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Copyright © 2006 Elsevier Inc. All rights reserved.
Cell Metabolism, Volume 3, Issue 6, 417-427, 1 June 2006
Add/View Comments (0)Genipin inhibits UCP2-mediated proton leak and acutely reverses obesity- and high glucose-induced β cell dysfunction in isolated pancreatic islets
Chen-Yu Zhang1, 2, 5, , , Laura E. Parton1, 5, Chian Ping Ye1, 5, Stefan Krauss1, 4, Ruichao Shen3, Cheng-Ting Lin3, John A. Porco3 and Bradford B. Lowell1, ,
1 Department of Medicine, Division of Endocrinology, Beth Israel Deaconess Medical Center and Harvard Medical School, 99 Brookline Avenue, Boston, Massachusetts 02215
2 State Key Laboratory of Pharmaceutial Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing, Jiangsu, China
3 Department of Chemistry and Center for Chemical Methodology and Library Development, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215
Ph: 617 667 5954; Fax:617 667 2927
4 Present address: Department of Cancer Biology and Therapeutics, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, Massachusetts 02215
5 These authors contributed equally to this work.
- Uncoupling protein 2 (UCP2) negatively regulates insulin secretion. UCP2 deficiency (by means of gene knockout) improves obesity- and high glucose-induced β cell dysfunction and consequently improves type 2 diabetes in mice. In the present study, we have discovered that the small molecule, genipin, rapidly inhibits UCP2-mediated proton leak. In isolated mitochondria, genipin inhibits UCP2-mediated proton leak. In pancreatic islet cells, genipin increases mitochondrial membrane potential, increases ATP levels, closes KATP channels, and stimulates insulin secretion. These actions of genipin occur in a UCP2-dependent manner. Importantly, acute addition of genipin to isolated islets reverses high glucose- and obesity-induced β cell dysfunction. Thus, genipin and/or chemically modified variants of genipin are useful research tools for studying biological processes thought to be controlled by UCP2. In addition, these agents represent lead compounds that comprise a starting point for the development of therapies aimed at treating β cell dysfunction.
Uncoupling protein 2 (UCP2) is a mitochondrial carrier protein with homology to other uncoupling proteins like UCP1 and UCP3 (Fleury et al., 1997). UCP2 is expressed in a number of tissues, including pancreatic islets (Krauss et al., 2003,Zhang et al., 2001). In previous studies, it has been demonstrated that UCP2 is a negative regulator of insulin secretion (Chan et al., 2001,Zhang et al., 2001). UCP2 mediates proton leak across the inner mitochondrial membrane. This has been demonstrated in studies using proteoliposomes (Echtay et al., 2001,Jaburek et al., 1999), isolated mitochondria (Echtay et al., 2002), and in intact cells (Krauss et al., 2002). In pancreatic β cells, UCP2-mediated proton leak decreases the yield of ATP from glucose (Chan et al., 2001,Zhang et al., 2001). Consequently, UCP2 negatively regulates glucose-stimulated insulin secretion (Chan et al., 2001,Zhang et al., 2001).
As UCP2 is a negative regulator of insulin secretion, it has been proposed that increased UCP2 expression in β cells could result in β cell dysfunction and the development of type 2 diabetes. Consistent with this idea, UCP2 mRNA and protein were found to be markedly increased in pancreatic islets of animal models of type 2 diabetes (Kassis et al., 2000,Laybutt et al., 2002,Winzell et al., 2003,Zhang et al., 2001). There is also evidence that increased amounts of UCP2 expression in humans can downregulate insulin secretion and increase the risk of type 2 diabetes (Krempler et al., 2002,Sasahara et al., 2004,Sesti et al., 2003). It has been shown that UCP2 levels in islets are increased by high glucose and high lipids in vitro (Laybutt et al., 2002,Patane et al., 2002). Of note, gene knockout of UCP2 restores first-phase insulin secretion, increases serum insulin levels, and greatly decreases levels of glycemia in ob/ob mice (Zhang et al., 2001). Furthermore, deficiency of UCP2 prevents obesity-, high glucose-, and high lipid-induced β cell dysfunction (Joseph et al., 2002,Joseph et al., 2004,Krauss et al., 2003). Taken together, these findings indicate that UCP2 is an important mediator of β cell dysfunction. Consequently, a UCP2 inhibitor lacking adverse side effects could be a potentially useful drug for treatment of β cell dysfunction and type 2 diabetes. It is important to note that, at present, no therapy exists for type 2 diabetes that is specifically aimed at a pathogenic cause of β cell dysfunction.
The "classical" regulators of UCPs are fatty acids and purine nucleotides. Purine nucleotides (ATP, ADP, GTP, and GDP) inhibit and fatty acids activate UCP2 and 3 when these are expressed in E. coli and reconstituted into liposomes (Echtay et al., 2001,Jaburek et al., 1999,Zackova et al., 2003). Recently, using mitochondria isolated from yeast and various rodent tissues, Echtay et al., 2002 showed that UCP2 and 3 catalyze proton leak when activated by exogenously added superoxide. In isolated mitochondria, purine nucleotides have been shown to inhibit superoxide-induced, UCP2- or 3-dependent proton leak (Echtay et al., 2002). However, purine nucleotides are not cell permeable and therefore are unable to inhibit UCPs when added to intact cells. The goal of the present study was to discover a UCP2 inhibitor capable of working in intact cells. Such an agent would be an extremely useful investigational tool for studying a number of aspects of UCP2 biology. In addition, a UCP2 inhibitor could theoretically represent a lead compound for agents aimed at improving β cell function in type 2 diabetes.
Stimulation of insulin secretion by Gardenia extract and genipin
The extract of Gardenia jasminoides Ellis fruits has been used over the years in traditional Chinese medicine to treat symptoms of type 2 diabetes (Danbuo, 1984,Wang, 1991). We evaluated Gardenia extract and observed that it dose dependently stimulated insulin secretion by pancreatic islets derived from wild-type (WT) but not from UCP2-deficient mice (data not shown). Because UCP2 was required for this effect, we assumed that Gardenia extract contained a UCP2 inhibitor. To identify the active molecule, Gardenia extract was fractionated using silica gel chromatography (1:1 hexane and ethyl acetate as eluant). Fractions were assessed for UCP2-dependent stimulation of insulin secretion. Using proton NMR and mass spectroscopic analysis, genipin was identified as a molecule within the active fraction. The chemical structure of genipin was confirmed by comparison with an authentic sample (Wako Chemicals USA, Inc.). Genipin, obtained from the commercial supplier noted above, was further tested for stimulation of insulin secretion (detailed studies for genipin presented later). Genipin stimulated insulin secretion, and this stimulation occurred with wild-type but not UCP2-deficient islets. Figure 1A shows the chemical structure of genipin (Djerassi et al., 1961).
Genipin inhibits superoxide-activated, UCP2-mediated mitochondrial proton leak
In order to assess the effect of genipin on superoxide-induced UCP2-mediated proton leak, mitochondria were isolated from kidneys of WT and UCP2-deficient mice, and proton leak was measured in the presence or absence of exogenous superoxide and genipin (Krauss et al., 2003). Exogenous superoxide was generated in situ by adding the enzyme xanthine oxidase and its substrate xanthine (+X/XO). In WT mitochondria, superoxide activated proton leak as indicated by the leftward shift of the proton leak titration curve (Figure 1B, +X/XO [□] versus control [▴]). Addition of genipin completely abolished superoxide-induced proton leak (Figure 1B, +genipin, +X/XO [♦] versus +X/XO [□]). In mitochondria lacking UCP2, addition of superoxide had no effect on proton leak (Figure 1D, +X/XO [□] versus control [▴]). Genipin was without effect on proton leak when either UCP2 (Figure 1D, +genipin, +X/XO [♦] versus +X/XO [□]), or superoxide (Figure 1C, +genipin [♦] versus control [▴]), or both were absent (Figure 1E, + genipin [♦] versus control [▴]). These results demonstrate that genipin inhibits superoxide-induced, UCP2-mediated proton leak. However, unknown from these studies is whether genipin inhibits UCP2 activity directly or whether genipin interferes in some way with the pathway by which superoxide activates UCP2.
Genipin does not scavenge superoxide
Genipin could inhibit UCP2-mediated proton leak by scavenging superoxide. To evaluate this possibility, we assessed the effects of genipin on an in vitro, cell-free superoxide generating system (xanthine + xanthine oxidase plus salmon sperm DNA plus hydroethidine) (Zhao et al., 2003). Genipin did not decrease levels of superoxide as observed by ethidium fluorescence, whereas addition of the enzyme superoxide dismutase (MnSOD) completely abolished this signal (data not shown). Therefore, genipin does not act as a superoxide scavenger.
Genipin inhibits HNE-activated, UCP2-mediated mitochondrial proton leak
Recently, it has been proposed that superoxide-dependent activation of UCP-mediated proton leak occurs via aldehydic lipid peroxidation intermediates such as 4-hydroxynonenal (HNE) (Murphy et al., 2003), which has been shown to directly stimulate UCP-dependent proton leak in isolated mitochondria (Echtay et al., 2003). According to this model, aldehydic peroxidation products such as HNE are generated as downstream products in an activation cascade, proximal to UCP2, and act as activators of UCP2-mediated proton leak (Echtay et al., 2003,Murphy et al., 2003). Thus, genipin could inhibit UCP2-mediated proton leak by interfering in a step in the pathway by which superoxide activates UCP2. In order to assess this possibility, we studied the effect of genipin on HNE-induced UCP2-mediated proton leak in mitochondria isolated from kidney of WT and UCP2-deficient mice (Echtay et al., 2003). In WT mitochondria, HNE activated proton leak (Figure 2A, +HNE [□] versus control [▴]). Preincubation of mitochondria with genipin completely abolished HNE-induced proton leak (Figure 2A, +genipin, +HNE [♦] versus +HNE [□]). In mitochondria lacking UCP2, addition of HNE had no effect on proton leak (Figure 2B, +HNE [□] versus control [▴]). Consistent with experiments shown in Figure 1, genipin had little or no effect on proton leak in mitochondria lacking UCP2 (Figure 2B, +genipin, +HNE [♦] versus +HNE [□]). Taken together, these results demonstrate that genipin inhibits HNE-induced, UCP2-mediated proton leak. Thus, genipin does not interfere with the pathway of activation of UCPs by superoxide but most likely inhibits UCP2 activity directly.
Genipin does not affect other mitochondrial transport functions in liver mitochondria
In order to assess the potential effect of genipin on other mitochondrial carrier proteins, many of which are 20%–30% identical at the amino acid level to UCP2, and to assess effects on mitochondrial integrity in general, we measured respiration in isolated mouse liver mitochondria using various respiratory substrates. During state 3 respiration, fuel oxidation is coupled by ATP synthase to the availability of exogenously added ADP (ADP stimulates proton transport by ATP synthase, reducing backpressure on proton pumps in the electron transport chain, hence stimulating respiration). Therefore, rates of respiration in state 3 are dependent upon continued entry of fuels via various mitochondrial carriers as well as the exchange of extramitochondrial ADP for matrix ATP by the adenine nucleotide carriers. Note that UCP1 and its homologs, UCP2 and 3, are not expressed in liver under normal physiological conditions (Pecqueur et al., 2001,Vidal-Puig et al., 1997), therefore, any observed effects on liver mitochondrial activity would be UCP-independent. Using succinate (Figure 2C) or malate-glutamate (Figure 2D) as substrate, respiration in states 3 and 4 was assessed. In addition, total mitochondrial respiratory capacity was determined by measuring respiration following addition of 1 μM FCCP (a chemical uncoupler that stimulates proton leak). Our results demonstrate that genipin does not acutely affect state 3, state 4, or total (FCCP-stimulated) respiration, indicating that genipin does not affect the activity of the adenine nucleotide carrier or other mitochondrial carriers involved in the metabolism of the substrates tested.
Genipin increases mitochondrial membrane potential, increases ATP levels, closes KATP channels, and stimulates insulin secretion in a UCP2-dependent manner
We previously demonstrated that UCP2 regulates mitochondrial membrane potential in intact cells. This was shown by demonstrating that lack of UCP2, or removal of endogenous superoxide (a UCP2 activator), increases mitochondrial membrane potential in dispersed islet cells (Krauss et al., 2003). In the present study, we investigated whether genipin induced changes in mitochondrial membrane potential in dispersed islet cells, as measured by the mitochondrial membrane potential–sensitive fluorescent dye, TMRM (Figure 3A). Addition of genipin to dispersed islet cells isolated from WT animals induced a rapid increase in mitochondrial TMRM fluorescence over time. In contrast, addition of genipin to dispersed islet cells lacking UCP2 had no effect on mitochondrial TMRM fluorescence. The detected TMRM signal was greatly reduced by addition of the chemical uncoupler, FCCP, indicating that it accurately reported mitochondrial membrane potential. Thus, addition of genipin induced a rapid increase in mitochondrial membrane potential in dispersed islet cells, and this effect required the presence of UCP2. These findings demonstrate that genipin inhibits UCP2-mediated proton leak, which then leads to an increase in mitochondrial membrane potential.
An increase in mitochondrial membrane potential is predicted to stimulate ATP production by ATP synthase and increase levels of ATP. This was tested by assessing effects of genipin on ATP levels in incubated islets. As is shown in Figure 3B, genipin increased ATP levels in WT islets but not in islets isolated from UCP2-deficient mice. In contrast, 20 mM glucose increased ATP levels in both WT and UCP2-deficient islets. Thus, acute inhibition of UCP2 increases ATP levels in islet cells.
In pancreatic β cells, ATP closes plasma membrane KATP channels which depolarizes the cell, opening voltage gated calcium channels, ultimately stimulating insulin secretion. In the present study, we assessed whether acute inhibition of UCP2 by genipin would, as predicted, close KATP channels. To address this issue, currents (Figure 3C(ii), bottom tracing) were recorded in response to alternate ±20 mV voltage steps (Figure 3C(ii), top tracing) from a holding potential of −70 mV, using the "perforated-patch" configuration (Sakura et al., 1998). To evaluate the density of KATP channels, currents (pA) were recorded in the presence of zero glucose and were normalized to cell capacitance (pF) to control for possible differences in cell size. These studies indicated that the density of KATP channels is similar in wild-type versus UCP2-deficient cells (pA/pF, mean ± SE, WT = 12.8 ± 1.8 [n = 5], UCP2−/− = 13.2 ± 2.2 [n = 5]). Representative current traces showing the effects of glucose, genipin, and tolbutamide (specific KATP channel blocker) are shown in Figure 3C(i) and the summary of these recordings is shown in Figure 3C(iii). As can be seen, genipin closed KATP channels in WT but not in UCP2-deficient cells. The absence of an effect of genipin in cells lacking UCP2 argues against a direct effect of genipin on KATP channels. In contrast, both 10 mM glucose and tolbutamide closed KATP channels in both WT and UCP2−/− cells, indicating that KATP channels function normally in UCP2-deficient cells. Thus, inhibition of UCP2 by genipin increases mitochondrial membrane potential, increasing the level of ATP, which then closes KATP channels.
UCP2 is a negative regulator of insulin secretion (Zhang et al., 2001). Thus, deficiency of UCP2 increases insulin secretion in vitro and in vivo (Krauss et al., 2003,Zhang et al., 2001). In the studies above, we show that genipin inhibits UCP2 activity in isolated mitochondria and intact cells. Thus, it is predicted that genipin would stimulate insulin secretion. We therefore assessed whether genipin affects insulin secretion in islets (Figure 4). Genipin dose dependently increased insulin secretion in WT islets (Figure 4, left panel). In contrast, genipin had no effect in islets deficient in UCP2 (Figure 4, right panel). Taken together, these studies show that genipin acutely inhibits UCP2 activity, which then increases mitochondrial membrane potential, increases ATP levels, closes KATP channels, and consequently, stimulates insulin secretion.
Genipin reverses high glucose- and obesity-induced β cell dysfunction
Impaired secretion of insulin by pancreatic β cells, together with resistance to insulin action, causes type 2 diabetes. A large body of work has established that chronic hyperglycemia and obesity cause loss of glucose-stimulated insulin secretion (GSIS) (Matthews and Clark, 1997,Robertson, 2000). We have shown that deficiency of UCP2, or removal of endogenously produced superoxide (a UCP2 activator), prevents high glucose- and obesity-induced loss of GSIS (Krauss et al., 2003). Here, we assessed the effect of genipin on high glucose- and obesity-induced β cell dysfunction. Pancreatic islets were isolated from WT (Figure 5A) or UCP2 KO mice (Figure 5B) and subjected to chronic incubations (48 hr) in low (5.5 mM) or high (25 mM) glucose. Following chronic incubation, islets were washed and insulin-secretion studies were performed using three different concentrations of glucose (5.5, 12.5, and 25 mM), with or without genipin present (5 μM).
WT islets incubated chronically at low glucose released increasing amounts of insulin in response to increasing concentrations of glucose (Figure 5A, left panel). Following chronic incubation in high glucose, islets increased basal insulin release but were completely unresponsive to glucose stimulation (Figure 5A, middle panel). These data are consistent with other reports and our previous results (Krauss et al., 2003) showing that chronic high glucose increases basal insulin secretion and, at the same time, causes complete loss of glucose responsiveness. To assess whether genipin could restore high glucose-induced loss of glucose responsiveness, we acutely added 5 μM genipin to islets during the insulin-release portion of the study. Acute addition of genipin reversed the high glucose-induced block in GSIS (Figure 5A, right panel). To see whether this effect of genipin was mediated by UCP2, islets were isolated from UCP2 KO mice and then incubated in chronic high glucose. Following chronic incubation in low glucose (5.5 mM), UCP2 KO islets, like WT islets, released increasing amounts of insulin in response to increasing concentrations of glucose (Figure 5B, left panel). Unlike WT islets, however, islets from UCP2 KO mice, when incubated chronically at 25 mM glucose, retained glucose responsiveness (Figure 5B, middle panel). In addition, genipin reduced the elevation in basal insulin secretion. Their pattern of response was similar to that observed in WT islets incubated with genipin (Figure 5A, right panel). Importantly, in UCP2 KO islets, genipin had no additional beneficial effect on GSIS (Figure 5B, right panel), indicating that improved glucose responsiveness is caused by genipin-dependent inhibition of UCP2.
Because absence of UCP2 in ob/ob islets restores glucose sensing, and because removing the activator of UCP2 (superoxide) in dysfunctional islets similarly restores islet function (Krauss et al., 2003,Zhang et al., 2001), we reasoned that genipin (as an inhibitor of UCP2) would reverse obesity-induced β cell dysfunction. Thus, we studied insulin secretion in islets from ob/ob mice and from ob/ob mice lacking UCP2 in the absence or presence of genipin. Islets from ob/ob mice showed elevated basal insulin secretion and loss of glucose responsiveness compared with WT islets (Figure 5C, first set of bars, versus Figure 5A, left panel), similar to what was observed in WT islets following chronic high-glucose treatment (Figure 5A, middle panel). Genipin acutely restored glucose sensing in ob/ob islets (Figure 5C, second set of bars). In addition, genipin reduced the elevation in basal insulin secretion. Islets isolated from ob/ob mice lacking UCP2 were responsive to glucose (Figure 5C, third set of bars), similar to ob/ob islets treated with genipin, and also similar to WT islets treated with genipin (Figure 5A, right panel) or UCP2-deficient islets during exposure to high glucose (Figure 5B, middle panel). Of note, islets from ob/ob mice lacking UCP2 treated with genipin did not improve glucose sensing above the level seen in islets lacking UCP2 alone (Figure 5C, last set of bars). Therefore, genipin acutely improves the significant obesity-induced impairment of GSIS.
A genipin derivative, 1, 10-anhydrogenipin (AG), inhibits proton leak, inhibits KATP channels, and stimulates insulin secretion in a UCP2-dependent manner
Genipin is a naturally occurring protein cross-linking agent (Fujikawa et al., 1988,Huang et al., 1998). To investigate if this action of genipin is required for inhibition of UCP2, we set out to synthesize a derivative of genipin that lacks cross-linking activity. It has been reported that genipin can be dimerized by equimolar amounts of glycine to form the blue pigment, genipocyanin G1, a highly conjugated dimeric adduct (Figure 6A, structure 1) (Fujikawa et al., 1987). It has also been reported that cytochrome c can be cross-linked by genipin to form oligomers and that the cross-linking process likely involves formation of complexes in which two primary amine groups from separate proteins react with genipin (Figure 6A, structure 2) (Fujikawa et al., 1988). As the C10 primary alcohol and the C1 hemiacetal of genipin are likely required for the formation of the oligomeric products (Touyama et al., 1994), it is conceivable that genipin derivatives lacking these two active sites may lose cross-linking activity. To confirm this prediction, a dehydrated derivative, 1,10-anhydrogenipin (AG, Figure 6B) was prepared from genipin. We performed a simple color assay by treating the synthetic derivative, AG, or genipin, with an equimolar amount of glycine in pH 7 buffer at 80°C. After 4 hr, the AG solution, but not the genipin solution, was still clear, which indicates that dimerization to form conjugated genipocyanin G1-type blue pigments did not occur with AG. To confirm that AG lacks protein cross-linking activity, the previously described cytochrome c cross-linking assay was performed (Fujikawa et al., 1988) with genipin and AG. This study, shown in the Supplemental Data available with this article online, revealed that genipin but not AG oligomerizes cytochrome c. Thus, as predicted, AG lacks protein cross-linking activity.
We then assessed the effects of AG on (1) UCP2-mediated proton leak in isolated mitochondria, on (2) KATP channel activity in dispersed islet cells, and on (3) insulin secretion from isolated islets. As previously observed, superoxide stimulates proton leak (Figure 6C, +X/XO [□] versus control [▴]). As with genipin, AG inhibited superoxide-stimulated proton leak (Figure 6C, +AG, +X/XO [♦] versus +X/XO [□]). Also, like genipin, AG had no effect on proton leak when superoxide was absent (Figure 6D, +AG [♦] versus control [▴]). As shown in Figure 7A, AG inhibited KATP channel activity in wild-type cells but not in UCP2-deficient cells. Finally, as shown in Figure 7B, AG stimulated insulin secretion in wild-type islets but not in UCP2-deficient islets. Comparison between the effects of genipin and AG, at the same doses, on KATP channel activity (10 μM) and on insulin secretion (5 μM, genipin data from Figure 4 plotted again in Figure 7B to facilitate comparison) raises the possibility that AG, while having the same activities as genipin, may be less potent. This apparent decrease in potency could be due to loss of cross-linking activity but more likely is related to other properties of AG that may affect its solubility, its ability to cross membranes, or possibly its interaction with UCP2. Additional studies are necessary to differentiate these possibilities. Despite these issues, these findings suggest that protein cross-linking activity is not required for inhibition of UCP2 by genipin-related compounds.
In the present study, we identified a molecule, genipin, which inhibits UCP2-mediated proton leak. In isolated kidney mitochondria, genipin specifically inhibits UCP2-mediated proton leak. In pancreatic islets, genipin increases mitochondrial membrane potential, increases ATP levels, closes plasma membrane KATP channels, and stimulates insulin secretion in a UCP2-dependent manner. Importantly, acute inhibition of UCP2 by genipin reverses high glucose- and obesity-induced β cell dysfunction.
Prior to this discovery, purine nucleotides (ATP, ADP, GTP, and GDP) were the only known inhibitors of UCPs (Echtay et al., 2001,Jaburek et al., 1999,Zackova et al., 2003). However, purine nucleotides are impermeable to cell membranes and therefore are not useful for studies in whole cells or animals. Superoxide is an important, physiologically relevant regulator of UCPs (Echtay et al., 2003). Recently, it has been proposed that superoxide-dependent activation of UCP-mediated proton leak occurs via aldehydic lipid peroxidation intermediates such as 4-hydroxynonenal (HNE) (Murphy et al., 2003), which have been directly shown to stimulate UCP-dependent proton leak in isolated mitochondria (Echtay et al., 2003). Our results show that genipin inhibits HNE-induced, UCP2-mediated proton leak, suggesting that genipin does not inhibit UCP2 by interfering with upstream components of the suggested superoxide activation pathway. Thus, we speculate that genipin inhibits UCP2-mediated proton leak by directly interacting with UCP2. Given the high degree of homology between UCP2 and UCP3, genipin may also inhibit UCP3-mediated proton leak. Our preliminary studies indicate that this could be the case. However, additional work is required to confirm this possibility.
Genipin is a naturally occurring cross-linking agent. It is theoretically possible that the cross-linking activity of genipin could be required for inhibition of UCP2. In addition, the cross-linking activity could produce adverse, nonspecific effects due to interaction with other proteins. For these reasons, we synthesized a genipin derivative, AG (Figure 6B), that lacks protein cross-linking activity. Of interest, AG inhibits UCP2-mediated proton leak, closes KATP channels, and stimulates insulin secretion in a UCP2-dependent fashion. These results suggest that the cross-linking activity of genipin is not required for its biological activity as a UCP inhibitor.
A PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) search for papers on genipin suggests a variety of activities for this compound. It is possible that these other activities are secondary to inhibition of UCP2. This possibility could be critically tested by using UCP2-deficient animals and cells to determine if the presence of UCP2 was required for these effects. Alternatively, and more likely given what is known about the biology of UCP2, these other activities are probably unrelated to inhibition of UCP2. This contention is supported by the fact that most of these other activities are observed at concentrations of genipin significantly higher than those employed in the present study. This raises the possibility that these other actions may be mediated by the cross-linking activity of genipin. If this is the case, then derivatives of genipin, like AG, that lack cross-linking activity but retain UCP2-inhibiting activity, could be useful UCP2 inhibitors.
β cells sense glucose through its metabolism and the resulting increase in ATP, which closes KATP channels depolarizing the cell, increasing Ca2+ influx, ultimately stimulating insulin secretion. Uncoupling protein 2 (UCP2) mediates mitochondrial proton leak, decreasing ATP production. It has previously been established that UCP2 negatively regulates glucose-stimulated insulin secretion (Chan et al., 2001,Zhang et al., 2001). Of pathophysiologic significance, UCP2 was markedly upregulated in islets of ob/ob mice (Zhang et al., 2001), a model of obesity-induced diabetes. Importantly, ob/ob mice lacking UCP2 had restored first-phase insulin secretion, increased serum insulin levels, and greatly decreased levels of glycemia (Zhang et al., 2001). These results establish UCP2 as a critical link between obesity, β cell dysfunction, and type 2 diabetes. Recently, we have demonstrated that endogenously produced superoxide activates UCP2 (Krauss et al., 2003). Interruption of the superoxide-UCP2 pathway improves high glucose- and obesity-induced impairment of glucose-stimulated insulin secretion (GSIS) (Krauss et al., 2003). In the present study, we demonstrate that acute exposure to genipin reverses high glucose- and obesity-induced loss of GSIS. A novel feature of the present study is the rapidity of this effect. Importantly, genipin is without effect in islets lacking UCP2, indicating that genipin's ability to reverse high glucose- and obesity-induced β cell dysfunction is explained by its ability to inhibit UCP2.
Exposure to either chronic high glucose or the obese state produces an elevation in basal insulin secretion, in addition to causing loss of glucose sensing. This has previously been observed by us (Krauss et al., 2003) and others (Bjorklund et al., 2000,Patane et al., 2002). Chronic exposure to free fatty acids similarly elevates basal insulin secretion while causing loss of glucose sensing (Joseph et al., 2004). The reason why basal secretion is elevated in these states of β cell dysfunction is not known. It is interesting to note that inhibition of UCP2 by means of gene knockout (Joseph et al., 2004,Krauss et al., 2003), removal of superoxide (Krauss et al., 2003), or genipin (present study) improves both abnormalities (i.e., the increased basal insulin secretion and the loss of glucose-stimulated insulin secretion). Just as it is unknown why these states cause an increase in basal insulin secretion, it is similarly unknown why inhibition of UCP2 corrects this abnormality. One possible explanation is that increased UCP2 activity, associated with states of β cell dysfunction, impairs the ability of the cell to keep cytosolic Ca2+ concentrations low. This could happen because increased UCP2 activity reduces ATP levels, which powers Ca2+-ATPases to pump Ca2+ out of the β cell and also into the endoplasmic reticulum (Rizzuto and Pozzan, 2006). In addition, increased UCP2 activity reduces the mitochondrial membrane potential, the driving force for Ca2+ entry into mitochondria. Consistent with this possibility, it has previously been shown that chronic hyperglycemia markedly elevates basal cytosolic Ca2+ concentrations (Bjorklund et al., 2000,Taylor et al., 2005). If this hypothesis is correct, then inhibition of UCP2 in states of β cell dysfunction would be expected to reduce basal cytosolic Ca2+ concentrations and hence basal insulin secretion while, at the same time, restoring glucose-stimulated insulin secretion. This hypothesis, regarding the role of increased UCP2 activity in augmenting basal insulin secretion in states of β cell dysfunction, will need to be evaluated in future studies.
Impaired glucose-stimulated insulin secretion by pancreatic β cells, together with resistance to insulin action, causes type 2 diabetes. Current treatment for diabetes mellitus begins with diet and exercise. However, compliance can be poor and, as the disease progresses, treatment with sulfonylureas, thiazolidinediones, or metformin, is often necessary. Of interest, none of these pharmacologic treatments are specifically directed at a pathogenic cause of pancreatic β cell dysfunction. Also, in many patients, these pharmaceutical agents prove inadequate for maintaining blood glucose at an acceptable level and injection with insulin is necessary. Therefore, a need exists for new therapies, and in particular, agents that specifically target pancreatic β cell dysfunction. Given that increased UCP2 activity in pancreatic β cells causes high glucose- and obesity-induced loss of glucose sensing, and that genipin inhibits UCP2 activity and acutely improves high glucose- and obesity-induced β cell dysfunction, genipin or analogs of genipin represent lead compounds that comprise a novel starting point for the development of therapies aimed at a pathogenic cause of pancreatic β cell dysfunction.
Experimental procedures
Isolation of mitochondria, proton leak titrations, and mitochondrial respiration studies
See Supplemental Data for detailed methodology.
Preparation of dispersed islet cells and imaging of mitochondrial membrane potential
See Supplemental Data for detailed methodology.
Patch-clamp electrophysiology
Experiments were carried out on dispersed islet cells, using the perforated-patch configuration, which retains cell metabolism intact (Sakura et al., 1998). Currents flowing through KATP channels were monitored using alternate 20 mV pulses of 200 ms duration from a holding potential of −70 mV. Pulses were applied every 2 s. Whole-cell currents were recorded using a MultiClamp 700B Amplifier and pClamp 9.2 software (Axon Instrument). Recording electrodes had resistances of 2.5–4 MΩ when filled with the following solution: containing 70 mM K2SO4, 10 mM NaCl, 10 mM KCl, 21 mM MgCl2, 10 mM HEPES, and 40 mM sucrose (pH 7.2 with KOH). For perforated-patch recordings, 0.15–0.2 mg/ml amphotericin B was added to the pipette solution. The standard external solution consisted of: 140 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 2.6 mM CaCl2, 10 mM HEPES, and 2 mM glucose (pH 7.4 with NaOH). Bath solutions containing either 2 mM glucose, 10 mM glucose, or 2 mM glucose plus 10 μM genipin, 10 μM AG (1,10-anhydrogenipin), or 0.2 mM tolbutamide were applied to the recording chamber through perfusion. All experiments were carried out at room temperature (22°C –24°C).
ATP measurements
Following overnight culture in RPMI 1640 (11 mM glucose, 7.5% FCS, and 1% penicillin-streptomycin), batches of five size-matched islets were preincubated for 30 min in 1 ml KRHB containing 5 mM glucose and then incubated for 1 hr in 1 ml KRHB containing either 5 or 20 mM glucose or 5 mM glucose plus 10 μM genipin. At the end of this incubation, islets were disrupted with trichloracetic acid (5% vol/vol final) and vortexed for 10 s. Following a 5 min incubation on ice, extracts were centrifuged (10,000 × g, 5 min) at 4°C. The supernatant (400 μl) was mixed with 1.5 ml of diethyl ether and the ether phase containing the trichloroacetic acid was discarded. This step was repeated a further three times. Extracts were diluted with 400 μl of buffer containing 20 mM HEPES, 3 mM MgCl2, (pH 7.75), and stored at −80°C. ATP was measured in duplicate using a luciferin-luciferase bioluminescent assay (Sigma). Light emission was recorded for 30 s using a photon counting luminometer (Victor2; PerkinElmer, Wellesley, Massachusetts). ATP concentration was obtained by comparison to known standards (0–100 pmol) prepared in parallel with the cell extracts.
Islet insulin secretion
Islets were isolated as previously described (Krauss et al., 2003) and incubated overnight in RPMI 1640 (11 mM glucose, supplemented with 7.5% FCS and 1% penicillin-streptomycin). On the next day, islets were transferred into DMEM (5.5 mM glucose plus 0.1% gelatin) with or without genipin (0 nM, 50 nM, 0.5 μM, or 5 μM) or the derivative AG (5 μM). After 1 hr of incubation, medium was removed for measurement of insulin concentrations. Insulin levels in the medium were determined as described previously (Zhang et al., 2001) except that a rat-insulin ELISA kit (Crystal Chem Inc., Chicago, Illinois) was used.
Islet incubation in chronic high glucose
Isolation of islets and incubation conditions
Islets were isolated and incubated overnight as described above. On the next day, all islets were transferred to RPMI 1640 (5.5 or 25 mM glucose, 7.5% FCS) and incubated for 48 hr at 37°C. After chronic incubation, all islets were washed three times with DMEM. Medium-sized islets (of the same size) were used for determination of insulin release. For each of the different conditions (chronic incubation at 5.5 and 25 mM glucose), three islets were transferred to an Eppendorf tube containing 1 ml of fresh DMEM (5.5, 12.5, and 25 mM glucose plus 0.1% gelatin) with or without 5 μM genipin and incubated at 37°C for 1 hr. For each condition in every experiment, 5–8 replicates were studied (i.e., 5–8 Eppendorf tubes, each containing 3 islets). After 1 hr of incubation, an aliquot of medium was removed for insulin assay.
Insulin-secretion studies in ob/ob islets
ob/ob mice lacking UCP2 were created as described previously (Zhang et al., 2001,Krauss et al., 2003). Male mice aged 16–20 weeks were used for islet studies. For each genotype, islets from three mice were pooled for insulin-release studies. After being isolated and incubated overnight in RPMI, 11 mM glucose, the islets were washed three times with DMEM and then placed into Eppendorf tubes (1 islet per tube), with each tube containing 1 ml of fresh DMEM (5.5, 12.5, and 25 mM glucose plus 0.1% gelatin) with or without 5 μM genipin, and incubated at 37°C for 1 hr. Islets of similar size were used for determination of insulin release with 5–10 replicates analyzed per condition. Multiple experiments were performed. The results of a representative experiment are shown in Figure 5C.
Genipin and 1,10-anhydrogenipin
Genipin was obtained from Wako Chemicals USA (http://www.wakousa.com, catalog # 078-0302). Preparation of 1,10-anhydrogenipin (AG): Genipin (5 g, 22.1 mmol) and triphenylphosphine (6085 mg, 23.2 mmol) were dissolved in 110 ml of distilled methylene chloride. To the solution at 0°C was added diisopropyl azodicarboxylate (4.60 ml, 23.2 mmol) dropwise. The resulting mixture was stirred at the same temperature for 30 min. Concentration in vacuo and purification on silica gel using hexane/EtOAc (10:1–4:1) as eluant afforded 1.9 g of AG as a white solid (41% yield, >99% purity). IR (neat): 2950, 1700, 1634, 1437, 1287, 1189, 1075, 1038, 891 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.48 (1 H, s), 5.54 (1H, br s), 5.30 (1H, d, J = 4.4 Hz), 4.45 (1H, br d, J = 12.8 Hz), 4.36 (1H, br d, J = 12.8 Hz), 3.71 (3H, s), 3.62 (1H, br s), 3.27 (1H, dd, J = 14.4, 6.8 Hz), 3.22 (1H, br d, J = 16.0 Hz), 2.69 (1H, br d, J = 16.0 Hz) ppm; 13C NMR (100 MHz, CDCl3): δ 167.7, 153.0, 143.3, 121.6, 107.7, 96.0, 64.7, 54.5, 51.0, 46.2, 32.1 ppm; HRMS (CI): calculated. for C11H12O4 208.0736, found 208.0696.
This work was supported by grants from the National Institutes of Health (to B.B.L.) and the American Diabetes Association (Junior Faculty Award), National Natural Science Foundation of China (Outstanding Young Scientist Award 30225037), IRP0430, and National Basic Research Foundation of China (973 Program, 2004CB518603) (to C.-Y.Z). J.A.P, Jr. thanks the National Institutes of Health (GM-62842) and Bristol-Myers Squibb (Unrestricted Grant in Synthetic Organic Chemistry) for research support.
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Publication Information
Received: September 1, 2005
Revised: December 3, 2005
Accepted: April 10, 2006
Published: June 6, 2006


text:
MSN Program Requirements
Students are eligible to graduate after fulfilling the requirements described below. Students who enroll in another college or university without prior permission of the Dean of Graduate Studies are considered to have withdrawn from SBU. Students wishing to transfer work from another institution and not withdraw from SBU must obtain prior approval from the Director of Graduate Studies within the College of Business and Computer Science.
Comprehensive Examination
Each student is required to pass a comprehensive examination at the end of the study for the degree and must be completed at the conclusion of the course during the last semester of study.. The examination will test the extent to which the student can analyze, synthesize, evaluate and apply that which has been learned from the various graduate study experiences. Responses on the examination will be evaluated by a committee of graduate faculty. The examination will be administered by the College of Nursing faculty.
Program of Study and Academic Regulations
The MSN degree program will consist of 36-39 credit hours. The direct care specialty of nursing education will be 39 hours. The MSN program is based on the belief that graduate education builds on previous knowledge, experience and degrees. The following plan of study provides the MSN student with core knowledge and concepts from which to build a specialty practice in nursing education and administration.
MSN Core Requirements
Master of Science in Nursing Core Classes (15 hours)
Course | Hours |
NUR 5013 Nursing Science | 3 |
NUR 5033 Concepts of Advanced Practice Nursing | 3 |
NUR 5043 Ethics and Policy Issues in Advanced Practice | 3 |
NUR 5083 Nursing Research | 3 |
NUR 5123 Information Systems | 3 |
Master of Science in Nursing Education (24 hours)
Course | Hours |
NUR 5053 Foundational Perspectives of Education | 3 |
NUR 5213 Teaching and Learning Principles of Nursing Education . | 3 |
NUR 5223 Evaluation in Nursing Education | 3 |
NUR 5313 Advanced Pathophysiology | 3 |
NUR 5323 Advanced Health Assessment & Promotion | 3 |
NUR 5333 Advanced Pharmacology | 3 |
NUR 5503 Clinical Practicum | 3 |
NUR 5523 Project/Practicum in Nursing Education | 3 |
Nurse Education Specialty Outcomes
- Utilize critical thinking skills to integrate education theories, technology, and professional standards into the design, implementation and evaluation of curriculum
- Determine interventions to evaluate learning and curricular outcomes in nursing education
- Analyze ethical, political, organizational, cultural, technological, and professional influences on nursing
education- Develop research based educational strategies for multicultural diverse students
- Integrate the principles of Christian discipleship and a Christian worldview into the development of
nursing education systems
Master of Science in Nursing Administration (21 hours)
Course | Hours |
NUR 5023 Health Care Finance | 3 |
NUR 5073 Human Resource Management in Health Care | 3 |
NUR 5093 Organizational Analysis and Innovation | 3 |
NUR 5103 Health Care Systems | 3 |
NUR 5113 Advanced Health Care Leadership & Administration | 3 |
NUR 5153 Quality and Safety in Health Care | 3 |
NUR 5523 Practicum in Nursing/Administration | 3 |
Nursing Administration Specialty Outcomes
- Utilize critical thinking skills to determine effective strategies that stimulate change within nursing that
lead to a more effective management of health care delivery systems- Collaborate with interdisciplinary teams using advance communication to promote health care systems
growth and effectiveness for diverse populations- Apply the principles of nursing and leadership theories, economic and finance principles, business
management practices, and cultural competency to the delivery of health care- Demonstrate leadership qualities and administrative expertise in making value based accountable decisions in the management, coordination, and delivery of nursing care
- Integrate the principles of Christian discipleship and a Christian worldview into the management of
personnel, health care resources, and access to care and attention to vulnerable populations

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Steve Boxer: "They once wanted to emulate films. But now it is the movie industry that is adopting the technology of video games" ...
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The traditional Indian bread. This video shows you the easiest way to make the standard whole wheat Indian flour bread known as the chapati. This is a signature bread used in almost all Indian households and is the primary bread which can be taken with meat,... - 126.902
Indian Chapati is a staple of every north Indian household. This flat bread made from wheat flour is not easy to make the first time, but when you get the hang of it, you will love to make it again and again. The soft texture will linger in your mouth all the... - 113.942
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Chapati is a staple in much of India and Pakistan, and also in other parts of SE Asia and Africa as well. It is often made fresh for each meal and even children learn to make this basic and easy bread. Often, the finished chapatis are brushed with ghee... - 42.2697
GETTING READY 1) Combine and sift the flours. 2) Add enough water to make a stiff dough. Set aside for 1 hour. 3) Knead the dough with a little water until soft. 4) Divide into small balls and roll into very thin pancakes, 4 to 6 inches in diameter. (You may... - 41.0519
GETTING READY 1) In a bowl, add all ingredients to combine thoroughly. MAKING 2) Place a well oiled skillet to heat up over a medium flame. 3) Pour in by 3 T of batter and spread in a round shape with 6" in diameter. 4) Cook to brown on both... - 40.1918
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Finely chop the fresh ginger root. Heat the oil in a medium skillet. Saute onions with ginger root or ground ginger. Add black pepper, turmeric powder, red chili pepper, and coriander seed powder. With a fork, mash the potatoes, and place in frying pan with... - 39.386
GETTING READY 1) In your food processor, use METAL BLADE setting. 2) In the work bowl, add flour and salt. Pulse on/off to mix. 3) Pour in oil to process to a coarse meal. 4) Pour in water in a slow steady stream, while the machine is running. 5) Form a stiff... - 37.9249
Chapatti With Onion has a fine taste. Chapatti With Onion gets its taste from flour mixed with onions and green chilies. Chapatti With Onion is inspired by many food joints worldwide. - 37.9058
Sift the flour into a deep bowl. Make a well in the centre, add the salt and water, and mix together to form a soft dough. Gather the dough from the sides of the bowl and knead with the back of your fist until the dough is pliable. Cover and leave to stand... - 36.9971
MAKING 1. Take a bowl and add maida and water into it. Shape the mixture into stiff dough and set it aside. 2. Take a skillet and melt ghee in it. Add semolina and fry it until it turns golden. 3. Stir in 1 cup water and cook until the semolina dries. 4.... - 36.89
MAKING 1. Take a skillet, add cleaned til to it and roast until it turns brown in color. 2. Add gram flour to the skillet and roast until it turns red. 3. Grate the jaggery on fine grater and remove all its lumps. 4. Take a bowl and add roasted til, gram... - 36.7608
MAKING 1 In a bowl, sift flour and mix it with salt. 2 Gradually pour in water, enough to make a soft, but not sticky dough. 3 Knead well and cover with a wet cloth. 4 Keep aside for an hour. 5 Knead it again. 6 Form small balls from the dough. 7 Roll... - 35.2031
Combine the flour and salt in a bowl. Gradually mix in the water until the dough binds together, but is not sticky or wet. Knead well for about 10 minutes, until firm and elastic. Oil a bowl, turn the dough in it twice, then cover with a damp tea-towel. Leave... - 35.1518
Chapatis a popular Indian bread that goes well with vegetarian and non vegetarian curry. Chapathi is an essential part of Indian diet and is perfect for your dinner! Try them! - 35.104
Place the flour in a bowl. Slowly add up to 1/2 cup water and mix until all the flour adheres and you can knead it. (You will probably need a little less than the 1/2 cup water.) Now knead it for 7 to 8 minutes. Roll into a ball. Cover with a damp cloth and... - 34.8378
MAKING 1. Take a medium-sized bowl and add salt and flour into it. Add some water and stir until the flour is moist and dough becomes stiff and blended. 2. Spread it over lightly floured board or pastry cloth and knead for another 5 minutes until the dough is... - 34.6981
Sift together flours and salt. Gradually add water as for pie crust, to make a dough of pie crust consistency. Turn out onto a floured board. Divide into about eight equal portions. Roll very thin. Heat a dry cast-iron skillet to medium hot. Cook, one at a... - 33.6925
In a large bowl, sift flours together. Add enough water to make a stiff dough and set aside 1 hour. Knead dough with a little water until soft. Divide in to twelve small balls. Roll into very thin pancakes 4 to 6 inches in diameter, or use a tortilla... - 33.5735
Blend flour, salt, and butter or margarine in Bosch bowl. Add enough water to make a stiff dough. Knead until smooth and elastic. Cover with a damp cloth, and let rest 30 minutes. Form dough into 1 inch balls. Roll out paperthin, and bake on a lightly greased... - 33.274
Cook channa daal in the pressure cooker or a big pan .and close the lid till water is goes keep on stiring ,then add jaggery to it and cook till it become soft, Grind them without using water. Combine cardamom with it and mix well with hand to get a soft... - 32.6847
Mix together all the ingredients in a mixing bowl until a firm dough is formed. Cover and refrigerate overnight. Divide the dough into walnut-sized balls. On a lightly floured surface roll out each ball about 1/4-inch thick. On a hot iron griddle, grill each... - 31.9047
Put the flour and salt into a large mixing bowl and make a hollow in the centre. Add the water gradually, working in the flour to make a firm dough. Knead well for about 15 minutes, until the dough is smooth. Cover with a damp cloth and leave to stand for... - 31.2659
mix all ingredients and make dough.Beat the dough for about 1 minute, until it becomes somewhat smooth and elastic. Divide the dough into 16 equal parts. Roll each part into an 8" round on a floured surface. Bake each chapati in an ungreased skillet over... - 30.9222
Mix the flour in a bowl with as little of the water as possible to make a stiff dough. Knead the dough very thoroughly so that it becomes evenly blended and pliable. If necessary, a little more water may be worked in. Replace the dough in the bowl, cover with... - 30.6995
Pinch off two small pieces of dough, each about the size of a walnut. Roll out to a small circle about 3" in diameter. On one side of one circle, smear a little oil. Put the other circle down on the oily side. Press down lightly. Dust with dry flour. Roll out... - 29.9622
Chapatis is a form of bread but different in shape and taste. The flour and perfect quantity of water gives the Chapatis a prefect shape and taste. Must catch it. - 29.3862
Pinch off a golf-ball-sized piece of the bread dough. Roll it out very thin on a well-floured surface. Cook over low heat in skillet (try cast iron with no oil) until browned. Turn and cook the other side. Refrigerate the dough so you can make just as many... - 27.8289
Mix whole wheat flour, corn flour and salt together. Add water gradually, adding just enough to keep dough from sticking to hands (about 1 cup). Divide into 8 portions and roll out each portion as thin as possible, with a rolling pin. Toast over medium high... - 25.0065
Place flour in a bowl. Make a well in the center of the flour and add water gradually, mixing until a soft dough is formed. Turn onto a lightly floured smooth surface. Knead the dough a few minutes until it is pliable. Cover with a clean damp cloth and let... - 24.8151
1. Sift the flour into a mixing bowl with the salt. Make a well in the centre and gradually stir in the measured water, mixing well with your fingers to form a supple dough. Knead for about 7-10 minutes then cover and set aside for 15-20 minutes. 2. Divide... - 19.724
Sift the flour and salt into a bowl. Add enough of the water to make a soft dough and knit together with your fingertips. Knead well for 10 minutes. Leave, covered with a damp cloth, for at least 1 hour. Knead well again, then divide into eight pieces. Roll... - 17.9497
This carrot multigrain chapati is high in nutrition. The carrot is extremely rich in vitamin A, which provides antioxidant effects to the body. It promotes good vision and is necessary for the formation and maintenance of healthy skin and tissues. The... - 0
This is one of the most common breads made in India. It is made of whole wheat flour and can be served with any form of curry dry or gravy. - 0
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In the first of 3 videos, Barry heads to the Sweet Cumin Indian Cookery School in Somerset and learns how to make some delicious chapatis. Also known as roti, they are made with simply 3 ingredients - warm water, vegetable oil & chapati flour. They're... - 0
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text:
Probation / Parole
ADVOCATING FOR VICTIMS OF VIOLENCE AGAINST WOMEN IN DEPARTMENTS OF PROBATION AND PAROLE (English and Español), Women's Justice Center, Santa Rosa, CA. Copyright © Marie De Santis.
CoLLABORATIVE BEHAVIORAL MANAGEMENT: INTEGRATION AND INTENSIFICATION OF PAROLE AND OUTPATIENT ADDICTION TREATMENT SERVICES IN THE STEP'N OUT STUDY, Peter D. Friedmann, Anne G. Rhodes, Faye S. Taxman and for the Step'n Out Research Group of CJ-DATS, Journal of Experimental Criminology (volume 5 [3], pages 227-243), Springer, New York, NY: September 1, 2009. Copyright © 2009 Springer.
CoMMUNITY CORRECTIONS RESPONSE TO DOMESTIC VIOLENCE: GUIDELINES FOR PRACTICE, American Probation and Parole Association, Lexington, KY: May 2009.
COURT OFFERED A SECOND CHANCE, AND THREE NOW ARE DEAD, Marna Anderson, Minneapolis-St. Paul Star Tribune, Minneapolis-St. Paul, MN: September 20, 2004. Copyright © 2004 Star Tribune.
DOMESTIC VIOLENCE AND PROBATION, Fernando Mederos, Denise Gamache and Ellen Pence, Battered Women's Justice Project, Minneapolis, MN.
DOMESTIC VIOLENCE: FUNDAMENTALS FOR COMMUNITY CORRECTIONS PRACTICE (online training), American Probation and Parole Association, Lexington, KY.
DOMestic violence intervention system: a model for response to woman abuse, Barbara J. Hart, 1990, Barbara J. Hart's Collected Writings, Minnesota Center Against Violence and Abuse, St. Paul, MN. Copyright © 1995-2004 Minnesota Center Against Violence and Abuse.
DUE SOUTH: TEXAS: JUSTICE REINVESTMENT INITIATIVE, Justice Policy Institute, Washington, DC: May 2011.
EFFECTIVE RESPONSES TO OFFENDER BEHAVIOR: LESSONS LEARNED FROM PROBATION AND PAROLE (executive summary and full report), American Probation and Parole Association, in partnership with National Center for State Courts and The Pew Charitable Trusts, 2013.
eLECTRONIC MONITORING REDUCES RECIDIVISM, Toward Criminal Justice Solutions, National Institute of Justice, Washington, DC: September 2011.
EVALUATION OF THE STATEWIDE AUTOMATED VICTIM INFORMATION AND NOTIFICATION PROGRAM, Seri Irazola, Erin Williamson, Emily Niedzwiecki, Sara Debus-Sherrill and Julie Stricker, October 2013.
GPS MonitorING OF OffenderS, Safety Net Project, Washington, DC. Copyright © 2008 National Network to End Domestic Violence, Safety Net Project.
GPS MonitorING TECHNOLOGIES AND DOMESTIC VIOLENCE: AN EVALUATION STUDY, Edna Erez, Peter R. Ibarra, William D. Bales and Oren M. Gur, June 2012.
GPS MONITORING: AN EFFECTIVE, COST-SAVING OPTION, Doris Wells, Corrections Today, Alexandria, VA: January / February 2014.
GPS to Monitor Parolee; Sex Offender – the State Plans to Outfit the Target of Inland Protests with the Device this Week, Lisa O'Neill Hill and Douglas Quan, Press-Enterprise, Riverside, CA: June 21, 2005. Copyright © 2005 The Press Enterprise Co.
HALF OF WOMEN ON PROBATION OR PAROLE EXPERIENCE MENTAL ILLNESS, Data Spotlight, Center for Behavioral Health Statistics and Quality, Rockville, MD: March 6, 2012.
"HOPE, ILLUSION, AND IMAGINATION: THE POLITICS OF PAROLE AND REENTRY IN THE ERA OF MASS INCARCERATION" ~ THE 19TH ANNUAL ROSE SHEINBERG SCHOLAR-IN-RESIDENCE LECTURE (video), Kathy Boudin, New York School of Law, New York, NY: March 4, 2013.
HOPE: A SWIFT AND CERTAIN PROCESS FOR PROBATIONERS, Kevin McEvoy, NIJ Journal (issue 269), National Institute of Justice, Washington, DC: March 2012.
Mom's Slaying Highlights Repeated Domestic Abuse, Valerie Kalfrin, Tampa Tribune, Tampa, FL: May 23, 2005.Copyright © 2005 The Tribune Co.
NOVEL CONDITION OF PROBATION: DON'T GET PREGNANT, Susan P. Raine, Health Law Perspectives, Houston, TX: October 20, 2008.Copyright © 2008 The University of Houston.
PAROLE OFFICER SURVEY, Texas Criminal Justice Coalition, Austin, TX: 2012.
PREDICTING RECIDIVISM RISK: NEW TOOL IN PHILADELPHIA SHOWS GREAT PROMISE, Nancy Ritter, NIJ Journal (issue 271), National Institute of Justice, Washington, DC: February 2013. More about new tool.
PROBATION AND PAROLE IN THE UNITED STATES, ..., Bureau of Justice Statistics, Washington, DC.
...2012 Bulletin, Laura M. Maruschak and Thomas P. Bonczar, December 2013 ...2011 Bulletin, Laura M. Maruschak and Erika Parks, November 2012 ...2010 Bulletin, Lauren E. Glaze and Thomas P. Bonczar, November 2011
...2012 Bulletin, Laura M. Maruschak and Thomas P. Bonczar, December 2013
...2011 Bulletin, Laura M. Maruschak and Erika Parks, November 2012
...2010 Bulletin, Lauren E. Glaze and Thomas P. Bonczar, November 2011
Probation Review Hearings Aim to Increase Victim Safety, Paul Dedinsky, NOTICE (volume II, number 2), National Center on Domestic and Sexual Violence, Austin, TX: Fall 2004.
recidivism predictions and parole decisions, Barbara J. Hart, Barbara J. Hart's Collected Writings, Minnesota Center Against Violence and Abuse, St. Paul, MN. Copyright © 1995-2004 Minnesota Center Against Violence and Abuse.
REPLICATING HOPE: CAN OTHERS DO IT AS WELL AS HAWAII?, Beth Pearsall, NIJ Journal (issue 273), National Institute of Justice, Washington, DC: February 2014.
RESPONDING TO STALKING ~ A GUIDE FOR COMMUNITY CORRECTIONS OFFICERS, Stalking Resource Center and American Probation and Parole Association, Washington, DC: 2010.
A TEN-STEP GUIDE TO TRANSFORMING PROBATION DEPARTMENTS TO REDUCE RECIDIVISM, Tony Fabelo, Geraldine Nagy and Seth Prins, Council of State Governments Justice Center, New York, NY. Copyright © 2011 Council of State Governments Justice Center.


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Wynaut/Corphish/Mienfoo/Scraggy(Scarf)/Missy/Fletchling sound good? It's my Offensive team that I'm finalizing now. Once I'm done, I'll post it if you think this'd be good :)
hi fite for SPL timezone is GMT-6. fite on smogtours server. I don't think I can battle tomorrow, but if this changes I'll let you know asap. I can battle on Thursday at 3-3:30 PM or 7-7:30 PM, or on Friday at 7-7:30 PM.
Sure. After perusing your list here are a few I would recommend off the top of my head: Hoshi no Samidare (Seriously, read it already) Sanctuary Ratman
lol yeah i kinda wanna put another animu girl there but i think that might just be my sig instead cause this one looks nice lol


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Refined Vines Cake
Difficulty: Somewhat Easy
This natural beauty stacks up nicely as a focal point at your formal affair. Use flowers, tendrils and leaves, cut from Sugar Sheets!, to build vines that climb from tier to tier.
Linked Forever Cake
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Using our punch and cutting insert, flower chains made from Sugar Sheets! are the perfect link to a sweet anniversary celebration.
Bouncy Butterfly Cake
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A colorful butterfly takes flight on this picture-perfect cake. The broad smile and fun colors are sure to charm any birthday girl or boy!
Bountiful Blossom Cake
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Lushly Layered Blooms Cake
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Carnation Celebration Cake
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Ruffled ribbon roses add a springtime touch to this pretty tiered cake. Flowers and stripes are easily made using Sugar Sheets!, giving this cake a professional touch.
In-the-Pink Flower Cake
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Our "pretty in pink and pearls" cake is perfect for every special occasion. Simply punch flower chains from Light Pink and Bright Pink Sugar Sheets to set a white-iced cake abloom.
Petaled Personality Cake
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A colorful petaled cake, made with Sugar Sheets!, is a fun way to perk up a birthday, wedding shower, school function or other special occasion!


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Truth & Rumors > MLB
Phillies after Ludwick and Bell?
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08:10 AM ET 07.15 | How happy will Ruben Amaro Jr. make his manager this trade deadline season? There is Michael Cuddyer in Minnesota. His .369 average and .470 on-base percentage against lefties would be an incredible addition. In case you have not noticed, however, the Twins are very much in the AL Central race even after a miserable start. There is Carlos Beltran of the New York Mets. ... There is Ryan Ludwick in San Diego. He's hitting .276 with three home runs against lefties this season but is only a .236 career hitter against lefthanders. Amaro, of course, is on the record as saying he'd rather add a bullpen arm than a righthanded bat. So maybe the general manager can acquire Ludwick and Padres closer Heath Bell.
You, too, can be a sports writer! - "The [insert team] are [wanting, desiring, craving, looking at, lusting after] a [insert position] [insert any living player] to [insert reason]". Just fill in the blanks and repeat ad nauseum.
Total Comments (39)
I am a fan of Bell. I think he would be a welcome addition to our bullpen!
Total Comments (4)
Would the Phillies actually give up prospects for Ludwick?
yes but not very good ones
Oh please, this guy writes all kinds of crap all the time.
as a matter of fact we'll give you prospects to take Hawpe
I heard that the Phillies are actually interested in Carlos Quentin on the ChiSox, actually I read it in the Tribune yesterday.
Doc looked great at the All-Star game.
Would the Phillies actually give up prospects for Ludwick?
let's hope not
If the Phillies need a right handed bat, they can take Alfonso Soriano off our hands. Please.
heath bell and josh willingham...thats who our phils are gettin
Pudres aren't trading anyone; they're dumping salary. Their tightwad mumbly mouthed carpetbagging conman of an 'owner' can't afford to eat at Jack In the Box, let alone run a major league club. That's why they are little more than a farm club. Nothing will change until they get someone in charge with real money, no payment plan. At least we have Comic-Con & the weather.
amaro jr will try to whatever it takes to get some offense and bullpen help, regardless if the phillies do or don't sign this type of players to a long term!!!
everybody talking about who?
the fastball,
the curve,
the changeup,
the slider,
cliff lee gives away nothing!!!
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CONVOCATORIA / SQUAD LIST: Atlético de Madrid-Real Madrid
Real Madrid - 10 months ago
Watching: Porteros: Casillas, Diego LĂłpez y Adán. Defensas: Varane, Pepe, Albiol, Carvalho y Nacho. Centrocampistas: Xabi Alonso, Essien, Khedira, Ă–zil, Di MarĂa, Modr...
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Dorothy always said, "There's no place like home!"
Bios, Profiles, Celebrity Interviews and more
Your gay guide to style
Looking to get away...
Red carpet coverage of LGBT nightlife and events
DJ Mixes and Artist Performances
Articles and Headlines
Fred Frick creates fabulous fashion for some of the most celebrated drag queens in the world. His glamtastic designs can be ogled at the biggest drag events of the year, including "Night of a Th...
The mother of all NYC lesbian parties is unexpectedly closing! And just after celebrating their 10th anniversary! Thanks to "The L Word," girls around the world learned what NYC girls have alway...
Hollywood's hottest ladies came out for "An Evening with Women," an annual benefit for the L.A. Gay & Lesbian Center. On hand were L Word stars Leisha Hailey, Katherine Moennig, Clement...
What a year! We're rewinding 2009 – 12-months filled with triumphs and tragedies, of gains and losses in the race for marriage equality, musical moments that brought down the house and amazi...
Cheyenne Jackson Christine Quinn Cyndi Lauper GLBT LGBT Matt Maggiacomo New York Pride 2010 activism bisexual event gay gay rights hisscene influencer influencers interview lesbian nightlife nyc ourscene ourscenetv ourscenetv.com party politics prop 8 protest today's influencers transgender video
I can't get enough of celebrities interviews.
I love the nightlife - gimme more on clubs and parties.
Let's see more from the red carpet!
I don't care, as long the the guys are cute.
Girls, girls and more girls.


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TSA nixes naked images from body scanners
WASHINGTON (AP) - The Transportation Security Administration says it's installing new technology in some U.S. airports so when a traveler goes through checkpoint security, a generic outline of a person's body will be shown instead of the image of a naked body.
The agency says the change is intended to protect travelers' privacy rights while securing commercial air travel. It will be used in 40 airports, including in Chicago, Dallas, Detroit, Miami and Newark.
The new software is designed to recognize items on the passenger that could pose a security threat.
The agency plans to eventually use this technology for more machines at more airports. The whole body imaging machines have sparked outrage among passengers and privacy advocates because they reveal images of naked bodies.
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01-29-2011 04:45 AM - edited 01-29-2011 04:47 AM
I'm using a Cheetah 15k.6 SAS (ST3146356SS) connected to a RocketRaid 2640 SAS controller, configured as JBOD (one single drive). OS is Windows XP x64. Two SATA drives are connected to the same controller (one as JBOD, one as "legacy"). As the drive shows 38 relocated sectors I want to use Seatools to test it. The drive seems to be OK (it's the OS drive) and all sectors are readable.
Using Seatools i can see thre (logical ?) drives connected to the SAS controller but it cannot access them (all tests fail or Seatool crashes). It does not display any meaningful information about the two drives configured as JBOD, just "DISK". Not even the logical name. On other SATA drives on another controller in a non-RAID configuration Seatools works as it should.
What is the supposed way to use Seatools on an SAS drive?
©2012 Seagate Technology LLC


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Place matters: metropolitics for the twenty-first centuryThree distinguished scholars challenge us to put the urban crisis back on the agenda, both as a moral challenge to our conscience and an economic challenge to prosperity. Focusing on the growing concentration of poverty in cities and older suburbs and the mounting costs of suburban sprawl, they argue that these problems have political origins and can thus be resolved through political means. |
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Review: Place Matters: Metropolitics for the Twenty-First Century (Studies in Government & Public Policy)
User Review - EW Lewis - GoodreadsHad to read this book for a summer course in my masters program. My biggest issue with the text is that it is highly biased. The tone used to describe Republicans and Conservatives at times verges on ... Read full review
Review: Place Matters: Metropolitics for the Twenty-First Century (Studies in Government & Public Policy)
User Review - Goodreadseh. textbook in summer.
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Former SKID ROW singer Sebastian Bach revealed on his Facebook page that he was in the studio yesterday (Friday, November 9) recording with a "top-secret project that will revolutionize popular music as we know it." No further details are currently available.
A photo from the sessions can be seen below. Bach in July posted a letter on his Facebook page in which he said he was willing to reunite with SKID ROW. "For the fans, I would be willing to put my ego aside and do something that would be special for the people who put us where we are today," he wrote. "I do not personally want it myself. But it's not always about 'what I want.' If the fans really want to see us together, I would do it for the fans. Sometimes you can do things for people other than yourself." Bach said he wanted to write the letter in response to current SKID ROW singer Johnny Solinger's interview in which Johnny said a recent tweet by Bach about a possible SKID ROW reunion was an attempt to "get attention." Bach's most recent solo album, "Kicking & Screaming", sold 6,600 copies in the United States in its first week of release to land at position No. 73 on The Billboard 200 chart. The singer's previous CD, "Angel Down", opened with 6,400 units back in November 2007 to debut at No. 190.SEBASTIAN BACH Recording 'Top-Secret Project That Will Revolutionize Popular Music'
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My nutrition/fitness/critical thinking blog:
A Post-Primal PrimalPat
Do not allow yourself to become wrapped up in a food 'lifestyle'. That is ego, and you are not that.
A Post-Primal PrimalPat
Do not allow yourself to become wrapped up in a food 'lifestyle'. That is ego, and you are not that.
Eat the fruit. You want the fruit, then eat it. Have some and enjoy it if it treats you well.
I was doing high fat in the winter, now with summer I've been eating so much fruit. I have some fruit meals too because it works really good for me and I love it. Sometimes I have fruit for dinner. I'm loving the varieties this summer and all the good ripeness. However, I have been craving less fat, thus my fat intake has been lower with the higher fruits.
Still looking lean with same caloric intake
Absolutely eat as much fruit as you want. "Listen to your body," even if listening to it got you fat in the first place. Eat 1000 calories in carbs per day. That's current wisdom, so it must be right. Sing like no one is listening and eat like it won't affect your body in any way.
Me, I'm going to go get some of the gold my unicorn is shooting out of its ass.
"Right is right, even if no one is doing it; wrong is wrong, even if everyone is doing it." - St. Augustine
All of that.
Why can't there be any moderation?
Why does it have to be that if any one says "use some moderation" everyone freaks out.
If you want to lose some weight and gain some muscle AND can do that eating ALL THE FRUIT, and it works for you. Great.
If not. Dial it back. Up the protein. Eat some more veggies.
For some people eating a lot of fruit or high carb stimulates their hunger making them consume more calories overall, or at least making maintaining lower calories more difficult because of the hunger signals... if this becomes an issue you might want to deal with that.
As you can tell, for SOME people eating lots of fruits works very well... for others it really doesn't.
Only you can figure out where you fit in this scheme.
But it doesn't have to be one extreme or the other.
You have your way. I have my way. As for the right way, the correct way, and the only way, it does not exist.
~Friedrich Nietzsche
And that's why I'm here eating HFLC Primal/Paleo.


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Fudgy Brownies Recipe
- 1-1/3 cups butter, softened
- 2-2/3 cups sugar
- 4 eggs
- 3 teaspoons vanilla extract
- 2 cups all-purpose flour
- 1 cup baking cocoa
- 1/2 teaspoon salt
- Confectioners' sugar, optional
- In a large bowl, cream butter and sugar until light and fluffy. Beat in eggs and vanilla. Combine the flour, cocoa and salt; gradually add to the creamed mixture.
- Spread into a greased 13-in. x 9-in. baking pan. Bake at 350° for 25-30 minutes or until the top is dry and the center is set. Cool completely. Dust with confectioners' sugar if desired. Yield: 2-1/2 dozen.
Reviews for Fudgy Brownies(16)
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Just as the name says - they are fudgy! Very easy to make too, which makes me want to make them again. I'll have no problem devouring these! I did half the recipe and put it in an 8x8 pan, put mini M&M's on top.
These were really good, and easy to make! I split them into two batches and put walnuts in half. Thank you for sharing!
So yummy! I had to bake them 10 minutes longer, as well, but what flavor! Super easy to make too! Thanks for the recipe!
The best brownies I have ever made.Because the recipe makes so many, I froze half since there is only my husband and I to enjoy them.They are just as good the second time around out of the freezer.
These are the best brownies I have ever made. Because the recipe made so much, I froze half since there is only my husband and myself.They were just as good the second time around.My husband loves them.
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Why Do We Resist God's Plan?
I am a planner. I know my plans seldom work out, but I keep on planning. Planning helps relieve my anxiety about a little reality I'd like to ignore: I don't have control . . . but it feels good to think I do.
So God comes along, and he says:
"Frankie, my boy, would you like to know what I have planned for you today? I think you'll find this really exciting."
"Not really God. You see, I'm busy making my own plans, and frankly, I'm in charge of my day. You did after all give me the freedom to plan my own day, so forgive me, but I'm doing exactly that."
Looking over my shoulder from an infinitely high perspective, God asks, "Where am I in today's planning schedule?"
"God, excuse me, but I have a full day, and I really don't have time to relax, clear my mind, and lounge around with you. Things have to get done you know!"
"Sounds like your in charge here, and I'm not really needed. So, I guess I'll just get back to running the universe while you manage your own life. Oh, by the way Frankie, that little matter of your death, you might what to include managing that in your daily schedule too. Could happen at any time, you know."
Criado há quase 2 anos atrás


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Want to set your photography apart from the crowd and get images that can be even more compelling? Lose the color!
Think "wildlife" and you'll likely think "color"—vivid plumage, multitone fur, brilliant scales, all against backdrops of verdant green and sky blue. So naturally everyone shoots wildlife in color. It's all the rage these days—particularly in nature shooting—to crank up color saturation to make photos stand out. But while vivid colors certainly catch the eye, sometimes taking the saturation in the other direction can have just as much, if not more, impact.
When you drain the color from an image, it allows the eye to focus on form, textures, shapes, and composition. With wildlife photography, you can use monochrome to highlight the patterns of an animal's fur or the textures of its skin, and to turn a distracting background into a neutral gray. Probably the biggest advantage of black-and-white is that it gives you a far greater leeway for exploring tonality. Viewers have certain expectations, for example, of how intense or what shade of yellow a lion should be. But in a monochrome image, you gain the freedom to choose to render the lion in high-key, or brooding dark, or anywhere in between, without making it appear as a grand departure from reality.
Years ago, when color film came onto the scene, black-and-white imaging began to fade in popularity—especially in wildlife photography, with its emphasis on naturalism. But monochrome has returned with a vengeance with the advent of digital. The digital camera's most powerful tool here is RAW capture, a format that allows you to "develop" images in a wide variety of color palettes—or with no color at all—from the same file.
Seeing in B&W
So many times I hear people say, "The image didn't work in color, so I tried it in black-and-white." This tactic can sometimes salvage an image, but to create successful b&w images consistently, you need to learn what to look for.
Contrast is a major force behind black-and-white imagery. A bright animal on a dark background is an obvious choice, but also look for color contrasts that can be taken advantage of in conversion. Take, for example, the cheetah image on this page. The cheetah was yellow and among some green grasses; in conversion, I was able to push the grasses darker and the cheetah lighter, though in reality they were relatively the same luminosity. This left a moodier image than the reality—an afternoon shot with lackluster light.
Which brings up another advantage of b&w: You can shoot longer in the day. Because additional contrast usually improves a monochrome image, successful captures can be taken well outside the "golden hour." Shadows or strong sidelight can be used in composition to emphasize the shape of an animal.
Underexpose a bit with backlighting to highlight the outline of a furry creature, or go the other direction and overexpose a portrait for a high-key effect. For the giraffe on page 108, for example, I positioned myself so that a cloud was behind it, and overexposed by 1.33 stop in order to blow out the sky for a studio portrait look. This graphic approach works very well in b&w and allows you to shoot in conditions that may never work in color—the eye is more forgiving of blown highlights in b&w than in color, where they can really distract.
Textures can also be emphasized through b&w. The rhino on the opening spread is a perfect example: Naturally dull in color, it's already on its way to a monochrome image. By increasing I contrast, I was able to enhance the detail in the hide, and the repeating lines of the ribs. I was also able to emphasize the tonal difference between the rain-soaked areas and those that were still dry. The bright background shrouded in rain also enhanced the image's mood in b&w, whereas in color it looked lifeless and washed out.
The patterns of the zebras at right were also brought out in conversion. Already monochrome, they were perfect candidates, but the background was a bright, distracting tan and green. The conversion allowed me to darken the background and highlight the dust being kicked up, allowing the high-contrast animals to really pop.
Shooting B&W
If you already have sufficient gear for shooting successful wildlife images in color, you have everything you need for b&w. And many of the same rules apply. You want your subject to stand out, so look for a complementary background, without clutter or distracting elements. You can most often achieve this through the use of long telephoto lenses, which limit the angle of view behind your subject. You can then fine-tune the background by moving yourself up, down, left, or right.
Another benefit of teles is the inherent lack of depth of field that comes with high magnification. A patchy, high-contrast background can be greatly smoothed out with a shallow focus on your subject, and can be evened out more with selective dodging and burning later on. For small creatures, a macro lens can be used for the same effect. While photographing the snake above right, I used a 180mm macro to isolate its head as it went through a sandy patch. An image of it in the grass may have worked well, but for me the simpler graphic image worked better. Without distracting blades of grass, it allowed smaller details, like the reflection in the eye and the flick of the tongue, to pop.


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Technical Data |
Interferon beta 1 (beta 1a), Recombinant, Human (IFNb1, IFNb1a) |
2ug 10ug |
Growth Factors, Cytokines | Storage: -20°CShipping: RT |
One of the type I interferons produced by fibroblasts in response to stimulation by live or inactivated virus or by double-stranded RNA. It is a cytokine with antiviral, antiproliferative, and immunomodulating activity. The synthesis of IFN-beta can be induced by common inducers of interferons, including viruses, double-stranded RNA, and micro-organisms. It is induced also by some cytokines such as TNF and IL-1 . In contrast to IFN-alpha, IFN-beta is strictly species-specific. IFN-beta of other species is inactive in human cells. IFN-beta is involved in the regulation of unspecific humoral immune responses and immune responses against viral infections. IFN-beta increases the expression of HLA class I antigens and blocks the expression of HLA class II antigens stimulated by IFN-gamma. Recombinant Human Interferon-Beta 1a is a single, glycosylated polypeptide chain containing 166 amino acids and having a molecular mass of 22500D. Sequence: The sequence of the first five N-terminal amino acids was determined and was found to be Ser-Tyr-Asn-Leu-Leu. Biological Activity: Interferon Beta-1a is fully biologically active when compared to standard. The specific activity as determined in a viral resistance assay (human "Wish" cell line and VSV virus or the monkey VERO cell line with EMCV virus) was found to be 270 x 10e6 IU/ mg. Dimers and Aggregates: < 1% (by silver-stained SDS-PAGE). Protein Content: Protein quantitation was carried out by two independent methods: 1. UV spectroscopy at 280nm. 2. Analysis by RP-HPLC, using a standard solution of IFN-beta as a Reference Standard. Storage and Stability: Lyophilized powder may be stored at -20°C. Stable for 12 months at -20°C. Reconstitute with sterile ddH2O. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Reconstituted product is stable for 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer Molecular Weight: 22.5kD |
Source: Recombinant, Human (CHO cells) Purity: Chromatographically purified (> 98% by RP-HPLC, FPLC, or reducing/non-reducing SDS-PAGE Silver Stain). Endotoxin: 0.1ng/ug (IEU/ug). Concentration: ~0.1mg/ml Form: Supplied as a lyophilized powder from 91mg Human albumin, 620mg mannitol, 9mg sodium acetate. Reconstitute with sterile ddH2O to a concentration of .0.1mg/ml. Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. |
1. Debat Zoguereh, D., et al., Rev. Neurol. (Paris) 160(11): 1081-1084 (2004). 2. Kalanie, H., et al., Eur. Neurol. 52(4): 202-206 (2004). 3. Filippi, M., et al., Lancet 364(9444): 1489-1496 (2004). 4. Lim, E.T., et al., J. Negat. Results Biomed. 3(1): 4 (2004). 5. Villa, A.M., et al., Arq. Neuropsiquiatr. 62(3B): 892-894 (2004). 6. Prosser, L.A., et al., Value Health 7(5): 554-568 (2004).