The mammalian abasic-endonuclease1/redox-factor1 (APE1/Ref1) is an essential protein whose subcellular distribution depends on the cellular physiological status. However, its nuclear localization signals have not been studied in detail. We examined nuclear translocation of APE1, by monitoring enhanced green fluorescent protein (EGFP) fused to APE1. APE1's nuclear localization was significantly decreased by deleting 20 amino acid residues from its N-terminus. Fusion of APE1's N-terminal 20 residues directed nuclear localization of EGFP. An APE1 mutant lacking the seven N-terminal residues (ND7 APE1) showed nearly normal nuclear localization, which was drastically reduced when the deletion was combined with the E12A/D13A double mutation. On the other hand, nearly normal nuclear localization of the full-length E12A/D13A mutant suggests that the first 7 residues and residues 8–13 can independently promote nuclear import. Both far-western analyses and immuno-pull-down assays indicate interaction of APE1 with karyopherin alpha 1 and 2, which requires the 20 N-terminal residues and implicates nuclear importins in APE1's nuclear translocation. Nuclear accumulation of the ND7 APE1(E12A/D13A) mutant after treatment with the nuclear export inhibitor leptomycin B suggests the presence of a previously unidentified nuclear export signal, and the subcellular distribution of APE1 may be regulated by both nuclear import and export.

Increased glucose utilization by aldose reductase (AR) has been implicated in the development of diabetes complications. However, the mechanisms that regulate AR during diabetes remain unknown. Herein we report that several nitric oxide (NO) donors prevent ex vivo synthesis of sorbitol in erythrocytes obtained from diabetic or nondiabetic rats. Compared with erythrocytes of nondiabetic rats, the AR activity in the erythrocytes of diabetic rats was less sensitive to inhibition by NO donors or by AR inhibitors-sorbinil or tolrestat. Treatment with N G -nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, enhanced AR activity and sorbitol accumulation in tissues of nondiabetic rats. Application of transdermal nitroglycerin patches or treatment with L-arginine did not inhibit AR activity or sorbitol accumulation in the tissues of nondiabetic animals. Treatment with L-NAME increased, whereas treatment with L-arginine or nitroglycerine patches decreased AR activity and sorbitol content in tissues of diabetic rats. These observations suggest that NO maintains AR in an inactive state and that this repression is relieved in diabetic tissues. Thus, increasing NO availability may be a useful strategy for inhibiting the polyol pathway and preventing the development of diabetes complications. Diabetes 51: 3095-3101, 2002A ldose reductase (AR) is the first and ratelimiting enzyme of the polyol pathway (1). Under euglycemic conditions, AR plays a minor role in glucose metabolism; however, during diabetes, its contribution is significantly enhanced (1-3). The increase in AR activity by hyperglycemia has been proposed to be the underlying metabolic cause of secondary diabetes complications such as cataractogenesis, retinopathy, neuropathy, and nephropathy (1-3). Because AR utilizes NADPH, it has been suggested that the activation of this enzyme depletes reducing equivalents, which may be otherwise required for the detoxification of oxidants (3). An increase in AR activity also results in sorbitol accumulation. This could potentially disrupt cellular integrity and function by imposing osmotic stress. Therefore, inhibiting AR could be useful in preventing oxidative and osmotic changes that accompany the excessive metabolism of glucose via the polyol pathway (2).The etiological role of AR in diabetes complications is supported by extensive evidence demonstrating that inhibition of this enzyme prevents hyperglycemic changes in the lens, kidney, and nerve (1-3). Nonetheless, in clinical trials AR inhibitors have been found to be only moderately effective, and issues related to their nonselectivity and nonspecific toxicity have remained unresolved (4,5). In addition, the efficacy of these drugs in inhibiting AR during diabetes may be compromised by changes in AR protein.Previous studies have shown that AR isolated from diabetic or hyperglycemic tissues is less susceptible to inhibition and is kinetically different from the enzyme purified from normal or euglycemic human or animal tissues (6 -8). Similar changes in the...

Diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) represent the most common and most aggressive forms of Non-Hodgkin lymphoma (NHL) respectively. Traditional chemotherapy includes (C)yclophosphamide, (H)ydroxydaunorubicin, (O)ncovin (vincristine) and (P)rednisone or (P)rednisolone (CHOP); or (R)ituximab-CHOP. However, in the past 10 years, other more targeted drugs have been developed for treatment of NHLs that includes Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib (IBN) and proteasome inhibitors such as carfilzomib (CFZ). Presently, there is a high interest in the endocannabinoid system, with specific interest in CB1 and CB2 ligands as potential therapeutic targets for both DLBCL and MCL. Cannabidiol (CBD) is a natural cannabinoid analog that has mixed affinity across CB1 and CB2 receptors; known in the literature as a CB1 antagonist and a CB2 agonist. Our previous work has demonstrated that CB1 antagonists has activity against DLBCL and MCL cell lines. Our study is aimed at comparing CBD against conventional chemotherapeutics, individually and in combination, in DLBCL and MCL cell lines. Here, we demonstrate that CBD in combination with known chemotherapeutics, ibrutinib (IBN), carfilzomib (CFZ), zanubrutinib (BGB) and tumorex (TMX), has synergistic potential in treating DLBCL and MCL cell lines. To determine the activity of CBD in comparison to IBN, cells from representative DLBCL (RC) and MCL (Mino) cell lines were plated at 5,000 cells per well. The cells were incubated for 72 hours in 20 µL medium with 10% FBS and 25 µM CBD, IBN or a combination of both. Viability assays were conducted using Celltiter-Glo Luminescent Cell Viability Assay. Experiments were performed 2-3 times independently, and each concentration was tested in triplicate. We next recreated the experiment using a different BTK inhibitor, BGB, instead of IBN. Likewise we next compared CBD to a proteasome inhibitor where cells from representative DLBCL (RC) and MCL (Mino) cell lines were plated at 5,000 cells per well. The cells were incubated for 72 hours in 20 µL medium with 10% FBS and 6 µM CBD, 10 nM CFZ, or a combination of both. Viability assays were conducted using Celltiter-Glo Luminescent Cell Viability Assay. Experiments were performed 2-3 times independently, and each concentration was tested in triplicate. We then followed by recreating the experiment using 12.5 µM of CBD, 250 nM TMX or both. Synergistic potential of CBD with conventional treatment IBN was examined at concentrations of 25μM. Data shows a marked reduction in viability when drugs are used in combination as compared to individual drug response. Combining CBD with IBN lowered the viability to under 25% as compared to control whereas, individually the drugs never fall under 75% of control. A similar pattern of synergy was demonstrated amongst CBD with BGB, CFZ and TMX respectively. This synergist effect more than likely is the result of the drugs effecting two or more different biochemical pathways. However, within keeping of our previous studies, it is a further indication of the viability of targeting the cannabinoid pathway for therapeutic intervention of malignant lymphoma cancer. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Guanosine-dependent cyclic nucleotide second messenger signaling has been implicated as a pivotal mediator of vascular function under both homeostatic eutrophic conditions as well as in the inimical environs of injury and/or disease. This biological system is highly regulated through reciprocal, complimentary, and often redundant upstream and downstream molecular and cellular elements and feedback controls. Key endogenous factors of the guanosine-dependent cyclic nucleotide cascade include upstream gaseous activating ligands (nitric oxide, carbon monoxide), downstream substrates (cGMP-gated ion channels, cGMP-dependent protein kinases), and cGMP hydrolyzing phosphodiesterases. This intricate system also has capacity to "cross-talk" with parallel adenosine-dependent cyclic nucleotide machinery. Numerous complexes of ligands, enzymes, cofactors, and substrates present significant targets for pharmacologic modulation at the cellular, genetic, and/or molecular level eventuating therapeutically as constructive functional responses observed in vascular physiology and/or pathophysiology. Interestingly, emerging evidence based largely on transgenic mouse models challenges the historically accepted concept that this signaling system functions principally as a therapeutic modality in cardiac and vascular tissues. The general purpose of this update is to provide current information on recently described neoteric agents that impact multifaceted and critical cGMP-dependent signaling in the vascular system. Emphasis will be placed on novel agents that exert significant and often multiple actions on upstream and downstream sites and are capable of eliciting robust effects on guanosine-dependent cellular actions. Individual sections will be devoted to agents that rely on an intact and functional cyclase heme and those that operate independently of the sGC heme. Attention will be placed on the physiologic and pathophysiologic clinical manifestations of these pharmacologic regimens. This review will conclude with some thoughts for future directions for study and continued discovery of novel sGC/cGMP controllers in the vascular system at the basic science and clinical levels.

Guanosine-dependent cyclic nucleotide second messenger signaling has been implicated as a pivotal mediator of vascular function under both homeostatic eutrophic conditions as well as in the inimical environs of injury and/or disease. This biological system is highly regulated through reciprocal, complimentary, and often redundant upstream and downstream molecular and cellular elements and feedback controls. Key endogenous factors of the guanosine-dependent cyclic nucleotide cascade include upstream gaseous activating ligands (nitric oxide, carbon monoxide), downstream substrates (cGMP-gated ion channels, cGMP-dependent protein kinases), and cGMP hydrolyzing phosphodiesterases. This intricate system also has capacity to "cross-talk" with parallel adenosine-dependent cyclic nucleotide machinery. Numerous complexes of ligands, enzymes, cofactors, and substrates present significant targets for pharmacologic modulation at the cellular, genetic, and/or molecular level eventuating therapeutically as constructive functional responses observed in vascular physiology and/or pathophysiology. Interestingly, emerging evidence based largely on transgenic mouse models challenges the historically accepted concept that this signaling system functions principally as a therapeutic modality in cardiac and vascular tissues. The general purpose of this update is to provide current information on recently described neoteric agents that impact multifaceted and critical cGMP-dependent signaling in the vascular system. Emphasis will be placed on novel agents that exert significant and often multiple actions on upstream and downstream sites and are capable of eliciting robust effects on guanosine-dependent cellular actions. Individual sections will be devoted to agents that rely on an intact and functional cyclase heme and those that operate independently of the sGC heme. Attention will be placed on the physiologic and pathophysiologic clinical manifestations of these pharmacologic regimens. This review will conclude with some thoughts for future directions for study and continued discovery of novel sGC/cGMP controllers in the vascular system at the basic science and clinical levels.