations of (A) CoCl2, (B) MnSO4, or (C, D) NiCl2 at pH 9.0 (in BTP-buffered arginine). At least two experiments were conducted for each sample in triplicate. Data are presented as mean arginase activity (nmol L-orn/min/mg protein) ± standard deviation.
Complementation of the H. pylori arginase mutant with the B. anthracis rocF gene
Since we were unsuccessful in several attempts to disrupt the rocF gene in B. anthracis, we instead transformed the B. anthracis rocF gene into an H. pylori rocF mutant devoid of arginase activity. This was achieved using the suicide plasmid complementation system developed previously [18], but with an improved vector backbone, pLSU0005 (see Methods). Expression of the B. anthracis rocF gene was driven off the strong H. pylori urease promoter. Transformation of the 26695 rocF mutant with the empty vector control was previously shown not to complement arginase activity, as expected [18]. H. pylori complemented clones 4a and 4b acquired arginase activity that exhibited B. anthracis properties, namely, optimal arginase activity at pH 9 with nickel (Fig. 9), but the overall activity was substantially lower than what is observed for native B. anthracis.
Figure 9. Complementation of the H. pylori rocF mutant with the B. anthracis rocF gene. Cellular extracts from wild type H. pylori 26695 (WT), the 26695 rocF mutant (RocF) and the rocF complemented with the B. anthracis rocF gene (clones 4a and 4b) were assayed for arginase activity at pH 6.0 (MES-arginine buffer) or 9.0 (Tris-arginine buffer) in the presence of cobalt or nickel with heat activation (50°C for 30 minutes). Representative of two experiments conducted in triplicate.
In this study arginase from B. anthracis was investigated under a variety of variables using six different models: i) extracts from B. anthracis; ii) extracts from E. coli (pBS-barocF); iii) viable cells of B. anthracis; iv) viable cells of E. coli (pBS-barocF); v) purified B. anthracis RocF (as His6-BaRocF); and vi) extracts from the H. pylori rocF complemented with B. anthracis rocF. In nearly every case, all models lead to the same conclusions. These conclusions are: i) B. anthracis arginase displays highest catalytic activity with nickel as the metal cofactor, when extracts or the purified protein is examined; ii) B. anthracis arginase is active without heat activation and without addition of metals, although cobalt, manganese, and nickel all improve activity; iii) the enzyme has much higher activity at pH 9.0 than at pH 6.0; iv) a viable cell arginase assay was developed in both B. anthracis and in an E. coli model and the characteristics of the enzyme mostly overlapped in the two models; v) viable cells exhibit far greater arginase activity when grown with manganese than with nickel or cobalt; vi) the B. anthracis arginase displays up to 1000-fold more specific activity than the H. pylori enzyme, depending on the conditions; vii) the B. anthracis rocF gene complements an H. pylori rocF mutant and confers B. anthracis-like arginase properties; and viii) the B. anthracis arginase gene is necessary and sufficient to confer arginase activity to E. coli.
Most bacteria that possess arginase have a complete urea cycle for eliminating excess nitrogen as urea. In striking contrast, B. anthracis contains arginase, but lacks the other enzymes of the urea cycle [23]. The role of arginase in B. anthracis pathogenesis and cellular physiology is only recently becoming understood [15,27], but our knowledge is far from complete. This current study uncovered novel characteristics about the enzyme as expressed in E. coli and in B. anthracis, and therefore lays important groundwork for future experiments in these areas. For the first time, it is demonstrated that the rocF gene from B. anthracis is the gene responsible for arginase activity, since E. coli containing B. anthracis rocF possessed arginase activity, while a kanamycin insertion into rocF abolished arginase activity. Thus, rocF was necessary and sufficient to confer arginase activity to E. coli. These results do not, however, rule out the possibility that B. anthracis contains genes that influence arginase expression or activity. Indeed, the B. anthracis genome contains a homologue of RocR, a positive transcriptional activator of the arginase-containing operon in B. subtilis [21].
Based on previous studies using the purified B. anthracis arginase [23], it was expected that the enzyme would be most active with manganese as its metal cofactor. In contrast, we identified nickel as the optimum metal cofactor for arginase activity in B. anthracis extracts, with purified His6-BaRocF, and with extracts from E. coli (pBS-barocF). Peak B. anthracis arginase activity was reached with much lower nickel concentrations than with manganese or cobalt. This suggests that in a cell-free system, the arginase has highest activity with nickel. The reason for the differences between our studies and those published previously [23] may be the use of an improved enzyme assay, coupled with our ability to assay the enzyme in multiple model systems that were unavailable 25 years ago. To our knowledge, this is the first arginase to have optimal activity with nickel. However, it is recognized that the actual metal found in vivo in B. anthracis native arginase is critical to study in the near future by inductively coupled plasma-mass spectrometry analysis. Such work cannot be accomplished with the current purified protein due to presence of the His6 tag and use of a nickel-containing column to purify the protein.
B. anthracis arginase was active with cobalt, manganese, and nickel. Therefore, depending on the pH and growth conditions, B. anthracis arginase can promiscuously utilize various metals to achieve catalytic activity. This metal promiscuity may allow the arginase to utilize the metal that is most readily available under a given environmental situation or in vivo condition encountered by B. anthracis. The only other reported bacterial arginase that has good activity with multiple metals is B. caldovelox (Table 1) [28], which has an arginase that is active with manganese, cobalt, cadmium and nickel. However, the B. caldovelox arginase protein (NCBI Accession Number: S68863), which is 73% identical to B. anthracis RocF, has optimal activity with manganese [28].
Table 1. Summary of arginase properties from Bacillus spp. and H. pylori.
In viable cells we showed that arginase activity can be directly assayed from E. coli expressing B. anthracis rocF or in native B. anthracis. This implies that the substrate, L-arginine, was transported into the cells, and was converted to urea and L-ornithine; the latter was measured in this study. Viable B. anthracis cells have up to ten-fold higher arginase activity with manganese than with cobalt or nickel (Fig. 6). This finding seemingly contradicts our earlier finding that the B. anthracis arginase in cell-free systems has highest activity with nickel. Two possible interpretations of this discrepancy are that i) B. anthracis arginase actually uses manganese rather than nickel in vivo and ii) B. anthracis arginase uses nickel, manganese or cobalt, depending on the situation. Since viable B. anthracis displayed a remarkable increase in arginase activity when the organisms were grown with extremely low levels of manganese (200 nM), we propose that B. anthracis expresses a very high affinity and specificity manganese transporter under our experimental conditions; this transporter would not be expected to transport nickel or cobalt, since addition of these metals did not increase B. anthracis arginase activity in viable cells (Fig. 6). It is speculated that such a transporter would be able to scavenge manganese from sites expected to be very low in manganese concentrations, such as would exist in vivo. Additional experiments are warranted to directly determine the metal cofactor found in the B. anthracis arginase active site.
The heat-activation step, which involves heating arginase at 50–60°C in the presence of metal, is required to obtain arginase activity in H. pylori and many other previously characterized arginases [11,24,26,29-31]. Previous studies have shown that heat-activation decreases B. anthracis arginase activity if manganese is absent [23]; we observed this with the purified protein as well. In contrast, we found that heating purified arginase in the presence of nickel dramatically improves activity over that of arginase that had not been heat-activated (Fig. 3). This result suggests that arginase may fold in a more ideal conformation with heat treatment in the presence of nickel than with cobalt or manganese, raising the possibility that nickel is the physiologic metal cofactor used by B. anthracis arginase. Despite these observations, however, substantial B. anthracis arginase activity still remains without heat-activation (Fig. 3, 8), unlike the case for most previously characterized arginases. Moreover, B. anthracis arginase has substantial activity without exogenously added metal (Fig. 3, 4), suggesting that sufficient levels of the residual metal cofactor may already be tightly bound to the enzyme's active site. This raises the possibility that the metal cofactor is more tightly bound to B. anthracis RocF than it is in other arginases. All of the other arginase have been proposed to have at least one of the two metal ions loosely bound to the active site [24,32,33].
We demonstrated that the H. pylori rocF mutant devoid of arginase activity could regain arginase activity with B. anthracis-like properties when the rocF mutant was complemented with the B. anthracis rocF gene (Fig. 9), suggesting that the B. anthracis RocF protein itself harbors at least some of the properties that allow it to have optimal activity with nickel. However, the B. anthracis rocF gene did not fully restore arginase activity to H. pylori even though a strongly active H. pylori urease promoter was used. This lack of full restoration may be due to codon usage differences between H. pylori and B. anthracis or to species-specific proteins that affect arginase activity. For example, H. pylori may have an arginase metal-delivering chaperone that does not recognize the B. anthracis enzyme. It is also possible that H. pylori has mechanisms to prevent arginase activity from becoming too high, which could lead to arginine starvation, since it was previously shown that wild type H. pylori carrying two copies of the arginase gene only have 25% more arginase activity than the wild type strain with a single copy [18]. Arginine is an essential amino acid for H. pylori but not B. anthracis. B. anthracis can therefore have much higher arginase activity without starving itself for arginine, since B. anthracis can synthesize more arginine intracellularly.
Table 1 summarizes the properties of a subset of eubacterial arginases. It is readily apparent that even within the genus Bacillus, whose arginases range from 66–99% identical at the amino acid level to B. anthracis, unique arginase properties exist. For example, multiple metals can be used for the arginase of B. caldovelox [28], while sporulation induces arginase in B. licheniformis [34], and the exosporium of spores from B. anthracis contain arginase activity [27]. While the putative metal-binding region is nearly completely conserved among the Bacillus species, there are a number of differences compared to the H. pylori arginase that may serve as important residues to target for site-directed mutagenesis.
The H. pylori arginase displays unique pH (6.0) and metal (cobalt) optima within the arginase superfamily. From the results of this study, the B. anthracis arginase likely displays a unique metal (nickel) optimum in the arginase superfamily and can potentially use multiple metals (cobalt, nickel, or manganese) to achieve catalytic activity suitable for its niche. Characteristic differences in arginases of B. anthracis versus H. pylori may reflect distinct in vivo niches occupied by these organisms. This study provides the foundation for further examination of the role of arginase in B. anthracis pathogenesis and cellular physiology.
Bacterial strains, growth conditions, and plasmids
Escherichia coli strain DH5α [F-, deoR, thi-1, gyrA96, recA1, endA1, relA1, supE44, Δ (lacZYA-argF) U169, hsdR17 (r-, m+), φ 80d lacZΔM15, λ-] (Stratagene, La Jolla, CA) was used for standard cloning and transformation procedures. Strain XL1-Blue MRF' [F-, thi-1, gyrA96, recA1, endA1, relA1, supE44, lac, hsdR17 (r-, m+), F' [proAB, lacIqZ)M15, Tn10 [tetr]] E. coli was grown on Luria (L) agar and in L broth plus appropriate antibiotics (ampicillin, 100 μg/mL; kanamycin, 25 μg/mL; tetracycline 15 μg/mL) at 37°C for ~24 hours; if grown in broth, the cultures were aerated (225 rpm).
Bacillus anthracis Sterne toxigenic acapsulate strain 7702 (pXO1+, pXO2-) [7,35] was grown in Brain Heart Infusion (BHI) broth, L broth, or on Campylobacter agar with 10% (v/v) defibrinated sheep blood (CBA) at 37°C for ~24 hours in 5% CO2 in air or in 10% CO2, 5% O2, 85% N2; when grown in broth, cultures were aerated (225 rpm).
Helicobacter pylori 43504 was grown from frozen stocks for 72 hours on CBA at 37°C, passaged to fresh CBA for an additional 48 hours and then transferred to 25 mL Ham's F-12 plus 2% fetal bovine serum (HyClone, Logan, UT) and incubated for 24 hours at 37°C. All conditions were microaerobic (10% CO2, 5% O2, 85% N2). The bacteria were then centrifuged for 10 min (6,000 × g) and resuspended in 600 μL 0.9% NaCl. Aliquots (100 μL) were spread onto 6 CBA plates and incubated at 37°C for 24 hours. The resultant cultures were used in the assays as described below.
Plasmid pBS [pBluescript II SK (+), Stratagene], pBS-rocF [11], and pBS-barocF (see below) were used in this study.
Molecular biology techniques
Plasmid DNA was isolated by the alkaline lysis method [36], or by using a column chromatography kit (Qiagen, Valencia, CA) for sequencing-grade plasmid. Restriction endonuclease digestions, ligations, and other enzyme reactions were conducted according to the manufacturer's instructions (Promega, Madison, WI or New England Biolabs, Beverly, MA). PCR reactions (50 μL) contained 10 to 100 ng of DNA, PCR buffer, 2.0 to 2.5 mM MgCl2, dNTPs (each nucleotide at a concentration of 0.20 to 0.25 mM), 200 pmol of each primer, and 2.5 units of thermostable DNA polymerase. E. coli was transformed by the calcium chloride method or electroporation.
Cloning of B. anthracis rocF into pBS
The B. anthracis arginase gene, rocF (1104 bp), including the 893 bp coding region, 184 bp upstream, and 27 bp downstream was PCR-amplified from the chromosome using primers DM55(5'-cg
- ggatcc
- atcgat
Construction of pBS-barocF::kan
To disrupt the B. anthracis arginase gene, the plasmid pBS-kan [37] was digested with SmaI and HincII to generate the blunt-ended non-polar kanamycin resistance cassette (~1.3 kb), and pBS-barocF was digested with HindIII (~4.2 kb). The pBS-barocF linearized fragment was rendered blunt-ended by Klenow and ligated to the kanamycin cassette using the Roche Rapid DNA Ligation Kit (Roche Diagnostics Corporation, Indianapolis, IN). The ligation mixture was transformed into E. coli DH5α and doubly antibiotic resistant (kanamycin, ampicillin) transformants were verified by restriction digestion and arginase assay.
Construction of E. coli XL1-Blue MRF' (pQE30-barocF), overexpressing B. anthracis RocF
Plasmid pQE30 was digested with BamHI and KpnI, purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The DNA was dephosphorylated using shrimp alkaline phosphatase. The B. anthracis rocF coding region was amplified from the chromosome using DM184, 5'-
Complementation of the H. pylori arginase mutant with the B. anthracis rocF gene
The B. anthracis rocF coding region was PCR-amplified from pBS-barocF using DM278 (5'-GCGCTGCAGGGATGAAAAAAGAAATTTCGG-3' and DM279 (5'-GCCCATGGCTTCTCAGTTGCTTTTCTTAC-3'). The ~900 bp product was digested with PstI and NcoI, and cloned downstream of the strong H. pylori urease promoter in pLSU0005 to yield pW7. Plasmid pLSU0005 is a derivative of suicide plasmid pIR203C04 [18] that has an improved multi-cloning site, the ureA promoter and a chloramphenicol resistance cassette and is used for complementation in H. pylori. Plasmid W7 (5 μg) was electroporated into the rocF mutant of H. pylori 26695 [38] and two chloramphenicol resistant transformants were confirmed by PCR analysis using flanking primers (data not shown); clones 4a and 4b were subsequently used.
Purification of the Bacillus anthracis arginase
E. coli XL1-Blue MRF' (pQE30-barocF) was grown overnight, diluted 1:100 in 500 mL of L broth plus ampicillin and tetracycline and the culture was incubated at 37°C, 225 rpm for 3–5 h (OD600 nm = ~0.7). Isopropyl thio-β-D-galactopyranoside (2.5 mM, final concentration) was added and the culture incubated for an additional 3–5 h at 37°C, 225 rpm. The resulting culture was centrifuged at 5,000 rpm (Sorval, SH-3000) for 15 min, 4°C. The supernatant was discarded and the pellet was resuspended in 1/20th to 1/40th the original culture volume in Wash/Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 15 mM imidazole, pH 8.0). The bacteria were lysed by two passages through a French Press (16,000 psi) and maintained at 4°C throughout the remainder of the purification procedure. The lysates were then centrifuged and the supernatant containing arginase activity was retained. For every 680 μL of cytosol, 320 μL of nickel-nitrilotriacetic acid/ethanol agarose resin (Ni-NTA, Qiagen) was added. This solution was gently mixed end-over-end for 1–2 h at 4°C. The cytosol/Ni-NTA agarose mixture was equally distributed among two, 8.5 by 2.0 cm polypropylene columns. The flow through was reserved in sterile polypropylene test tubes, and the columns were washed 6–8 times with ~10 mL Wash/Lysis buffer per wash. These washes were retained for SDS-PAGE analysis. The protein was eluted using Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) at 1 mL per elution, for a total of 12 elutions per column. The flow through fractions, washes, and elutions were analyzed for protein by SDS-PAGE, and the elutions were monitored for arginase activity (data not shown). All samples were stored at 4°C or at -20°C in 50% glycerol.
Preparation of arginase-containing extracts
Bacteria were harvested from solid medium using a sterile swab and resuspended in 0.9% NaCl or phosphate-buffered saline (PBS). Broth-grown cells were centrifuged at 16,000 × g for 5 minutes. The pelleted cells were resuspended in 150 μl of 0.9% NaCl or PBS. For whole cell assays, bacteria were directly assayed. For extracts, the suspensions were sonicated (25% intensity, Sonic Dismembrator Model 500, Fisher Scientific, Pittsburgh, PA) in an ice bath twice for 30 sec with a minimum 30 sec rest on ice between pulses. Following sonication, the lysate was clarified by centrifugation at 16,000 × g for 5 minutes. The resulting supernatant was retained on ice and used the same day for determination of arginase activity.
Arginase Assay
Extracts or viable bacteria were characterized using the standard arginase assay described previously [11] or with variations (described below). The sample (25 μl) was added to 25 μL of CoCl2, MnSO4, NiCl2, FeSO4, CaCl2, MgSO4, ZnCl2, or CuSO4 (final concentration of 5 mM, except as noted) or 25 μL of deionized distilled H2O (ddH2O, no metal control). An L-ornithine (0 to 3125 μM) standard curve was generated (extinction coefficient was typically 0.00045 to 0.00070 μM-1). The samples and standards were heat-activated (50–55°C, 30 min) or were maintained on ice for 30 min. Next, 200 μL of buffered 10 mM L-arginine (15 mM MES [2-(N-morpholino)ethanesulfonic acid], pH 6.0; 15 mM Tris, pH 9.0; or 15 mM Bis-Tris Propane [BTP], pH 6.3 or 9.0) was added, and the samples were incubated at 37°C for 1 hour. Enzyme rates were linear at this time point. The reaction was stopped by the addition of acidified ninhydrin (4 mg/mL). After heating for one hour at 90–95°C, the standards and samples were measured spectrophotometrically at 515 nm (Biomate 3, Thermo Spectronic, Rochester, NY) in 1.5 ml cuvettes. Representative data were normalized for protein and are presented as specific activity in pmol or nmol L-ornithine/min/mg protein ± standard deviation with a minimum of two experiments conducted in duplicate or triplicate. Some graphs were converted to log scale since the enzyme activity is much higher under certain experimental conditions. Some experiments were performed using BTP as a buffer, because it had a broad buffering capacity of pH 6.3 to 9.5, which would eliminate any potential buffer effects. It was determined that biochemical intermediates and end products, such as putrescine, spermidine, spermine and urea, did not react with ninhydrin (data not shown).
Protein determinations
Protein determinations were performed by the Bicinchoninic Acid assay (Pierce Chemical Company, Rockford, IL), following the manufacturer's 30 minute method. NaCl or PBS was used as the negative control. The results were calculated by a standard curve using bovine serum albumin.
SDS-PAGE analysis
Proteins were electrophoresed through an SDS-polyacrylamide gel (12%) by standard methods.
Statistical Analyses
An unpaired two-tailed Welch's t test was used to determine statistical relationships (GraphPad Instat 3.05). p < 0.05 was considered significant.
Authors' contributions
RJV co-designed and conducted nearly all of the experiments and drafted the manuscript and figures in partial fulfillment of requirements for the Masters degree. EH conducted the experiments on complementation of H. pylori arginase with the B. anthracis rocF; RFR provided scientific advice and assisted with the writing; DJM directed the project and edited the manuscript.
This work was supported by Public Health Service grant R01 CA101931 (to DJM) and U54 AI57168 (to RFR) from the National Institutes of Health, and a grant from Drexel University College of Medicine (to RFR).
We thank Dr. John W. Foster for helpful discussions and Emily L. Watson for technical support.
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Jenkinson CP, Grody WW, Cederbaum SD: Comparative properties of arginases.
Comp Biochem Physiol B Biochem Mol Biol 1996, 114(1):107-132. PubMed Abstract | Publisher Full Text
Hirsch-Kolb H, Heine JP, Kolb HJ, Greenberg DM: Comparative physical-chemical studies of mammalian arginases.
Comp Biochem Physiol 1970, 37(3):345-359. PubMed Abstract | Publisher Full Text
Weaver J, Kang TJ, Raines KW, Cao GL, Hibbs S, Tsai P, Baillie L, Rosen GM, Cross AS: Protective role of Bacillus anthracis exosporium in macrophage-mediated killing by nitric oxide.
Infection & Immunity 2007, 75(8):3894-3901. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Patchett ML, Daniel RM, Morgan HW: Characterisation of arginase from the extreme thermophile 'Bacillus caldovelox'.
Biochim Biophys Acta 1991, 1077(3):291-298. PubMed Abstract | Publisher Full Text
Greenberg DM, Bagot AE, Roholt OA Jr.: Liver arginase. III. Properties of highly purified arginase.
Arch Biochem Biophys 1956, 62(2):446-453. PubMed Abstract | Publisher Full Text
Ramaley RF, Bernlohr RW: Postlogarithmic phase metabolism of sporulating microorganisms. II. The occurrence and partial purification of an arginase.
J Biol Chem 1966, 241(3):620-623. PubMed Abstract | Publisher Full Text
Schreier HJ, Smith TM, Bernlohr RW: Regulation of nitrogen catabolic enzymes in Bacillus spp.
J Bacteriol 1982, 151(2):971-975. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Brown GW Jr.: Studies in comparative biochemistry and evolution. I. Avian liver arginase.
Arch Biochem Biophys 1966, 114(1):184-194. PubMed Abstract | Publisher Full Text
Christianson DW: Arginase: structure, mechanism, and physiological role in male and female sexual arousal.
Acc Chem Res 2005, 38(3):191-201. PubMed Abstract | Publisher Full Text
Simon JP, Stalon V: Purification and structure of arginase of Bacillus licheniformis.
Biochimie 1976, 58(11-12):1419-1421. PubMed Abstract | Publisher Full Text
Cataldi A, Labruyere E, Mock M: Construction and characterization of a protective antigen-deficient Bacillus anthracis strain.
Mol Microbiol 1990, 4(7):1111-1117. PubMed Abstract | Publisher Full Text
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. 2nd edition. Edited by Laboratory CSH. Cold Spring Harbor, N. Y. ; 1989.
McGee DJ, Coker C, Testerman TL, Harro JM, Gibson SV, Mobley HL: The Helicobacter pylori flbA flagellar biosynthesis and regulatory gene is required for motility and virulence and modulates urease of H. pylori and Proteus mirabilis.
J Med Microbiol 2002, 51(11):958-970. PubMed Abstract | Publisher Full Text
McGee DJ, Radcliff FJ, Mendz GL, Ferrero RL, Mobley HL: Helicobacter pylori rocF is required for arginase activity and acid protection in vitro but is not essential for colonization of mice or for urease activity.
J Bacteriol 1999, 181(23):7314-7322. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Laishley EJ, Bernlohr RW: Regulation of arginine and proline catabolism in Bacillus licheniformis.
J Bacteriol 1968, 96(2):322-329. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Baumberg S, Harwood CR: Carbon and nitrogen repression of arginine catabolic enzymes in Bacillus subtilis.
J Bacteriol 1979, 137(1):189-196. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
De Hauwer G R. Lavelle, and J. M. Wiame.: Pyrroline dehydrogenase and catabolism regulation of arginine and proline in Bacillus subtilis.


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NCET Tech Bite – How to Use Gmail for Work and be an Email Ninja – Oct 28th
Wednesday, October 28, 2015
11:30 – Noon: Networking
Noon – 1 pm: Program
How to use gmail for work for and be an email ninja.
Approximately 10 years Google entered the arena of web based email, joining other brands like AOL, Yahoo, and Hotmail. Since then Gmail has surpassed those competitors by becoming the standard-bearer for ad supported, consumer email services. Did you know that in 2014, Gmail smartphone app had over 90 million users? Or that nearly 25% of Americans use Gmail during work hours?
So what makes Gmail so popular?
On October 28, Nicole Rose Dion will share facts, tips, and tricks about Gmail. By the time you leave the luncheon you will know how to use labels/folders in Gmail to help organize your email; manage contacts and groups, and how to strive (and hopefully achieve) for an empty inbox.
Join us for Tech Bite on Wednesday, October 28, from 11:30 am to 1:00 pm at the Atlantis Casino Resort Spa.
NCET members may bring an unlimited number of guests at member prices. Join before you RSVP for this event and your savings will pay for some of all of your membership!
Wednesday, October 28, 2015
11:30 am > 1:00 pm
Register Now
- $25 – NCET members
- $35 – non-members
NEW! Tables for 8 – includes premium location at front of room, table signage, and recognition from the podium during luncheon.
- $200 – NCET members
- $280 – non-members
NCET events have been selling out! RSVP early so you don't miss this great event.
The Fine Print:
- Advance reservations required. RSVP by 5:00 pm on the Tuesday before the lunch. (Sorry, walk-ins not available)
- We encourage you to pay in advance—there is a $10 service charge for payment at the door.
- No-shows will be charged the full amount of the reservation unless cancelled at least 48 hours before the event.
NCET members receive substantial discounts on NCET events. Your membership pays for itself quickly. Join NCET now and save!
Atlantis Casino Resort Spa
Paradise A Ballroom
3800 S. Virginia Street
Reno NV 89502
About Our Speaker
Nicole Rose Dion is the Co-VP of Social Media for NCET and Email/Web Manager for AAF Reno.
For more information, contact NCET at (775) 453-0130 or firstname.lastname@example.com
Photography courtesy of Debbie McCarthy – Visit her on Facebook
|Luncheon Sponsorships – $500 per luncheon
|Luncheon Display Tables
|• Logo and live link on four emails sent to 7,000 of Northern Nevada business, community and educational leaders.
• Logo and live link on NCET's Tech Bite webpage
• Mention on NCET's Facebook pages• Recognition at luncheon
• Logo displayed in onscreen before luncheon begins
• Opportunity to distribute literature and other handouts to attendees
• Sponsorships include a display table and two tickets to that month's luncheon.
|Looking for an inexpensive way to promote your business?
NCET offers display table space exclusively to NCET members at each Tech Bite luncheon for the bargain price of just $150 for a full table or $75 for a 1/2 table.
Use your table for displays, to hand out information, pass out pamphlets or to greet attendees.This is a great way to highlight your services and promote your business!
You can reserve your 1/2 table or a full table as you purchase your ticket to the luncheon.
|For more information, contact Melissa Marsh at (775) 771-2426 or firstname.lastname@example.com to take advantage of these opportunities.


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sunchoke salad with brussel sprout confetti
Yield: 4 Side dishes
Submitted by vsbrittan
Updated: November 4, 2010
IngredientsFor the Salad:
2 cups sunchokes, peeled and boiled until tender (about 4-5 large pieces)
1 cup sunchokes, peeled and rinsed
3-4 brussel sprouts
� shallot, thinly sliced
2 tablespoons sunflower seeds, lightly toasted
For the Vinaigrette:
1 tablespoon Dijon mustard
1 tablespoon apple cider or white wine vinegar
Juice of � lemon
3 tablespoons olive oil
Salt and Freshly Ground Pepper (white pepper is best, if you have it)
PreparationMake the vinaigrette by whisking together the mustard, vinegar, and lemon juice. Season with salt, and then slowly pour in the olive oil, whisking all the time. Season to taste.
Meanwhile, boil the two cups of peeled sunchokes until just tender, about 10 minutes.
Drain and slice in thin rounds. Slice the remaining 1 cup of raw sunchokes in the same way. Immediately mix with the dressing. Add the shallots and seeds.
Peel the outer leaves off the brussel sprouts, then grate or thinly slice, to create the "confetti" shreds. Add to the salad and toss.
Makes a side for 4 people.


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Reverend Maryum's talk title:
"From the Practical to the Mystical"
This week's video invitation link:
Sunday, October 4, 2015 - https://youtu.be/MsI8yL5IXRM
This week's quote: "Through surrender, spiritual energy comes into this world. If you suddenly feel very light, clear, and deeply at peace that is an unmistakable sign that you have truly surrendered. You then live in a state of nonresistance, a state of grace and lightness, free of struggle." Eckhart Tolle, The Power of Now
Reverend Maryum's Weekly Prayer
I Move Forward with Strength and Courage
There is only One-One Power, One Presence, and One Life. That Life is whole perfect, and complete; It is my life now. I love in a state of grace. My thoughts of peace and joy comfort me. Today, I know who and what I am. I am fully aligned with the Power and Presence of Spirit.
Whenever I feel challenged by a person or a situation in my life, I turn to the presence of God within me. I draw from that source of love, strength, and courage. I step away from the day's concerns, worry, doubt and stress to pray, I more fully center myself in God's presence and allow my faith to surface. I nurture it with love as I would a seedling. Any false idea or appearance of lack, loss or limitation is released to the nothingness from whence it came. I am clear and centered and able to focus on my good. I nourish this faith with affirmations of courage and shine the light of truth on the path ahead. I am strengthened and I move forward and act wisely. My faith fills me with peace as I face difficult circumstances. Faith helps me lovingly release any anxious thoughts about my life and I place my trust in God to guide me in discovering my inner strength. I have God's favor and it is my divine birthright. I claim and declare I am filled with power, courage and determination knowing I am one with the One.
I live, move, and have my being in the ever-present idea of wholeness and love. I experience Divine Love and surrender to the Truth, knowing I am deeply at peace, and anything false falls away. I am a master of my mind and my experience. I am grateful acknowledging this truth. I release this prayer into the creative medium knowing there is no sweeter or more sacred moment that now. This is a job for God! It is done. And so it is. Amen
Rev. Maryum Morse
Rev's Weekly Affirmation:
I rely upon the Infinite Power within me to guide my life!
Center of the Heart Affirmation:
Miracles follow miracles.
Wonders never cease.
I give thanks for this magnificent day!


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One of the things that New Democrats have been trying to hammer into the heads of those of us in the Parliamentary Press Gallery for this election and last is that "New Democrats beat Conservatives!". In other words, in most of the country, if you want to put a Conservative MP out of his or her parliamentary misery, then the real choice is a New Democrat and not those hated Liberals.
The argument holds up well in some regions — hello, Vancouver Island and Saskatchewan! — but not so well in others — hello Atlantic Canada!
Nonetheless, one of the more interesting strategic moves for both federal and provincial New Democrats in Ontario over the last few years, is their focus on southwestern Ontario, the rust belt of the province, if you will, that stretches west of Highway 6, that road that slices north to south from Owen Sound to Hamilton. Here, you will literally find cornfield that run right up to factory walls. Agriculture and manufacturing. Then there's some university towns with a slightly progressive bent like Guelph, Kitchener-Waterloo, London. Federal New Democrats hold one seat London. Their provincial cousins also hold a seat in London and stole a seat in Kitchener that was once a long-time Progressive Conservative seat.
So on Sunday, Thomas Mulcair and the NDP bus is barnstorming this region, hopeful of boosting what the polls have said is some sagging NDP fortunes in Ontario.
It all starts at 0800 ET at the campaign office of Mulcair's Brantford-Brant candidate Marc Laferriere. Go watch. The office is at 69 Erie Ave. in Brantford.
Diane Freeman is a real catch for the New Democrats. She used to a Liberal. But she's going all in on Team NDP this time around. Riding has a long Liberal history, though, and the smart money might be on the red team here. Still, for the first time in just about ever in Waterloo –it's a legitimate three-way race, partly because of Freeman's presence. Meet her and Mulcair at her campaign office at 105 University Ave East at 1000 ET.
Ethan Rabidoux was once a Tory. Now he's a New Democrat. Tough riding, though, for the NDP. Catch Mulcair at 1100 ET at Radiboux's campaign office at 184 Ontario Street in beautiful downtown Stratford.
The NDP holds its big rally of the day at the London Convention Centre at 1230 ET. The convention centre is in the riding of Conservative incumbent Susan Truppe who is definitely the Conservative who is most on the bubble in this election. Right now, it's looking slightly less perilous for here but both the Liberals and the New Democrats are not far back. If the anti-Harper vote collapses behind either the NDP or the Liberals, Truppe is a likely loser. But London West, were the Minister of State for Science and Tech Ed Holder is the Conservative incumbent is also in play. Over the last few weeks, I've been hearing different things about this race: Holder's safe; Holder's out; safe; out. My take: It's in play and the Liberals are charging hard. Meanwhile, in London-Fanshawe, the NDP are incumbents and Irene Mathyssen seems in a good spot to hold the seat she's held since 2006. Down in St. Thomas, Conservative Joe Preston retired but the Conservatives look like they'll hold this one.
Watch this riding on election night. Folks in Sarnia have put an MP on the government side of the House since 1968. That means they've always elected either a Liberal or someone of the Conservative/Progressive Conservative persuasion. New for this year: The NDP are in play here. Could a win by Jason McMichael, a border guard, mean Mulcair is off to 24 Sussex? Maybe. But right now, the Conservatives appear to be holding steady. Mulcair pumps up McMichael's campaign at the Dante Club at 1330 London Road in Sarnia at 4:30 pm.
It's autoworker vs autoworker in Essex. Incumbent Conservative Jeff Watson came from the Chrysler line to his seat in the Commons, a seat he's held since 2004. Now comes Tracey Ramsey to challenge him. Ramsey has spent 20+ years working on the line at Ford's Essex plant, the plant where they build engines for the best-selling F-150 pickup truck. I'll bet you can talk trucks and politics with Ramsey and Mulcair at her campaign office at 71 Talbot Street North at 7 pm ET.
For more about the Predictionator calls here — they're mine — and why and how I put these calls together, please visit: Predictionator? What the heck is that?


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iHerb Customer Reviews
Mrs. Meyers Clean Day, Scented Soy Candle Lavender Scent, 7.2oz
1 person found the following review helpful
Posted by 4619212150583864980 on Oct 15, 2014
氣味很舒服很清香, 但價錢比較貴點, 如如平點更好
fancy smell
1 person found the following review helpful
Posted by 4762160047647720862 on Jul 18, 2015
It looked very much to find a candle like this, the smell is strong and fancy I use it prayer time and give me a feeling happiness بحثت عن شمعه بهذه القوه كثيراً واخيراً وجدت هذه الرائحه خياليه وجميله واستخدمها في اوقات العباده وتعطيني شعور بالسعاده وبكل تأكيد سأعود لاشتريها مجدداً
Hmmmmmm ......
1 person found the following review helpful
Posted by Reviewer3041110 on Jan 03, 2013
This smell is far from lavender and when I first tried it among guests we all agreed that the smell was kind of repulsive like a WC cleaner product. Lavender smell lovers will be hardly dissapointed. Still it is a best quality natural candle and I'm always devoted to Mrs Meyers products except of this lanender candle.
Smells great, fills the room with scent, lasts a long time.
Posted by Reviewer1781635 on May 12, 2011
Reasonably priced compared to other candles on the market.
Mrs. Meyers Clean Day, Soy Candle, Lavender
Posted by Reviewer2612013 on May 29, 2011
Clean smelling, not over-powering like Yankee Candles. Fits nicely on bathroom countertop. Good value for the money.


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Beyond basic study skills: The use of technology for success in college
ABSTRACT Technology has become a fundamental component of both education and work. Yet regardless of perceived benefits, in many cases students do not use technology effectively. One challenge educators confront is how to motivate students to effectively use the technological mediums provided in their classes. The goal of the current study is twofold: to use the Technology Acceptance Model (TAM) to examine two motivators of behavior, ease of use and perceived need, and to assess how they affect students' likelihood of effectively using technology. Second, we evaluate how the match between expectations of the use of technology and the actual student use affect actual classroom performance. To test our hypotheses, college students (N = 384) in introductory psychology classes completed a survey. We also obtained the instructor's perceptions of the need for technology in their class and students' final class grades. Results showed that ease of use and perceived need of technology were related to the frequency of computer use and intentions to use technology. Additionally, findings suggested that technology use, specifically technology deemed important by the instructor, was related to academic success (i.e., final grade in class).
- SourceAvailable from: chih-hung Chung[Show abstract] [Hide abstract]
ABSTRACT: Coinciding with the development and growth of the Internet, there has been a dramatic increase in the application of the Learning Management Systems (LMS) in higher education. University and college campuses should consider evaluating each LMS to ensure that the system meets the requirements and demands of the institution. Therefore, the purpose of this study is to present a model which incorporates the concepts and findings from research on LMS application in higher education. The alternative model was modified based on the Technology Acceptance Model (TAM). In addition, five categories of LMS features for higher education are discussed including: (1) transmitting course content; (2) evaluating students; (3) evaluating courses and instructors; (4) creating class discussions; and (5) creating computer-based instruction. This study reviews and discusses prior research and provides several recommendations including a model of development and design of an LMS for future implementation in higher educational environments.
- [Show abstract] [Hide abstract]
ABSTRACT: This case study investigates the use of online blogs as a teaching tool. A collaborative blog was implemented in parallel classes on group processes in the United States and Germany. Our goal was to connect American and German graduate students by helping them to talk about group communication and meeting behaviors. Collected data included transcripts of the messages, as well as students' evaluations of the blog (collected at the end of the project). Quantitative analyses assessed students' participation rates and the content of their postings. Qualitative analysis examined the use of the blog as a teaching and learning tool. The results showed that students interacted more on the blog than was required by the instructor. Students valued blogging as a new learning experience. We discuss the pedagogical implications of blog usage for teaching about groups and provide recommendations for instructors interested in using blogs in their own courses.Small Group Research 06/2013; 44(4). DOI:10.1177/1046496413487020 · 1.35 Impact Factor
Article: Using technology in higher education: The influence of gender roles on technology self-efficacy[Show abstract] [Hide abstract]
ABSTRACT: The present study examines the relationship between technology self-efficacy among university students and gender roles. Previous research has based differences in technology self-efficacy on biological sex and found significant differences. University students were asked to complete a survey dealing with gender roles and technology self-efficacy. The current study shows that gender roles, specifically masculinity, is the source of this difference in technology self-efficacy, and not biological sex alone. Further, masculinity predicts technology self-efficacy above and beyond what can be explained by other contributing factors such as previous computer hassles and perceived structural technology support.Computers in Human Behavior 07/2013; 29(4):1779–1786. DOI:10.1016/j.chb.2013.02.012 · 2.69 Impact Factor


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The Latest News:
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Tag Archives: Consensus Report
Milwaukee Journal Sentinel: Preserve Soldiers Home; honor vets
Milwaukee Journal Sentinel November 9, 2012 Preserve Soldiers Home; honor vets By Stephanie Meeks and Bob Curry As Wisconsinites observe Veterans Day next week, they should take note of an important but often-overlooked place constructed to serve and honor the men … Continue reading
Posted in News Tagged #MySoldiersHome, Bob Curry, Consensus Report, Dry Hootch, Milwaukee Journal Sentinel, NTHP, Stephanie Meeks Leave a comment
A CAC update from the National Trust and MPA
As we continue to welcome home veterans returning from Iraq and Afghanistan, we are reminded of the critical role that medical, social and emotional services play in adjusting to civilian life after what has been, in many cases, multiple tours … Continue reading
Posted in News Tagged CAC, Consensus Report, Milwaukee Preservation Alliance, MPA, national trust for historic preservation, NTHP 1 Comment


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The Collins Mini Gem English-Norwegian & Norwegian-English Dictionary is an up-to-date, easy-reference dictionary, ideal for learners of Finnish and English of all ages. The dictionary covers about 20,000 words from each language with Collins pre-recorded audio pronunciation for each word.
A search tool
Featuring various search options to help you find the words you need more easily, including:
• A quick search of words while you type
• A 'fuzzy filter' for when you are not sure of the correct spelling of a word
• A 'keyword' search for finding key words within phrasal verbs
• A wild card search facility which can replace a letter or a group of letters
A learning tool
The dictionary app includes a number of features which help you to expand your vocabulary further:
• A 'Favourites' feature allows you to create your own categories and lists of words
• Your last visited words are all saved in your 'History', so that you can quickly look back at recently checked words
• Discover a new word every day with the 'Daily' feature
• Ability to have dictionary Word of the Day as a widget on the device home screen


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|Search | About | Preferences | Interact | Help
|Find any book at the best price.
by Cooper, Geoffrey M.
Publisher:Sinauer Associates
Because molecular and cellular biology is such an exciting and rapidly moving area of science, teaching it at the undergraduate level is a rewarding, yet challenging, task. Designed for use on introductory cell biology courses, this third edition presents current comprehensive science in a readable and cohesive text that students can master in the course of a semester. This edition retains the organization, themes and special features of earlier editions, but it has been updated to reflect scientific advances since publication of the second edition in 2000, including progress made in genome sequencing regulation of programmed cell death and potential medical applications of embryonic cells.
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Weird, ridiculous campaign ideas that we all secretly want to do...,
The best way to make an 'arcane archer' (doesn't have to use the prestige class),
Map choices,
Philosophy of Science and the Scientific Method,
1001 Rules of Golarion,
Game Props.,
Anti-Unchained Screed,
Player keeps pushing the boundaries,
Unsummoner rant thread,
Where does the fox live? (Kitsune),
>>Ask *James Jacobs* ALL your Questions Here!<<,
Lemmy's Custom Weapon Generation System!,
Agents of Shield,
By RAW, in Golarion, is abortion evil?,
Fleshwarped goblinoids,
Reign of Winter,
For The Glory of Icathia Discussion,
PFS CORE - PbP Gameday IV - #5-08 The Confirmation Gameplay,
Out of the Darkness; Into the Light Discussion,
PFS PbP GameDay IV: PFS 03-12 - Wonders in the Weave I,
JZ's Reign of Winter (The Snows of Summer) Gameplay,
"...to cover all the lands in a second darkness!",
KM Devon's Kingmaker {Omega Team},
The Isle of Dread - Discussion,
PFS - PbP Gameday IV: #6-05 Slaveships of Absalom (lvl 1-5),
PFS - PbP Gameday IV - #4-19 [CORE] The Night March Of Kalkamedes (1-5) Starts 19 September 2015,
Operation Typhoon- SpyCraft2 Discussion,
GMTrex's PFS [Normal Mode] 1-40 Hall of Drunken Heroes,
The World of Not-Pandora!,
Looking for a GM for a Solo (maybe Duo) Campaign.,


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Certified Service
Our Certified Service expert technicians can help make maintaining your vehicle simple and convenient. We are not only a one-stop destination for all your vehicle maintenance needs, but we also have convenient hours, competitive service & parts pricing and advanced technology to take the stress out of servicing your vehicle. From oil changes to tire rotations, brakes, batteries and multi-point vehicle inspections, we can handle it all. Simply click on the tab or call to schedule an appointment today.
Hours Of Operation
Chevrolet Service in Fort Wayne, IN
Looking for a trusted Chevrolet service center in Fort Wayne, IN? Drop by Kelley Chevrolet for world-class auto repair and maintenance at affordable rates. At our Fort Wayne, IN Chevrolet service department, we have a team of qualified technicians to help resolve any vehicle-related issues that you may have. From oil change to tire rotation, our well-equipped Fort Wayne Chevrolet service department can handle it all! Visit our website or stop by our showroom for a quick quote!


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United States & Canada Customers: Call 1-800-813-6897
Available 24 hours a day, 7 days a week.
International Customers: Call 1-512-837-4810
Available 24 hours a day, 7 days a week. English language only.
Based off of the Fubuki ZT, the Mitsubishi Rayon Fubuki Z Graphite Wood Shaft features a similar bend profile in a lighter weight option, with progressive torque, and a more standard balance point. All that makes it a very versatile shaft designed to fit a wide range of players and abilities.
Online Shipping Information
Golfsmith.com offers simple, flat-rate shipping to destinations in the continguous U.S. via FedEx.
Our policy is to strive for the best possible combination of price and service for your shipment.
In order to better serve our guests, we will occassionally offer discounted shipping promotions. Any shipping discounts will be applied in your shopping cart.
"Overnight" shipments will arrive the next business day (excluding weekends and holidays) after departing Golfsmith. "2-Day Air" shipments arrive in two business days after departing Golfsmith. On-time delivery for both methods is guaranteed by FedEx. Shipping delays may occur during busy Holidays or in the spring -- we will do our best to accommodate last minute and expedited orders placed during these times.
Most orders are shipped within 48 hours of online placement. Orders must be placed by 9am (CST) Monday-Friday in order to be eligible for that day's "Overnight" or "2-Day Air" shipments. Orders for residential delivery placed on Sunday or Monday will not be shipped until Tuesday and orders for business delivery placed on weekends will not ship until Monday, so please take this into consideration when specifying expedited shipping requests.
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Mitsubishi Rayon
Fubuki ZT 50 .335 Graphite Wood Shaft
Prolaunch 65 .335 Graphite Wood Shaft
Mitsubishi Rayon
Kuro Kage Black HBP 50 .335 Graphite Wood Shaft
Titleist 913 & 910 .335 Shaft Adaptor - RH$45.00
New Decade Multicompound Cord Midsize Black Grip (+1/16")$10.09
RIP Phenom 70 .335 Graphite Wood Shaft
Universal .600" 5" Shaft Extender (PKG Of 6)$15.99
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Unit Iron Ferrule 13.4 OD Black/Silver/Black Dozen$3.99
Radiused Hosel Ferrules-Iron Dozen$2.99
Proforce V2 55 .335 Graphite Wood Shaft
Diablo Hybrid Fluted Ferrule .370$4.00


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Justin T. Dimino Memorial Scholarship Fund established
The Justin T. Dimino Memorial Scholarship Fund has been established as a component charitable fund of the Community Foundation of Orange and Sullivan. This fund was created by Justin's family to honor and memorialize Justin who passed away suddenly on January 9, 2011 at the age of 16. "Justin will forever hold a special place in all our hearts," said Justin's mother, Stephanie Stack.
Justin Dimino was in his junior year at Monroe-Woodbury High School at the time of his passing. According to Justin's family, he was a boy who lit up the room with his smile and personality. Justin was the starting defensive back for the Championship Crusader Football Team at Monroe-Woodbury High School. Football was the love of his life, as was his family and friends.
Through the Justin T. Dimino Scholarship Fund, a scholarship will be awarded annually, beginning in June 2012, the year Justin would have graduated from Monroe-Woodbury High School. The scholarship will be awarded to a graduating member of the varsity football team at Monroe-Woodbury High School who has achieved a B average or better.
The Justin T. Dimino Scholarship Fund is facilitated through the Community Foundation of Orange and Sullivan, a 501(c)(3) public charity that was created to help individuals, organizations, and businesses establish charitable endowment funds to benefit their community. To make a gift to this Fund, please make check payable to CFOS-Justin T. Dimino Scholarship Fund, and mail to the Community Foundation at 30 Scott's Corners Drive, Suite 202, Montgomery, NY 12549. Secure on-line donations may be made to the Justin T. Dimino Scholarship Fund at cfoc-ny.org. For more information, please contact the Community Foundation at (845) 769-9393.
Upcoming Events
Jewish Family Service of Orange County invites you to our "Dine Around"
Come join us for an elegant and unusual evening of dining and entertainment.
Enjoy the company of friends as we traverse the Goshen area from home to home for appetizers, main courses and desserts.
Ellen Solomon and Arlene Solomon Roshko will welcome you for appetizers
Various hosts will serve main courses
Joel Finkelstein and Bonna Horovitz will host you for desserts
Date: Sunday, October 18, 2015
Time: 4:45 p.m.-9:00 p.m.
Minimum donation is $54.00 per person *
RSVP due September 25 (please indicate kashrut or other dietary observance)
Make all checks payable to: Jewish Family Service
Memo: Dine Around
In early October we will send you the address of the home serving appetizers.
Questions? Contact 341-1173 ext. 393 or email firstname.lastname@example.com
*Donations are tax deductable
Come join us for our Fall Ball Illuminate Goshen Fundraising Dinner!
The event will take place at the beautiful and recently expanded Villa Venezia
$75 Tickets include hors d'oeuvres, buffet dinner, and top shelf open bar
All proceeds will go towards purchasing lights to illuminate the downtown Village of Goshen and towards the New Year's Eve Ball Drop
Sponsorship Opportunities Include
$1000 Platinum Bulb Sponsor
$500 Gold Bulb Sponsor
$250 Silver Bulb Sponsor
$35 Glass Bulb
Tickets can be purchased on our paypal page:
Thank you for your continued support of our efforts!
8:30 – 10:30 – Breakfast (Awards and Keynote)
10:30 – 10:45 – Break & Networking
10:45 – 12:15 – Education Sessions #1 (2 simultaneous sessions)
12:15 – 12:30 – Break/Networking/Pick up Brown Bag Lunch
12:30 – 2:00 – Education Sessions #2 (2 simultaneous sessions)
Latest News
- Ted Petrillo Community Fund Established within the Community Foundation
- Job Posting – Director of Development
- Message from Community Foundation's Board Chairman Derrik Wynkoop
- Message from the President and CEO
- Community Foundation of Orange and Sullivan Announces Leadership Transition
- Community Foundation to honor Jeff Bank and Rich Rowley & Marianne Murray
- HONOR Endowment Fund Established within the Community Foundation
- Safe Space America Established within the Community Foundation
- Honigsbaum-Caplan-Bensley Scholarship Available to College Students
- Jocelyn Levner-Nissenbaum Memorial Scholarship Fund Established


text:
rogues--thus." I boxed his ear with my open hand, so that he fell against the wall. "I will now picture also the use of boots by kicking you into the inn yard which is adjacent." So saying I hurled him to the great front door which stood open, and then, taking a sort of hop and skip, I kicked for glory and the Saints.
I do not know that I ever kicked a man with more success. He shot out as if he had been heaved by a catapult. There was a dreadful uproar behind me, and I expected every moment to be stormed by the waiter-and-pot-boy regiment. However I could hear some of the gentlemen bystanding cry:
"Well done! Well kicked! A record! A miracle!"
But my first hours on English soil contained still other festivities. Bright light streamed out from the great door, and I could plainly note what I shall call the arc or arcs described by Forister. He struck the railing once, but spun off it, and to my great astonishment went headlong and slap-crash into some sort of an upper servant who had been
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text:
Recently after a chkdsk and reboot I lost the address bar in Windows Explorer and could not add it back. How do I bring the address bar back?
I have been searching for weeks across many posts and today found a solution that actually works so I wanted to share that:
Open up a new Windows Explorer, the Address Bar should now be visible.
In Windows 7 this seems to happen also in normal mode if "Auto-hide" is activated in full screen mode. Put explorer into full screen mode (F11) and open system menu (alt+space). Uncheck Auto-Hide and return to normal mode (F11).
protected by DragonLord May 8 at 19:28
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