Serendipitous identification of phenylhydrazine derivatives as potent inhibitors of carbapenem-resistant Acinetobacter baumannii.

Background: In the authors' previous study, 4-(2-((3-methyl-4-oxo-2-thioxo/dioxothiazolidin-5-ylidene) methyl) hydrazineyl) benzonitriles were found to demonstrate potent antibacterial activity against Acinetobacter baumannii. Interestingly, the aforementioned compounds contain a 4-cyanophenylhydrazine motif. Materials & methods: Intrigued by this observation, the authors focused on preparing a library of 4-cyanophenylhydrazine derivatives and studied their detailed antibacterial potential. Results: This study led to the identification of a 4-cyanophenylhydrazine with potent inhibitory activity against carbapenem-resistant A. baumannii BAA-1605, with minimum inhibitory concentration (MIC) of 0.25 µg/ml and highest selectivity index of 640. The compound also demonstrated potent inhibition against multidrug-resistant A. baumannii isolates (MIC: 0.25-1 µg/ml). Conclusion: The identified 4-cyanophenylhydrazine compound exhibited synergistic activity with amikacin, tobramycin and polymyxin B against carbapenem-resistant A. baumannii BAA-1605.

Synthesis, Biological Evaluation, Migratory Inhibition and Docking Study of Indenopyrazolones as Potential Anticancer Agents.

Some bioactive derivatives of indeno[1,2-c]pyrazolones were synthesized through the reaction of phenylhydrazine, different aldehydes and indan-1,2,3-trione at room temperature in acetonitrile. Analytical and spectroscopic studies have confirmed the structural characteristics of the synthesized compounds. In addition, the target compounds were screened for the in-vitro antiproliferative properties against the B16F10 melanoma cancer cell lines by the standard MTT assay. The effect on inflammatory marker cyclooxygenase 2 and matrix metalloproteinase 2, 9 was also checked to determine the anti-inflammatory and anti-cell migratory properties of these compounds. The final compounds were also tested for their tyrosinase inhibitory activity. Among all compounds, screened for anticancer activity, three compounds 4e, 4f and 4h reduced the cell proliferation significantly comparable to that of the positive standard drug erlotinib (IC50 =418.9±1.54 µM) with IC50 values ranging from 20.72-29.35 µM. The compounds 4c-4h decreased the COX-2 expression whereas the MMP 2, 9 expressions were significantly reduced by 4a, 4b and 4h. This was confirmed by molecular docking studies, as 4e, 4f and 4h displayed good interactions with the active site of BRAF protein. The compounds 4b, 4f and 4h exhibited moderate tyrosinase inhibition effect as compared to α-MSH. Collectively, compound 4h can be considered as a candidate for further optimization in the development of anticancer therapies based on the results of biological investigations in this study.

Role of Toll-like receptor 4 in intravascular hemolysis-mediated injury.

Massive intravascular hemolysis is a common characteristic of several pathologies. It is associated with the release of large quantities of heme into the circulation, promoting injury in vulnerable organs, mainly kidney, liver, and spleen. Heme activates Toll-like receptor 4 (TLR4), a key regulator of the inflammatory response; however, the role of TLR4 in hemolysis and whether inhibition of this receptor may protect from heme-mediated injury are unknown. We induced intravascular hemolysis by injection of phenylhydrazine in wildtype and Tlr4-knockout mice. In this model, we analyzed physiological parameters, histological damage, inflammation and cell death in kidney, liver, and spleen. We also evaluated whether heme-mediated-inflammatory effects were prevented by TLR4 inhibition with the compound TAK-242, both in vivo and in vitro. Induction of massive hemolysis elicited acute kidney injury characterized by loss of renal function, morphological alterations of the tubular epithelium, cell death, and inflammation. These pathological effects were significantly ameliorated in the TLR4-deficient mice and in wildtype mice treated with TAK-242. In vitro studies showed that TAK-242 pretreatment reduced heme-mediated inflammation by inhibiting the TLR4/NF-κB (nuclear factor kappa B) axis. However, analysis in liver and spleen indicated that TLR4 deficiency did not protect against the toxic accumulation of heme in these organs. In conclusion, TLR4 is a key molecule involved in the renal inflammatory response triggered by massive intravascular hemolysis. TLR4 inhibition may be a potential therapeutic approach to prevent renal damage in patients suffering from hemolysis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Repro-protective role of royal jelly in phenylhydrazine-induced hemolytic anemia in male mice: Histopathological, embryological, and biochemical evidence.

To estimate the repro-protective effect of royal jelly (RJ) on phenylhydrazine (PHZ)-induced anemia's detrimental effects, 24 mature mice were divided into control group (0.10 mL normal saline; intra-peritoneally), RJ group (100 mg/kg/day; orally), experimental anemia (EA) group that received only PHZ (6 mg/100 g/48 h; intra-peritoneally), and RJ + EA (according to the previous prescription) group. After 35 days, testicular histoarchitecture, RNA damage in germinal cells, sperm characteristics, testicular total anti-oxidant capacity and malondialdehyde as well as serum testosterone levels, pre-implantation embryo development and cyclin D1 and c-myc mRNA levels at two-cell, morula and blastocyst stages were analyzed. Spermatogenesis indices were ameliorated following RJ co-administration. Moreover, RJ co-treatment reduced germinal cells RNA damage, improved sperm characteristics, boosted pre-implantation embryo development and restored androgenesis, and oxidant/anti-oxidant status. Co-administration of RJ also decreased mRNA levels of cyclin D1 and up-regulated those of c-myc in two-cell embryos, morulas and blastocysts. The findings suggest that RJ can play a repro-protective role in PHZ-induced anemia in mice through anti-oxidant defense system reinforcement and androgenesis restoration as well as cyclin D1 and c-myc expressions regulation.

Synthesis, In-vitro evaluation and molecular docking studies of oxoindolin phenylhydrazine carboxamides as potent and selective inhibitors of ectonucleoside triphosphate diphosphohydrolase (NTPDase).

Members of the ectonucleoside triphosphate diphosphohydrolases (NTPDases) constitute the major family of enzymes responsible for the maintenance of extracellular levels of nucleotides and nucleosides by catalyzing the hydrolysis of nucleoside triphosphate (NTP) and nucleoside diphosphates (NDP) to nucleoside monophosphate (NMP). Although, NTPDase inhibitors can act as potential drug candidates for the treatment of various diseases, there is lack of potent as well as selective inhibitors of NTPDases. The current study describes the synthesis of a number of carboxamide derivatives that were tested on recombinant human (h) NTPDases. The most promising inhibitors were 2h (h-NTPDase1, IC50: 0.12 ± 0.03 µM), 2d (h-NTPDase2, IC50: 0.15 ± 0.01 µM) and 2a (h-NTPDase3, IC50: 0.30 ± 0.04 µM; h-NTPDase8, IC50: 0.16 ± 0.02 µM). Four compounds (2e, 2f, 2g and 2h) were associated with the selective inhibition of h-NTPDase1 while 2b was identified as a selective h-NTPDase3 inhibitor. Considering the importance of NTPDase3 in the regulation of insulin release, the NTPDase3 inhibitors were further investigated to elucidate their role in the insulin release. The obtained data suggested that compound 2a was actively participating in regulating the insulin release without producing any effect on NTPDase3 mRNA. Moreover, the most potent inhibitors were docked within the active site of respective enzyme and the observed interactions were in compliance with in vitro results. Hence, these compounds can be used as pharmacological tool to further investigate the role of NTPDase3 coupled to insulin release.

Central histaminergic transmission modulates the expression of chronic nicotine withdrawal induced anxiety-like and somatic behavior in mice.

The present study investigated the plausible modulatory role of central histaminergic transmission on the expression of nicotine withdrawal induced anxiety and somatic behavior in mice. Abrupt cessation of chronic nicotine (2 mg/kg, i.p. × 3/day) treatment for 12 days to mice, expressed increased anxiety in light & dark test and total abstinence (somatic) score at 24 h post nicotine withdrawal time. The somatic signs includes a composite score of all behaviors such as grooming, rearing, jumping, body shakes, forelimb tremors, head shakes, abdominal constrictions, scratching, empty mouth chewing or teeth chattering, genital licking, tail licking. Mice exhibited higher expression to nicotine withdrawal induced anxiety in light & dark test at 24 h post-nicotine withdrawal time on pre-treatment centrally (i.c.v) with histaminergic agents like histamine (0.1, 50 µg/mouse), histamine H3 receptor inverse agonist, thioperamide (2, 10 µg/mouse), histamine H1 receptor agonist, FMPH (2, 6.5 µg/mouse) or H2 receptor agonist amthamine (0.1, 0.5 µg/mouse) or intraperitoneally (i.p.) with histamine precursor, l-histidine (250, 500 mg/kg) as compared to control nicotine withdrawn animals. Furthermore, mice pre-treated with all these histaminergic agents except histamine H1 receptor agonist, FMPH shows exacerbated expression to post-nicotine withdrawal induced total abstinence (somatic) score in mice. On the other hand, central injection of selective histamine H1 receptor antagonist, cetirizine (0.1 µg/mouse, i.c.v.) or H2 receptor antagonist, ranitidine (50 µg/mouse, i.c.v) to mice 10 min before 24 h post-nicotine withdrawal time completely alleviated the expression of nicotine withdrawal induced anxiety and somatic behavior. Thus, it can be contemplated that the blockade of central histamine H1 or H2 receptor during the nicotine withdrawal phase could be a novel approach to mitigate the nicotine withdrawal associated anxiety-like manifestations. Contribution of endogenous histamine via H1 or H2 receptor stimulation in the nicotine withdrawal induced anxiety and somatic behavior is proposed.

Identification of the subtype-selective Sirt5 inhibitor balsalazide through systematic SAR analysis and rationalization via theoretical investigations.

We report here an extensive structure-activity relationship study of balsalazide, which was previously identified in a high-throughput screening as an inhibitor of Sirt5. To get a closer understanding why this compound is able to inhibit Sirt5, we initially performed docking experiments comparing the binding mode of a succinylated peptide as the natural substrate and balsalazide with Sirt5 in the presence of NAD+. Based on the evidence gathered here, we designed and synthesized 13 analogues of balsalazide, in which single functional groups were either deleted or slightly altered to investigate which of them are mandatory for high inhibitory activity. Our study confirms that balsalazide with all its given functional groups is an inhibitor of Sirt5 in the low micromolar concentration range and structural modifications presented in this study did not increase potency. While changes on the N-aroyl-ß-alanine side chain eliminated potency, the introduction of a truncated salicylic acid part minimally altered potency. Calculations of the associated reaction paths showed that the inhibition potency is very likely dominated by the stability of the inhibitor-enzyme complex and not the type of inhibition (covalent vs. non-covalent). Further in-vitro characterization in a trypsin coupled assay determined that the tested inhibitors showed no competition towards NAD+ or the synthetic substrate analogue ZKsA. In addition, investigations for subtype selectivity revealed that balsalazide is a subtype-selective Sirt5 inhibitor, and our initial SAR and docking studies pave the way for further optimization.

Novel carboxylated pyrroline-2-one derivatives bearing a phenylhydrazine moiety: Design, synthesis, antifungal evaluation and 3D-QSAR analysis.

Aiming to discover novel high-efficient antifungal leads that possess an innovative action mechanism, twenty-three carboxylated pyrroline-2-one derivatives, bearing a phenylhydrazine moiety, were rationally designed and firstly prepared in this letter. The in vitro bioassays showed that most of the compounds possessed excellent antifungal effects with the EC50 values of less than 1 µg/mL against the phytopathogenic fungi Fusarium graminearum (Fg), Botrytis cinerea (Bc), Rhizoctonia solani (Rs) and Colletotrichum capsici (Cc). The further bioassays showed that the compound 6u showed the comparable in vivo control effect with carbendazim against fusarium head blight and rice sheath blight. The 3D-QSAR model revealed the pivotal effects of a bulky electron-donating group at the 1-position of pyrrole ring, a bulky electron-withdrawing group at the 4-position of phenyl ring and a small alkyl at the carbonate group on the anti-Rs activities of target compounds. The abnormal mycelial morphology and delayed spore germination were observed in the treatments of compound 6u. Given the excellent and broad-spectrum antifungal effects the target compounds have, we unfeignedly anticipated that the above finding could motivate the discovery of high-efficient antifungal leads, which might possess an innovative action mechanism against phytopathogenic fungi.

In vitro Multistage Malaria Transmission Blocking Activity of Selected Malaria Box Compounds.

PURPOSE: Continuous efforts into the discovery and development of new antimalarials are required to face the emerging resistance of the parasite to available treatments. Thus, new effective drugs, ideally able to inhibit the Plasmodium life-cycle stages that cause the disease as well as those responsible for its transmission, are needed. Eight compounds from the Medicines for Malaria Venture (MMV) Malaria Box, potentially interfering with the parasite polyamine biosynthesis were selected and assessed in vitro for activity against malaria transmissible stages, namely mature gametocytes and early sporogonic stages. METHODS: Compound activity against asexual blood stages of chloroquine-sensitive 3D7 and chloroquine-resistant W2 strains of Plasmodium falciparum was tested measuring the parasite lactate dehydrogenase activity. The gametocytocidal effect was determined against the P. falciparum 3D7elo1-pfs16-CBG99 strain with a luminescent method. The murine P. berghei CTRP.GFP strain was employed to assess compounds activities against early sporogonic stage development in an in vitro assay simulating mosquito midgut conditions. RESULTS: Among the eight tested molecules, MMV000642, MMV000662 and MMV006429, containing a 1,2,3,4-tetrahydroisoquinoline-4-carboxamide chemical skeleton substituted at N-2, C-3 and C-4, displayed multi-stage activity. Activity against asexual blood stages of both strains was confirmed with values of IC50 (50% inhibitory concentration) in the range of 0.07-0.13 µM. They were also active against mature stage V gametocytes with IC50 values below 5 µM (range: 3.43-4.42 µM). These molecules exhibited moderate effects on early sporogonic stage development, displaying IC50 values between 20 and 40 µM. CONCLUSION: Given the multi-stage, transmission-blocking profiles of MMV000642, MMV000662, MMV006429, and their chemical characteristics, these compounds can be considered worthy for further optimisation toward a TCP5 or TCP6 target product profile proposed by MMV for transmission-blocking antimalarials.

Rats provide a superior model of human stress erythropoiesis.

Mouse models are widely used to study human erythropoiesis in vivo. One important caveat using mouse models is that mice often develop significant extramedullary erythropoiesis with anemia, which could mask important phenotypes. To overcome this drawback in mice, here we established in vitro and in vivo rat models for the studies of stress erythropoiesis. Using flow cytometry-based assays, we can monitor terminal erythropoiesis in rats during fetal and adult erythropoiesis under steady state and stress conditions. We used this system to test rat erythropoiesis under phenylhydrazine (PHZ)-induced hemolytic stress. In contrast to mice, rats did not have an increased proportion of early-stage erythroid precursors during terminal differentiation in the spleen or bone marrow. This could be explained by the abundant bone marrow spaces in rats that allow sufficient erythroid proliferation under stress. Consistently, the extent of splenomegaly in rats after PHZ treatment was significantly lower than that in mice. The level of BMP4, which was significantly increased in mouse spleen after PHZ treatment, remained unchanged in rat spleen. We further demonstrated that the bone marrow c-Kit positive progenitor population underwent a phenotype shift and became more CD71 positive and erythroid skewed with the expression of maturing erythroid markers under stress in rats and humans. In contrast, the phenotype shift to an erythroid-skewed progenitor population in mice occurred mainly in the spleen. Our study establishes rat in vitro and in vivo erythropoiesis models that are more appropriate and superior for the study of human stress erythropoiesis than mouse models.

Phenylhydrazides as inhibitors of Leishmania amazonensis arginase and antileishmanial activity.

Searching for new substances with antileishmanial activity, we synthesized and evaluated a series of α,α-difluorohydrazide and α,α-difluoramides against Leishmania amazonensis arginase (LaArg). Four α,α-difluorohydrazide derivatives showed activity against LaArg with Ki in the range of 1.3-26 µM. The study of the kinetics of LaArg inhibition showed that these substances might act via different inhibitory mechanisms or even by a combination of these. The compounds were tested against L. amazonensis promastigotes and the best result was obtained to the compound 4 (EC50 of 12.7 ±â€¯0.3 µM). In addition, in order to obtain further insight into the binding mode of such compounds, molecular docking studies were performed to obtain additional validation of experimental results. Considering these results, it is possible to conclude that α,α-difluorohydrazide derivatives are a promising scaffold in the development of new substances against the etiological agent of leishmaniasis by targeting LaArg.

Effect of a new cyclic derivative of GABA, RGPU-207, on the functions of cardiac and cerebral mitochondria of stressed animals.

OBJECTIVES: The objective of this study was to investigate the effects of a new derivative of GABA, RGPU-207 compound, on the mitochondrial functions of stressed animals. METHODS: RGPU-207 and the comparator drugs (phenibut and piracetam) were administered intraperitoneally to unstressed and stressed male rats at a dose of 9.4, 25 and 400 mg/kg, respectively. The oxygen consumption by cardiac and cerebral mitochondria in state 3 and 4 and Chance's respiratory control ratio (RCR) was investigated. The concentration of lipid peroxidation products (LPO) such as malondialdehyde (MDA), conjugated dienes (CD) and diketones was evaluated in the isolated mitochondria, as well as the activity of the antioxidant system (AOS) enzymes (superoxide dismutase (SOD), glutathione peroxidase (GP) and catalase). KEY FINDINGS: A new cyclic GABA derivative, RGPU-207 compound, at the dose of 9.4 mg/kg promotes a decline in MDA, diketone and CD concentrations in mitochondria and increases the levels of SOD, GP and catalase activity. Mitochondrial functional activity increases: oxygen consumption by cerebral mitochondria in state 4 decreases when complex I of the respiratory chain is activated, while malate-dependent state 3 respiration of cardiac mitochondria tends to increase. RCR of cardiac mitochondria increases when complexes I and II are involved. In cerebral mitochondria, malate-dependent and succinate-dependent RCR rise. CONCLUSIONS: Twenty-four-hour immobilization and pain stress activate LPO processes inhibit the activity of the aos enzymes and decrease the functional activity of cardiac and cerebral mitochondria. RGPU-207 restricts LPO, enhances the antioxidant enzyme activity and improves the mitochondrial respiration. The efficacy of RGPU-207 is comparable with phenibut and piracetam.

Trypanosoma carassii infection in goldfish (Carassius auratus L.): changes in the expression of erythropoiesis and anemia regulatory genes.

Trypanosoma carassii is a flagellated bloodstream parasite of cyprinid fish with pathogenesis manifesting primarily as anemia in experimentally infected fish. This anemia is characterized by decreases in the number of circulating red blood cells (RBCs) during peak parasitemia. We examined changes in the key blood metrics and expression of genes known to be important in the regulation of erythropoiesis. Increasing parasitemia was strongly correlated with an overall decrease in the total number of circulating RBCs. Gene expression of key erythropoiesis regulators (EPO, EPOR, GATA1, Lmo2, and HIFα) and proinflammatory cytokines (IFNγ and TNFα) were measured and their expressions differed from those in fish made anemic by injections of phenylhydrazine (PHZ). Significant upregulation of pro-erythropoietic genes was observed in PHZ-induced anemia, but not during peak parasitic infection. Previously, we reported on functional characterization of goldfish erythropoietin (rgEPO) and its ability to induce survival and differentiation of erythroid progenitor cells in vitro. Treatment of goldfish during the infection with rgEPO reduced the severity of anemia but failed to fully prevent the onset of the anemic state in infected fish. Proinflammatory cytokines have been implicated in the suppression of erythropoiesis during trypanosomiasis, specifically the cytokines TNFα, IFNγ, and IL-1ß. Analysis of key proinflammatory cytokines revealed that mRNA levels of IFNγ and TNFα were upregulated in response to infection, but only TNFα increased in response to PHZ treatment. Synergistic activity of the proinflammatory cytokines may be required to sustain prolonged anemia. These findings provide insight into the relationship between T. carassii and host anemia and suggest that T. carassii may directly or indirectly suppress host erythropoiesis.

Transferrin receptor 1 is required for enucleation of mouse erythroblasts during terminal differentiation.

Enucleation is the process whereby the nucleus is extruded from the erythroblast during late stage mammalian erythropoiesis. However, the specific signaling pathways involved in this process remain unclear. To better understand the mechanisms underlying erythroblast enucleation, we investigated erythroblast enucleation using both the spleens of adult mice with phenylhydrazine-induced anemia and mouse fetal livers. Our results indicated that both iron-bound transferrin (holo-Tf) and the small-molecule iron transporter hinokitiol with iron ions (hinokitiol plus iron) promote hemoglobin synthesis and the enucleation of mouse spleen-derived erythroblasts. Although an antitransferrin receptor 1 (TfR1) monoclonal antibody inhibited both enucleation and hemoglobin synthesis promoted by holo-Tf, it inhibited only enucleation, but not hemoglobin synthesis, promoted by hinokitiol plus iron. Furthermore, siRNA against mouse TfR1 were found to suppress the enucleation of mouse fetal liver-derived erythroblasts, and the endocytosis inhibitor MitMAB inhibited enucleation, hemoglobin synthesis, and the internalization of TfR1 promoted by both types of stimuli. Collectively, our results suggest that TfR1, iron ions, and endocytosis play important roles in mouse erythroblast enucleation.

QSAR studies of phenylhydrazine-substituted tetronic acid derivatives based on the 1 H NMR and 13 C NMR chemical shifts.

The quantitative structure-activity relationship models of 40 phenylhydrazine-substituted tetronic acid derivatives were established between the 1 H nuclear magnetic resonance (NMR) and 13 C NMR chemical shifts and the antifungal activity against Fusarium graminearum, Botrytis cinerea, Rhizoctonia cerealis, and Colletotrichum capsici. The models were validated by R, R2 , RA2 , variance inflation factor, F, and P values testing and residual analysis. It was concluded from the models that the 13 C NMR chemical shifts of C8, C10, C7, and the 1 H NMR chemical shifts of Ha contributed positively to the activity against Fusarium graminearum, Botrytis cinerea, Colletotrichum capsici, and Rhizoctonia cerealis, respectively. The models indicated that decreasing the election cloud density of specific nucleuses in compounds, for example, by the substituting of electron withdrawing groups, would improve the antifungal activity. These models demonstrated the practical application meaning of chemical shifts in the quantitative structure-activity relationship study. Furthermore, a practical guide was provided for further structural optimization of the antifungal phenylhydrazine-substituted tetronic acid derivatives based on the 1 H NMR and 13 C NMR chemical shifts.

Impairment of Nitric Oxide Pathway by Intravascular Hemolysis Plays a Major Role in Mice Esophageal Hypercontractility: Reversion by Soluble Guanylyl Cyclase Stimulator.

Paroxysmal nocturnal hemoglobinuria (PNH) patients display exaggerated intravascular hemolysis and esophageal disorders. Since excess hemoglobin in the plasma causes reduced nitric oxide (NO) bioavailability and oxidative stress, we hypothesized that esophageal contraction may be impaired by intravascular hemolysis. This study aimed to analyze the alterations of the esophagus contractile mechanisms in a murine model of exaggerated intravascular hemolysis induced by phenylhydrazine (PHZ). For comparative purposes, sickle cell disease (SCD) mice were also studied, a less severe intravascular hemolysis model. Esophagus rings were dissected free and placed in organ baths. Plasma hemoglobin was higher in PHZ compared with SCD mice, as expected. The contractile responses produced by carbachol (CCh), KCl, and electrical-field stimulation (EFS) were superior in PHZ esophagi compared with control but remained unchanged in SCD mice. Preincubation with the NO-independent soluble guanylate cyclase stimulator 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine (BAY 41-2272; 1 µM) completely reversed the increased contractile responses to CCh, KCl, and EFS in PHZ mice, but responses remained unchanged with prior treatment with NO donor sodium nitroprusside (300 µM). Protein expression of 3-nitrotyrosine and 4-hydroxynonenal increased in esophagi from PHZ mice, suggesting a state of oxidative stress. In endothelial nitric oxide synthase gene-deficient mice, the contractile responses elicited by KCl and CCh were increased in the esophagus but remained unchanged with the intravascular hemolysis induced by PHZ. In conclusion, our results show that esophagus hypercontractile state occurs in association with lower NO bioavailability due to exaggerated hemolysis intravascular and oxidative stress. Moreover, our study supports the hypothesis that esophageal disorders in PNH patients are secondary to intravascular hemolysis affecting the NO-cGMP pathway.

Role of histamine H1 receptor in caffeine induced locomotor sensitization.

The present study elucidated the role of histamine H1 receptor in the caffeine induced locomotor sensitization. Intermittent administration of caffeine (15 mg/kg, i.p.) on alternate days (induction phase) i.e. 1st, 3rd, 5th, 7th, 9th, 11th and 13th resulted in the development of locomotor sensitization. In addition, challenge with sub-stimulant dose of caffeine (10 mg/kg, i.p.) directly on 17th day to induction group animals resulted in expression to locomotor sensitization to caffeine. I.c.v. injection of histaminergic agents concomitantly with caffeine during induction phase i.e. histamine H1 receptor agonist, FMPH (6.5 µg/mouse) significantly potentiated while H1 receptor antagonist, cetirizine (0.1 µg/mouse) attenuated the locomotor sensitization induced by caffeine (15 mg/kg, i.p.). In addition, challenge with caffeine (10 mg/kg, i.p.) on the expression day (17th) to the induction group mice on FMPH + caffeine treatment showed enhanced, while those on cetirizine + caffeine treatment exhibited lesser expression to locomotor sensitization. Therefore, a possible contributory role of the central histaminergic system via H1 receptor stimulation or up-regulation in the caffeine-induced locomotor sensitizing effect is proposed.

Stress Erythropoiesis Model Systems.

Bone marrow steady-state erythropoiesis maintains erythroid homeostasis throughout life. This process constantly generates new erythrocytes to replace the senescent erythrocytes that are removed by macrophages in the spleen. In contrast, anemic or hypoxic stress induces a physiological response designed to increase oxygen delivery to the tissues. Stress erythropoiesis is a key component of this response. It is best understood in mice where it is extramedullary occurring in the adult spleen and liver and in the fetal liver during development. Stress erythropoiesis utilizes progenitor cells and signals that are distinct from bone marrow steady-state erythropoiesis. Because of that observation many genes may play a role in stress erythropoiesis despite having no effect on steady-state erythropoiesis. In this chapter, we will discuss in vivo and in vitro techniques to study stress erythropoiesis in mice and how the in vitro culture system can be extended to study human stress erythropoiesis.

Combined Parthenolide and Balsalazide Have Enhanced Antitumor Efficacy Through Blockade of NF-κB Activation.

Balsalazide is a colon-specific prodrug of 5-aminosalicylate that is associated with a reduced risk of colon cancer in patients with ulcerative colitis. Parthenolide, a strong NF-κB inhibitor, has recently been demonstrated to be a promising therapeutic agent, promoting apoptosis of cancer cells. In the current study, the antitumor effect of balsalazide combined with parthenolide in human colorectal cancer cells and colitis-associated colon cancers (CAC) was investigated. The results demonstrate that the combination of balsalazide and parthenolide markedly suppress proliferation, nuclear translocation of NF-κB, IκB-α phosphorylation, NF-κB DNA binding, and expression of NF-κB targets. Apoptosis via NF-κB signaling was confirmed by detecting expression of caspases, p53 and PARP. Moreover, treatment of a CAC murine model with parthenolide and balsalazide together resulted in significant recovery of body weight and improvement in histologic severity. Administration of parthenolide and balsalazide to CAC mice also suppressed carcinogenesis as demonstrated by uptake of 18F-fluoro-2-deoxy-D-glucose (FDG) using micro-PET/CT scans. These results demonstrate that parthenolide potentiates the efficacy of balsalazide through synergistic inhibition of NF-κB activation and the combination of dual agents prevents colon carcinogenesis from chronic inflammation. IMPLICATIONS: This study represents the first evidence that combination therapy with balsalazide and parthenolide could be a new regimen for colorectal cancer treatment. Mol Cancer Res; 15(2); 141-51. ©2016 AACR.

A novel role for nuclear factor-erythroid 2 in erythroid maturation by modulation of mitochondrial autophagy.

We have recently demonstrated that the transcription factor nuclear factor-erythroid 2, which is critical for erythroid maturation and globin gene expression, plays an important role in the pathophysiology of myeloproliferative neoplasms. Myeloproliferative neoplasm patients display elevated levels of nuclear factor-erythroid 2 and transgenic mice overexpressing the transcription factor develop myeloproliferative neoplasm, albeit, surprisingly without erythrocytosis. Nuclear factor-erythroid 2 transgenic mice show both a reticulocytosis and a concomitant increase in iron deposits in the spleen, suggesting both enhanced erythrocyte production and increased red blood cell destruction. We therefore hypothesized that elevated nuclear factor-erythroid 2 levels may lead to increased erythrocyte destruction by interfering with organelle clearance during erythroid maturation. We have previously shown that nuclear factor-erythroid 2 overexpression delays erythroid maturation of human hematopoietic stem cells. Here we report that increased nuclear factor-erythroid 2 levels also impede murine maturation by retarding mitochondrial depolarization and delaying mitochondrial elimination. In addition, ribosome autophagy is delayed in transgenics. We demonstrate that the autophagy genes NIX and ULK1 are direct novel nuclear factor-erythroid 2 target genes, as these loci are bound by nuclear factor-erythroid 2 in chromatin immunoprecipitation assays. Moreover, Nix and Ulk1 expression is increased in transgenic mice and in granulocytes from polycythemia vera patients. This is the first report implying a role for nuclear factor-erythroid 2 in erythroid maturation by affecting autophagy.