Assessment of DNA damage in women using oral contraceptives

The effect of the use of an oral contraceptive (OC) on the frequency of sister chromatid exchanges (SCEs) and on the response in the alkaline comet assay (single-cell gel electrophoresis (SCGE)) was investigated in 18 women taking contraceptive pills daily for 24 months. As controls, fertile women were included with regular menstrual cycles who received no OC drugs. A significant increase in the number of lymphocytes with DNA migration and an increased frequency of SCE per metaphase were observed in OC users as compared with their age-matched untreated controls (P<0.005). As higher incidences of spontaneous SCEs in peripheral blood lymphocytes have been reported to occur in females during pregnancy due to profound changes in the levels of certain sex hormones such as progesterone and estrogen, particularly during the last trimester, 17 pregnant women served as positive controls in this study in order to test the rate of genetic damage due to those changes. Higher frequencies of SCEs and comet responses were observed in pregnant women than in their matched controls. However, no statistically significant difference in DNA damage was observed between OC users and pregnant women (P>0.05). This study underscores the fact that prolonged and extensive use of these drugs in our daily life may be hazardous and also, that OC users should be aware of multifactorial risk factors (environmental, genetic and life style patterns) that may be responsible for additional DNA damage.     

Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

Kaposi''s Sarcoma (KS), caused by Kaposi''s Sarcoma Herpesvirus (KSHV), is a highly vascularised angiogenic tumor of endothelial cells, characterized by latently KSHV-infected spindle cells and a pronounced inflammatory infiltrate. Several KSHV proteins, including LANA-1 (ORF73), vCyclin (ORF72), vGPCR (ORF74), vIL6 (ORF-K2), vCCL-1 (ORF-K6), vCCL-2 (ORF-K4) and K1 have been shown to exert effects that can lead to the proliferation and atypical differentiation of endothelial cells and/or the secretion of cytokines with angiogenic and inflammatory properties (VEGF, bFGF, IL6, IL8, GROα, and TNFβ). To investigate a role of the KSHV K15 protein in KSHV-mediated angiogenesis, we carried out a genome wide gene expression analysis on primary endothelial cells infected with KSHV wildtype (KSHVwt) and a KSHV K15 deletion mutant (KSHVΔK15). We found RCAN1/DSCR1 (Regulator of Calcineurin 1/Down Syndrome critical region 1), a cellular gene involved in angiogenesis, to be differentially expressed in KSHVwt- vs KSHVΔK15-infected cells. During physiological angiogenesis, expression of RCAN1 in endothelial cells is regulated by VEGF (vascular endothelial growth factor) through a pathway involving the activation of PLCγ1, Calcineurin and NFAT1. We found that K15 directly recruits PLCγ1, and thereby activates Calcineurin/NFAT1-dependent RCAN1 expression which results in the formation of angiogenic tubes. Primary endothelial cells infected with KSHVwt form angiogenic tubes upon activation of the lytic replication cycle. This effect is abrogated when K15 is deleted (KSHVΔK15) or silenced by an siRNA targeting the K15 expression. Our study establishes K15 as one of the KSHV proteins that contribute to KSHV-induced angiogenesis.     

Carbonic anhydrase inhibitors. Inhibition of red blood cell ostrich (Struthio camelus) carbonic anhydrase with a series of aromatic and heterocyclic sulfonamides

The purification of red blood cell carbonic anhydrase (CA, EC 4.2.1.1) from ostrich (scCA) blood is reported, as well as an inhibition study of this enzyme with a series of aromatic and heterocylic sulfonamides. The ostrich enzyme showed a high activity, comparable to that of the human isozyme II, with kcat, of 1.2 x 10(6) s(-1) and kcat/KM of 1.8 x 10(7) M(-1)s(-1), and an inhibition profile quite different from that of the human red blood cell cytosolic isozymes hCA I and II. scCA has generally a lower affinity for sulfonamide inhibitors as compared to hCA I and II. The only sulfonamide which behaved as a very potent inhibitor of this enzyme was ethoxzolamide (KI = 3.9 nM) whereas acetazolamide and sulfanilamide behaved as weaker inhibitors (inhibition constants in the range 303-570 nM). Several other aromatic and heterocyclic sulfonamides, mostly derivatives of sulfanilamide, homosulfanilamide, 4-aminoethylbenzenesulfonamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide, showed good affinities for the ostrich enzyme, with KI values in the range 25-72 nM.     

Detection ofCandida albicans by culture, serology and PCR in clinical specimens from patients with ulcerative colitis: Re-evaluation of an old hypothesis with a new perspective

The relationship between inflammatory bowel disease and microorganisms was evaluated. The presence of Candida albicans-specific IgM and IgG antibodies in serum samples and the presence of C. albicans in stool and colonal mucosa samples of the patients did not exhibit any significant difference between 21 patients in active stage and 15 patients in remission of ulcerative colitis (UC) (compared with 19 control patients). The invasion of yeast cells to the colonal mucosa was demonstrated by detecting C. albicans DNA using specific PCon1, PCon2, and PspA2 primers in PCR assay. Eighteen of 36 patients (50%) were found to be DNA positive while in 19 controls only 4 (21%) were found to be positive. The presence of DNA in the association of the positive serological reactivity is suggested as an important diagnostic marker of UC.     

Some morphological and anatomical observations during alleviation of salinity (NaCI) stress on seed germination and seedling growth of barley by polyamines

In this work, the effects of polyamine (cadaverine, putrescine, spermidine, spermine) pretreatments on the germination of barley seeds, seedling growth under saline (NaCI) conditions and on leaf anatomy of the seedlings grown for 20 days in pots with perlite containing different concentrations of NaCI prepared with Hoagland solutions following the germination period of 7 days were studied. The inhibitive effect of salt on seed germination and seedling growth was alleviated in varying degrees, and dramatically, by polyamine pretreatments at the levels of NaCI studied; particularly with lower levels of salt, the control seeds were able to germinate with great difficulty. Also, the successes of these pretreatments continued, even decreased, with higher levels of NaCI such that the control seeds showed no germination. On the other hand, on the various parameters of leaf anatomy of barley seedlings, interactions between pretreatments of polyamine and salinity, with some of these being statistically important, were observed.     

Purification and characterization of an intracellular chymotrypsin-like serine protease from Thermoplasma volcanium

An intracellular serine protease produced by Thermoplasma (Tp.) volcanium was purified using a combination of ammonium sulfate fractionation, ion exchange, and alpha-casein agarose affinity chromatography. This enzyme exhibited the highest activity and stability at pH 7.0, and at 50 degrees C. The purifed enzyme hydrolyzed synthetic peptides preferentially at the carboxy terminus of phenylalanine or leucine and was almost completely inhibited by PMSF, TPCK, and chymostatin, similarly to a chymotrypsin-like serine protease. Kinetic analysis of the Tp. volcanium protease reaction performed using N-succinyl-L-phenylalanine-p-nitroanilide as substrate revealed a Km value of 2.2 mM and a Vmax value of 0.045 micromol(-1) ml(-1) min(-1). Peptide hydrolyzing activity was enhanced by >2-fold in the presence of Ca2+ and Mg2+ at 2-12 mM concentration. The serine protease is a monomer with a molecular weight of 42 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram activity staining.     

The effect of heme oxygenase-1 induction by octreotide on radiation enteritis

Radiation enteritis occurs as a response to abdominal radiation, which can cause mucosal damage in the gastrointestinal mucosal epithelium. The small intestine is one of the most radiosensitive organs in the abdomen. The present study was undertaken to investigate the effect of octreotide (OCT) administration on heme oxygenase-1 (HO-1) expression of the radiation enteritis model. Rats received 50 mg/kg/day OCT for 4 days before irradiation and continued for 3 days after irradiation. Intestinal myeloperoxidase (MPO) activities, malondialdehyde (MDA) levels are indicators of oxidative damage while caspase-3 activities reveal apoptosis degree of the small intestine. At histological examination, the terminal ileum tissue was analyzed for morphological changes. Irradiation significantly increased the intestinal MPO and caspase-3 activities, MDA levels and HO-1 expression in comparison to sham control group. OCT treatment was associated with increased HO-1 expression and caspase-3 activity, decreased MPO activity and MDA levels. Histological examination revealed that the intestinal mucosal structure was preserved in the OCT treated group. OCT appears to have protective effects against radiation-induced intestinal damage. This protective effect is, in part, mediated by modification of the inflammatory response and the induction of HO-1 expression.     

Antitrypanosomal cycloartane glycosides from Astragalus baibutensis

Baibutoside (5), a new cycloartane-type triterpene glycoside, has been isolated from the roots of Astragalus baibutensis along with four known glycosides, acetylastragaloside I (1), and astragalosides I, II, and IV (2-4, resp.). The structure elucidation of the compounds were achieved by a combination of one- and two-dimensional NMR techniques (DQF-COSY, HSQC, HMBC, and ROESY), and mass spectrometry (ESI-MS), where all the compounds were shown to have cycloastragenol (=(20R,24S)-3beta,6alpha,16beta,25-tetrahydroxy-20,24-epoxy-9,19-cyclolanostane) as aglycone. All compounds were tested for in vitro antiprotozoal activity. Compounds 1-4 displayed notable activity vs. Trypanosoma brucei rhodesiense, with acetylastragaloside I (1) being the most potent (IC50 9.5 microg/ml). Acetylastragaloside I (1) was also lethal to T. cruzi (IC50 5.0 microg/ml), and it is the first cycloartane-type triterpene with remarkable trypanocidal activity against both T. brucei rhodesiense and T. cruzi. However, it exhibits some cytotoxicity on mammalian cells.     

Synthesis of 4-aryl-3,4-dihydropyrimidin-2(1H)-thione derivatives as potential calcium channel blockers

Biginelli reaction which involves condensation of methyl 3-oxopentanoate, aromatic aldehydes and thiourea with a catalytic amount of HCl at reflux temperature has been used for the synthesis of 4-aryl-6-ethyl-5-(methoxycarbonyl)-3,4-dihydropyrimidin-2(1H)-thiones (1–16). In addition, Lewis acids such as FeCl₃·6H₂O and/or H₃BO₃ were also used as catalysts for one-pot synthesis of the products. Compared to the classical Biginelli reaction conditions, the usage of Lewis acids has the advantage of good yields and short reaction times. The calcium channel blocker activities of 1–16 were screened by the tests performed on isolated rat ileum and thoracic aorta. Although product 11, 2-nitrophenyl-derivative, has potent antispasmodic activity on BaCl₂-stimulated rat ileum, it does not have vasodilator activity on KCl-stimulated rat thoracic aorta. Product 15, 2-ethoxyphenyl-derivative, exhibited significant antispasmodic and vasodilator activities in both screening paradigms.     

Carbonic anhydrase inhibitors. Inhibition of red blood cell ostrich (Struthio camelus) carbonic anhydrase with a series of aromatic and heterocyclic sulfonamides

The purification of red blood cell carbonic anhydrase (CA, EC 4.2.1.1) from ostrich (scCA) blood is reported, as well as an inhibition study of this enzyme with a series of aromatic and heterocylic sulfonamides. The ostrich enzyme showed a high activity, comparable to that of the human isozyme II, with k_cat of 1.2·10⁶ s^{? 1} and k_cat/K_M of 1.8·10⁷ M^{? 1} s^{? 1}, and an inhibition profile quite different from that of the human red blood cell cytosolic isozymes hCA I and II. scCA has generally a lower affinity for sulfonamide inhibitors as compared to hCA I and II. The only sulfonamide which behaved as a very potent inhibitor of this enzyme was ethoxzolamide (K_I = 3.9 nM) whereas acetazolamide and sulfanilamide behaved as weaker inhibitors (inhibition constants in the range 303–570 nM). Several other aromatic and heterocyclic sulfonamides, mostly derivatives of sulfanilamide, homosulfanilamide, 4-aminoethylbenzenesulfonamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide, showed good affinities for the ostrich enzyme, with K_I values in the range 25–72 nM.