Unusual Relationship between Iron Deprivation and Organophosphate Hydrolase Expression.

Organophosphate hydrolases (OPH), hitherto known to hydrolyze the third ester bond of organophosphate (OP) insecticides and nerve agents, have recently been shown to interact with outer membrane transport components, namely, TonB and ExbB/ExbD. In an OPH negative background, Sphingopyxis wildii cells failed to transport ferric enterobactin and showed retarded growth under iron-limiting conditions. We now show the OPH-encoding organophosphate degradation (opd) gene from Sphingobium fuliginis ATCC 27551 to be part of the iron regulon. A fur-box motif found to be overlapping with the transcription start site (TSS) of the opd gene coordinates with an iron responsive element (IRE) RNA motif identified in the 5' coding region of the opd mRNA to tightly regulate opd gene expression. The fur-box motif serves as a target for the Fur repressor in the presence of iron. A decrease in iron concentration leads to the derepression of opd. IRE RNA inhibits the translation of opd mRNA and serves as a target for apo-aconitase (IRP). The IRP recruited by the IRE RNA abrogates IRE-mediated translational inhibition. Our findings establish a novel, multilayered, iron-responsive regulation that is crucial for OPH function in the transport of siderophore-mediated iron uptake. IMPORTANCE Sphingobium fuliginis, a soil-dwelling microbe isolated from agricultural soils, was shown to degrade a variety of insecticides and pesticides. These synthetic chemicals function as potent neurotoxins, and they belong to a class of chemicals termed organophosphates. S. fuliginis codes for OPH, an enzyme that has been shown to be involved in the metabolism of several organophosphates and their derivatives. Interestingly, OPH has also been shown to facilitate siderophore-mediated iron uptake in S. fuliginis and in another Sphingomonad, namely, Sphingopyxis wildii, implying that this organophosphate-metabolizing protein has a role in iron homeostasis, as well. Our research dissects the underlying molecular mechanisms linking iron to the expression of OPH, prompting a reconsideration of the role of OPH in Sphingomonads and a reevaluation of the evolutionary origins of the OPH proteins from soil bacteria.

Estimation of malignant transformation rate in cases of oral epithelial dysplasia and lichen planus using immunohistochemical expression of Ki-67, p53, BCL-2, and BAX markers.

BACKGROUND: The issue of a possible malignant transformation in the lesions like epithelial dysplasia and oral lichen planus (OLP) is a matter of serious controversy. The purpose of this study was to suggest the malignant transformation rate in OLP and oral epithelial dysplasia (OED) by using immunohistochemical expression of the tumor markers Ki-67, p53, BCL-2, and BAX. MATERIALS AND METHODS: This was a cross-sectional study carried out among 70 samples, including 10 samples from normal healthy mucosa categorized into Group 1, Group 2 (30 OLP), and Group 3 (30 OED) samples. Five sections (4 µm thick) were obtained and stained with monoclonal antibodies such as Ki-67, p53, BCL-2, and BAX and analyzed for number of positive cells and also for intensity of staining. Statistical analysis was done using Mann-Whitney U-test (P < 0.05). RESULTS: Significant results were found only for expressions of Ki-67, p53, BCL-2 markers in both study groups (P < 0.05). In these groups, the intensity of staining was mostly mild to moderate for all studied tumor markers. In this study, subjects with an average positive IHC expression of Ki-67, p53, BCL-2, and BAX markers in normal mucosa was about 22.5%, which was significantly lower when compared with OLP (54.9%) and OED (64.9%). CONCLUSION: The high propensity for malignant transformation in OED followed by OLP suggests that a wide range of inherent and extrinsic factors contribute to the disease progression and malignant transformation.

Simultaneous inhibition of cell-cycle, proliferation, survival, metastatic pathways and induction of apoptosis in breast cancer cells by a phytochemical super-cocktail: genes that underpin its mode of action.

Traditional chemotherapy and radiotherapy for cancer treatment face serious challenges such as drug resistance and toxic side effects. Complementary / Alternative medicine is increasingly being practiced worldwide due to its safety beneficial therapeutic effects. We hypothesized that a super combination (SC) of known phytochemicals used at bioavailable levels could induce 100% killing of breast cancer (BC) cells without toxic effects on normal cells and that microarray analysis would identify potential genes for targeted therapy of BC. Mesenchymal Stems cells (MSC, control) and two BC cell lines were treated with six well established pro-apoptotic phytochemicals individually and in combination (super cocktail), at bioavailable levels. The compounds were ineffective individually. In combination, they significantly suppressed BC cell proliferation (>80%), inhibited migration and invasion, caused cell cycle arrest and induced apoptosis resulting in 100% cell death. However, there were no deleterious effects on MSC cells used as control. Furthermore, the SC down-regulated the expression of PCNA, Rb, CDK4, BcL-2, SVV, and CD44 (metastasis inducing stem cell factor) in the BC cell lines. Microarray analysis revealed several differentially expressed key genes (PCNA, Rb, CDK4, Bcl-2, SVV, P53 and CD44) underpinning SC-promoted BC cell death and motility. Four unique genes were highly up-regulated (ARC, GADD45B, MYLIP and CDKN1C). This investigation indicates the potential for development of a highly effective phytochemical combination for breast cancer chemoprevention / chemotherapy. The novel over-expressed genes hold the potential for development as markers to follow efficacy of therapy.

A novel function of FoxO transcription factors in thrombin-stimulated vascular smooth muscle cell proliferation.

Thrombin exerts coagulation-independent effects on the proliferation and migration of vascular smooth muscle cells (SMC). Forkhead box-O (FoxO) transcription factors regulate cell proliferation, apoptosis and cell cycle arrest, but a possible functional interaction between thrombin and FoxO factors has not been identified to date. In human cultured vascular SMC, thrombin induced a time-dependent phosphorylation of FoxO1 and FoxO3 but not FoxO4. This effect was mimicked by an activating-peptide (AP) for protease-activated receptor (PAR)-1, and abolished by a PAR-1 antagonist (SCH79797). APs for other PARs were without effect. FoxO1 and FoxO3 phosphorylation were prevented by the PI3 kinase (PI3K) inhibitor LY294002 while inhibitors of ERK1/2 (PD98059) or p38MAPK (SB203580) were ineffective. LY294002 moreover prevented thrombin-stimulated SMC mitogenesis and proliferation. FoxO1 and FoxO3 siRNA augmented basal DNA synthesis and proliferation of SMC. Nuclear content of FoxO proteins decreased time-dependently in response to thrombin, coincided with suppressed expression of the cell cycle regulating genes p21CIP1 and p27kip1 by thrombin. FoxO1 siRNA reduced basal p21CIP1 while FoxO3 siRNA attenuated p27kip1 expression; thrombin did not show additive effects. LY294002 restored p21CIP1 and p27kip1 protein expression. Immunohistochemistry revealed that human native and failed saphenous vein grafts were characterised by the cytosolic presence of p-FoxO factors in co-localisation of p21CIP1 and p27kip1 with SMC. In conclusion, thrombin and FoxO factors functionally interact through PI3K/Akt-dependent FoxO phosphorylation leading to expression of cell cycle regulating genes and ultimately SMC proliferation. This may contribute to remodelling and failure of saphenous vein bypass grafts.

Suppression of ovalbumin-induced Th2-driven airway inflammation by ß-sitosterol in a guinea pig model of asthma.

In the present study, the efficacy of ß-sitosterol isolated from an n-butanol extract of the seeds of the plant Moringa oleifera (Moringaceae) was examined against ovalbumin-induced airway inflammation in guinea pigs. All animals (except group I) were sensitized subcutaneously and challenged with aerosolized 0.5% ovalbumin. The test drugs, ß-sitosterol (2.5mg/kg) or dexamethasone (2.5mg/kg), were administered to the animals (p.o.) prior to challenge with ovalbumin. During the experimental period (on days 18, 21, 24 and 29), a bronchoconstriction test (0.25% acetylcholine for 30s) was performed and lung function parameters (tidal volume and respiration rate) were measured for each animal. On day 30, blood and bronchoalveolar lavaged fluid were collected to assess cellular content, and serum was collected for cytokine assays. Lung tissue was utilized for a histamine assay and for histopathology. ß-sitosterol significantly increased the tidal volume (V(t)) and decreased the respiration rate (f) of sensitized and challenged guinea pigs to the level of non-sensitized control guinea pigs and lowered both the total and differential cell counts, particularly eosinophils and neutrophils, in blood and bronchoalveolar lavaged fluid. Furthermore, ß-sitosterol treatment suppressed the increase in cytokine levels (TNFα, IL-4 and IL-5), with the exception of IL-6, in serum and in bronchoalveolar lavaged fluid detected in model control animals. Moreover, treatment with ß-sitosterol protected against airway inflammation in lung tissue histopathology. ß-sitosterol possesses anti-asthmatic actions that might be mediated by inhibiting the cellular responses and subsequent release/synthesis of Th2 cytokines. This compound may have therapeutic potential in allergic asthma.

Immunosuppressive activity of ethanolic extract of seeds of Moringa oleifera Lam. in experimental immune inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditionally, the plant Moringa oleifera Lam. (Moringaceae) is used for the treatment of ascites and rheumatism, while the dried seeds of the plant are used as an 'anti-allergic' agent. AIM OF THE STUDY: The aim of the study was to assess the efficacy of ethanolic extract of seeds from Moringa oleifera Lam. in experimental immune inflammation. MATERIALS AND METHODS: Circulatory and splenic leukocyte counts, delayed-type hypersensitivity reactions and humoral antibody responses were measured in mice using SRBC as the antigen. In addition, macrophage phagocytosis was measured by the carbon clearance test. RESULTS: The extract dose-dependently (50, 100 and 200mg/kg) inhibited spleen weight as well as circulatory leukocyte and splenocyte counts. The delayed-type hypersensitivity reaction was significantly inhibited (P<0.01) by decreasing the mean foot pad thickness at 48 h. The production of the humoral antibody titer was significantly ameliorated at a dose of 100 and 200mg/kg (P<0.05 and P<0.01, respectively). Furthermore, the extract caused a down-regulation of macrophage phagocytosis due to carbon particles. CONCLUSION: Taken together, the above findings suggest that the seeds of Moringa oleifera have immunosuppressive activity.

Inhibitory effect of n-butanol fraction of Moringa oleifera Lam. seeds on ovalbumin-induced airway inflammation in a guinea pig model of asthma.

Moringaceae, which belongs to the Moringa oleifera Lam. family, is a well-known herb used in Asian medicine as an antiallergic drug. In the present study, the efficacy of the n-butanol extract of the seeds of the plant (MONB) is examined against ovalbumin-induced airway inflammation in guinea pigs. The test drugs (MONB or dexamethasone) are administered orally prior to challenge with aerosolized 0.5% ovalbumin. During the experimental period, bronchoconstriction tests are performed, and lung function parameters are measured. The blood and bronchoalveolar lavage fluid are collected to assess cellular content, and serum is used for cytokine (tumor necrosis factor-alpha, interleukin-4, and interleukin-6) assays. Histamine assays of lung tissue are performed using lung tissue homogenate. The results suggest that in ovalbumin-sensitized model control animals, tidal volume is decreased, respiration rate is increased, and both the total and differential cell counts in blood and bronchoalveolar lavage fluid are increased significantly compared with nonsensitized controls. MONB treatment shows improvement in all parameters except bronchoalveolar lavage tumor necrosis factor-alpha and interleukin-4. Moreover, MONB treatment demonstrates protection against acetylcholine-induced bronchoconstriction and airway inflammation. These results indicate that MONB has an inhibitory effect on airway inflammation. Thus, MONB possesses an antiasthmatic property through modulation of the relationship between Th1/Th2 cytokine imbalances.

Effect of Moringa oleifera Lam. seed extract on ovalbumin-induced airway inflammation in guinea pigs.

To determine the therapeutic potential of herbal medicine Moringa oleifera Lam. family: Moringaceae in the control of allergic diseases, the efficacy of the ethanolic extract of the seeds of the plant (MOEE) against ovalbumin (OVA)-induced airway inflammation in guinea pigs was examined. During the experimental period, the test drugs (MOEE or dexamethasone) were administered by oral route prior to challenge with aerosolized 0.5% OVA. Bronchoconstriction tests were performed and respiratory parameters (i.e., tidal volume and respiratory rate) were measured. At the end of experiment, blood was collected from each animal to perform total and differential counts and serum was used for assay of IL-4, IL-6, and TNFalpha. Lung lavage fluid (BAL) was collected for estimation of cellular content and cytokine levels. Lung tissue histamine assays were performed using the homogenate of one lobe from each animal; a separate lobe and the trachea were subjected to histopathology to measure the degree of any airway inflammation. The results suggest that in OVA-sensitized control animals that did not receive either drug, tidal volume (V(t)) was decreased, respiration rate (f) was increased, and both the total and differential cell counts in blood and BAL fluid were increased significantly. MOEE-treatment of sensitized hosts resulted in improvement in all parameters except BAL TNFalpha and IL-4. Moreover, MOEE-treatment also showed protection against acetylcholine-induced broncho-constriction and airway inflammation which was confirmed by histological observations. The results of these studies confirm the traditional claim for the usefulness of this herb in the treatment of allergic disorders like asthma.

Protective Effect of Ethanolic Extract of Seeds of Moringa oleifera Lam. Against Inflammation Associated with Development of Arthritis in Rats.

The present investigation was carried out to study the anti-arthritic activity of ethanolic extract of seeds of Moringa oleifera Lam. (MOEE) in adjuvant-induced arthritis in adult female Wistar rats. During the experimental period, body weight, paw edema volume (primary lesion) and arthritic index (secondary lesion) was observed. On the 21st day, serum from each animal was used for estimation of Rheumatoid Factor (RF) value and levels of selected cytokines (TNFalpha, IL-1, and IL-6). Whole blood was used for measurement of erythrocyte sedimentation rate (ESR). Liver homogenate was utilized for assessment of oxidative stress and histopathology was performed to measure degree of inflammation in synovial joint. Our results suggest that, percentage reduction in body weight was less, paw edema volume and arthritic index score was decreased significantly as compared to diseased control animals. Serum levels of RF, TNF-alpha, IL-1, and IL-6 also showed decreased levels as compared to those in the diseased control group. Treatment with MOEE also altered oxidative stress in relation to its anti-inflammatory activity. Histopathological observations showed mild or less infiltration of lymphocytes, angiogenesis and synovial lining thickening. From all above results and observations, it can be concluded that Moringa oleifera possesses promising antiarthritic property.

Effect of Moringa oleifera Lam. seed extract on toluene diisocyanate-induced immune-mediated inflammatory responses in rats.

Moringa oleifera Lam. is a small tree cultivated throughout India. We have investigated the effect of ethanolic extract of seeds of Moringa oleifera (MOEE, an herbal remedy) on the potential prevention of immune-mediated inflammatory responses in toluene diisocyanate (TDI as antigen)-induced asthma in Wistar rats. Rats were divided into five different groups (n = 8/group): Group-I = unsensitized control; Group-II = TDI control/vehicle; Group-III = dexamethasone (DXM) 2.5 mg/kg; and, Groups IV and V = 100 mg/kg and 200 mg/kg body weight [BW] of MOEE, respectively. All rats (except unsensitized controls) were sensitized by intranasal application of 10% TDI to induce airway hypersensitivity. Animals in Groups II-V were given their respective drug treatment per os from 1 wk prior to initiation of sensitization until the day of final provocation with 5% TDI. After this last challenge, all rats were examined for hyperreactivity symptoms and then sacrificed to determine their total and differential leucocytes in blood and bronchoalveolar (BAL) fluid and levels of TNF proportional, variant, IL-4, and IL-6 in their BAL and serum. Homogenates of one lung lobe from each animal were utilized to assess oxidative stress; a separate lobe underwent histologic examination to assess airway inflammatory status. The results suggest that asthmatic symptoms were found in TDI control rats only, while both MOEE- and DXM-treated rats did not manifest any airway abnormality. In MOEE- and DXM-treated rats, neutrophil and eosinophil levels in the blood were decreased significantly; levels of total cells and each different cell in their BAL fluid were markedly decreased as compared to those in TDI controls. TNF alpha, IL-4, and IL-6 were predominant in serum as well as in BAL fluids in TDI controls, but these levels were reduced significantly by MOEE treatment. The antioxidant activity in relation to antiinflammatory activity of the extract and histopathological observations also reflected a protective effect. Based on the above findings and observations, it can be concluded that Moringa oleifera may possess some beneficial properties that act against chemically stimulated immune-mediated inflammatory responses that are characteristic of asthma in the rat.

Inhibitory Action of Ethanolic Extract of Seeds of Moringa oleifera Lam. On Systemic and Local Anaphylaxis.

The current study characterizes the mechanism by which the seed extract of Moringa oleifera Lam (Moringaceae) decreases the mast cell-mediated immediate type hypersensitivity reaction. The immediate type hypersensitivity reaction is involved in many allergic diseases such as asthma and allergic rhinitis. Moringa oleifera, a shrub widely used in the traditional medicine in India, has been reported to possess anti-cancer, hypotensive, anti-arthritic, and anti-inflammatory activities. In the present study, the effects of the ethanolic extract of seeds of Moringa oleifera (MOEE-herbal remedy) on systemic and local anaphylaxis were investigated. The potential anti-anaphylactic effect of MOEE was studied in a mouse model of Compound 48/80-induced systemic anaphylactic shock. Passive cutaneous anaphylaxis activated by anti IgE-antibody was also used to assess the effect of MOEE. In addition, rat peritoneal mast cells (RPMC) were used to investigate the effect of MOEE on histamine release induced by compound 48/80. When administered 1 hr before 48/80 injection, MOEE at doses of 0.001-1.000 g/kg completely inhibited the inducible induced anaphylactic shock. MOEE significantly inhibited passive cutaneous anaphylaxis activated by anti-IgE antibody at a dose of 1 g/kg. When MOEE extract was given as pretreatment at concentrations ranging 0.1-100 mg/ml, the histamine release from the mast cells that was induced by the 48/80 was reduced in a dose-dependent manner. These results suggest a potential role for MOEE as a source of anti-anaphylactic agents for use in allergic disorders.