Nucleases play important roles in DNA synthesis, recombination and repair. We have previously shown that human exonuclease 1 (hEXO1) is phosphorylated in response to agents stalling DNA replication and that hEXO1 consequently undergoes ubiquitination and degradation in a proteasome-dependent manner. In the present study, we have addressed the identity of the pathway transducing stalled-replication signals to hEXO1. Using chemical inhibitors, RNA interference, ATM- and ATR-deficient cell lines we have concluded that hEXO1 phosphorylation is ATR-dependent. By means of mass spectrometry, we have identified the sites of phosphorylation in hEXO1 in undamaged cells and in cells treated with hydroxyurea (HU). hEXO1 is phosphorylated at nine basal sites and three additional sites are induced by HU treatment. Analysis of single- and multiple-point mutants revealed that mutation to Ala of the three HU-induced sites of phosphorylation partially rescued HU-dependent degradation of hEXO1 and additionally stabilized the protein in non-treated cells. We have raised an antibody to pS714, an HU-induced site of the S/T-Q type, and we provide evidence that S714 is phosphorylated upon HU but not IR treatment. The antibody may be a useful tool to monitor signal transduction events triggered by stalled DNA replication.

Cinnamon is used to flavor most foods in Arabian countries. The aim of this study was to evaluate the medicinal importance, reflecting an important trend in research. The hepatoprotective activity of aqueous and ethanolic extracts of cinnamon was investigated against carbon tetrachloride (CCl 4 ) induced lipid peroxidation and hepatic injury in rats. The elevated serum AST and ALT enzymatic activities induced by CCl 4 were significantly restored to near normal by oral administration of 200 mg/kg of either extracts once daily for 7 days, as compared to untreated rats. There was a significant elevation in the level of liver malondialdhyde (MDA), while the activities of antioxidant enzymes superoxide dismutase and catalase (SOD and CAT) were significantly decreased in CCl 4 intoxicated rats. The results obtained indicated that ethanolic extract has more potent hepatoprotective action than water extract against CCl 4 by lowering the MDA level and elevating antioxidants enzymes activities (SOD and CAT). The possible mechanism of this activity may be free radical-scavenging polyphenol compounds. The hepatoprotective properties were documented by the histopathological data obtained. Consequently, this extract can be used as a therapeutic regime in treatment of some hepatic disorders without any side effects. Further study will be done for separation and identification of active components and for testing antitumor activity.

Breast cancer is the principle cause of death among women worldwide. In this study, we investigated the anti-tumor potential of lycopene (Lyco) alone or combined with melatonin (Lyco + Mel) for 120 days against a single oral dose of (50 mg/kg B.W.) 7,12-dimethylbenz(a)anthracene (DMBA)-induced oxidative stress and mammary carcinogenesis in female rats. The treatment protocol started from the day immediately after DMBA administration. Results obtained indicated that there was an elevation in the levels of malondialdhyde and nitric oxide in serum and breast tissues of DMBA injected rats. The combined treatment (Lyco + Mel) group showed a potential reduction of these parameters more than lyco individually. The activities of SOD, CAT, and GPx were found to be significantly high than lyco alone treated rats. In DMBA group a negative significant correlation between weight and serum nitric oxide (r = -0.59), and a positive significant correlation between NO and MDA (r = 0.81) was observed. Histopathological examination revealed the formation of tumor and angiogenesis in DMBA-induced rats and these abnormal changes were ameliorated by combined treatment with Lyco + Mel. In conclusion, these results suggested that supplementation of diet with lycopene with melatonin provided antioxidant defense with strong chemo preventive activity against DMBA-induced mammary tumors.

Studying the pharmacokinetics of antiretroviral drugs in breast milk has important implications for the health of both the mother and the infant, particularly in resource-poor countries. Breast milk is a highly complex biological matrix, yet it is necessary to develop and validate methods in this matrix, which simultaneously measure multiple analytes, as women may be taking any number of drug combinations to combat human immunodeficiency virus infection. Here, we report a novel extraction method coupled to high-performance liquid chromatography and tandem mass spectrometry for the accurate, precise, and specific measurement of 7 antiretroviral drugs currently prescribed to infected mothers. Using 200 µL of human breast milk, simultaneous quantification of lamivudine (3TC), stavudine (d4T), zidovudine (ZDV), nevirapine (NVP), nelfinavir (NFV), ritonavir, and lopinavir was validated over the range of 10-10,000 ng/mL. Intraday accuracy and precision for all analytes were 99.3% and 5.0 %, respectively. Interday accuracy and precision were 99.4 % and 7.8%, respectively. Cross-assay validation with UV detection was performed using clinical breast milk samples, and the results of the 2 assays were in good agreement (P = 0.0001, r = 0.97). Breast milk to plasma concentration ratios for the different antiretroviral drugs were determined as follows: 3TC = 2.96, d4T = 1.73, ZDV = 1.17, NVP = 0.82, and NFV = 0.21.

BackgroundBrucellosis is a common zoonosis that can cause a severe febrile illness in humans. It constitutes a persistent health problem in many developing countries around the world. It is one of the most frequently reported diseases in Saudi Arabia and incidence is particularly high in the Central region, and around the city of Riyadh. The aim of this study was to evaluate a two-stage PCR assay for detection of human brucellosis particularly in endemic areas.MethodsA total of 101 serum samples were collected from patients with acute febrile illness (AFI) of unknown cause from two different locations in the Western region of Saudi Arabia. The first location (Northern) is characterized by a nomadic rural population while the second (Central) is a modern urban city. All samples were subjected to DNA extraction and Brucella genus-specific PCR amplification using B4/B5 primers of the bcsp31 gene. Positive B4/B5 samples were subjected to multiplex species-specific Brucella PCR amplification.ResultsIn the Northern location, 81.9% of the AFI samples were confirmed Brucella positive, while all the samples collected from the Central region proved to be Brucella negative. Samples positive for Brucella were subjected to multiplex species-specific Brucella amplification. B. abortus was detected in 10% and B. melitensis in 8% of the samples, while the majority (82%) of samples showed both B. abortus and B. melitensis. As expected, B. suis was not detected in any of the samples.ConclusionsThis study concluded that a two-stage PCR assay could be useful as a rapid diagnostic tool to allow the consideration of brucellosis as a possible cause of AFI, particularly in non-urban locations. It also recommends the collection of epidemiological data for such patients to obtain further information that may help in rapid diagnosis.

Abstract:Reactive oxygen species play a significant role in the pathogenesis of retinopathy in diabetes patients. The current study aimed to assess the effect of ethyl acetate extract (EAE) from Balanites aegyptiaca (10, 25 or 50 mg/kg b.w.) in experimental diabetic rats. To achieve this aim, five groups of male rats were included: control, diabetic, and diabetic rats treated with 10, 25, and 50 µg/kg b.w. of EAE for eight weeks. Our results suggests a protective role of EAE against oxidative stress induced by streptozocine. EAE treatment produced a reduction in blood glucose levels, HbA1c, malondialdehyde and vascular endothelial growth factor (VEGF) in diabetic retina (p < 0.001), as well as an enhancement in antioxidant capacity against streptozocine-induced oxidative stress. Tumor necrosis factor alpha (TNF-α), interleukin (IL-1β) and vascular endothelial growth factor (VEGF) were OPEN ACCESSMolecules 2015, 20 14426 significantly reduced in diabetic rats treated with EAE, compared with untreated diabetic rats. Analysis of EAE by GC-MS indicated the presence of β-sistosterol. Overall, EAE modulates oxidative stress induced by streptozocine and enhances antioxidant activity, which may provide additional endothelial protection in retina of diabetic rats. These results hold great promise in the management of diabetic complications.

Hepatocellular carcinoma (HCC) remains one of the leading causes of death worldwide. The complex etiology is attributed to many factors like heredity, cirrhosis, hepatitis infections or the dysregulation of the different molecular pathways. Nevertheless, the current treatment regimens have either severe side effects or tumors gradually acquire resistance upon prolonged use. Thus, developing a new selective treatment for HCC is the need of the hour. Many anticancer agents derived from plants have been evaluated for their cytotoxicity towards many human cancer cell lines. Strigolactones (SLs)-a newly discovered class of phytohormones, play a crucial role in the development of plant-root and shoot. Recently, many synthetic analogues of SLs have demonstrated pro-apoptotic effects on different cancer cell lines like prostate, breast, colon and lung. In this study, we tested synthetic SLs analogues on HCC cell line-HepG2 and evaluated their capability to induce cell proliferation inhibition and apoptosis. Primary WST-1 assays, followed by annexin-V/7AAD staining, demonstrated the anti-proliferative effects. The SLs analogues TIT3 and TIT7 were found to significantly reduce HepG2 cell viability in a dose- and time-dependent manner and induce apoptosis. Interestingly, though TIT3 and TIT7 strongly affected cancer cell proliferation, both compounds showed moderate anti-proliferative effect on normal cells. Further, migration of cancer cells was suppressed upon treatment with TIT3 and TIT7 in a wound healing assay. In summary, these findings suggest that two SLs analogues TIT3 and TIT7 exert selective inhibitory effects on cancer cells most likely through targeting microtubules. SLs analogues could be used in future as potential anti-cancer candidates in chemotherapy.

Nicotine is a major pharmacologically active and addictive component of tobacco smoke, which is regarded to be a primary risk factor in the development of cardiovascular and pulmonary diseases. Epicatechin is one of the most potent antioxidants present in the human diet. Particularly high levels of this compound are found in tea, apples and chocolate. It has been reported that tea extracts and/or its constituents have antibacterial, antiviral, antioxidative, antitumor and antimutagenic activities. Vitamin E is a major lipid-soluble antioxidant vitamin and free radical scavenger, presents as an integral component of cellular membranes and has important biological functions. The primary mechanism by which vitamin E is proposed to prevent cancer is through their antioxidant properties. The goal of this study is to evaluate the effect of epicatechin alone or combined with vitamin E in inhibiting the oxidative stress induced by nicotine in rats. Results obtained indicated that there was a significant elevation in the levels of malondialdhyde (MDA) in nicotine injected rats. The combined treatment (epicatechin + Vit E) group showed a potential reduction of these parameters more than individual treatment. The activities of superoxide dismutase, catalase and glutathione peroxidase were found significantly higher in combined treated than untreated rats. In nicotine group, a negative significant correlation between reduced glutathione and MDA (r = -0.92) was observed. In conclusion, these results suggested that the supplementation of diet with epicatechin and vitamin E provided antioxidant defense with strong chemopreventive activity against nicotine-induced carcinogenesis.