Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats

This study aimed to investigate the effect of magnolol (5,5′-diallyl-2,2′-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca²⁺ currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3–100 μM). In the presence of Bay K8644 (100 nM), magnolol (10–100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and N_ώ-nitro-l-arginine methyl ester (l-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3–100 μM) inhibited the L-type Ca²⁺ currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca²⁺ channel activity.     

An in situ study of bioenergetic properties of human colorectal cancer: The regulation of mitochondrial respiration and distribution of flux control among the components of ATP synthasome

The aim of this study is to characterize the function of mitochondria and main energy fluxes in human colorectal cancer (HCC) cells. We have performed quantitative analysis of cellular respiration in post-operative tissue samples collected from 42 cancer patients. Permeabilized tumor tissue in combination with high resolution respirometry was used.Our results indicate that HCC is not a pure glycolytic tumor and the oxidative phosphorylation (OXPHOS) system may be the main provider of ATP in these tumor cells. The apparent Michaelis–Menten constant (K_m) for ADP and maximal respiratory rate (V_m) values were calculated for the characterization of the affinity of mitochondria for exogenous ADP: normal colon tissue displayed low affinity (K_m = 260 ± 55 μM) whereas the affinity of tumor mitochondria was significantly higher (K_m = 126 ± 17 μM). But concurrently the V_m value of the tumor samples was 60–80% higher than that in control tissue. The reason for this change is related to the increased number of mitochondria. Our data suggest that in both HCC and normal intestinal cells tubulin β-II isoform probably does not play a role in the regulation of permeability of the MOM for adenine nucleotides.The mitochondrial creatine kinase energy transfer system is not functional in HCC and our experiments showed that adenylate kinase reactions could play an important role in the maintenance of energy homeostasis in colorectal carcinomas instead of creatine kinase.Immunofluorescent studies showed that hexokinase 2 (HK-2) was associated with mitochondria in HCC cells, but during carcinogenesis the total activity of HK did not change. Furthermore, only minor alterations in the expression of HK-1 and HK-2 isoforms have been observed.Metabolic Control analysis showed that the distribution of the control over electron transport chain and ATP synthasome complexes seemed to be similar in both tumor and control tissues. High flux control coefficients point to the possibility that the mitochondrial respiratory chain is reorganized in some way or assembled into large supercomplexes in both tissues.     

Dual oxidase 2 generated reactive oxygen species selectively mediate the induction of mucins by epidermal growth factor in enterocytes

Dual oxidase 2 enzyme is a member of the reactive oxygen species-generating cell membrane NADPH oxidases involved in mucosal innate immunity. It is not known if the biological activity of dual oxidase 2 is mediated by direct bacterial killing by reactive oxygen species produced by the enzyme or by the same reactive oxygen species acting as second messengers that stimulate novel gene expression. To uncover the role of reactive oxygen species and dual oxidases as signaling molecules, we have dissected the pathway triggered by epidermal growth factor to induce mucins, the principal protective components of gastrointestinal mucus. We show that dual oxidase 2 is essential for selective epidermal growth factor induction of the transmembrane MUC3 and the secreted gel-forming MUC5AC mucins. Reactive oxygen species generated by dual oxidase 2 stabilize tyrosine phosphorylation of epidermal growth factor receptor and induce MUC3 and MUC5AC through persistent activation of extracellular signal-regulated kinases 1/2–protein kinase C. Knocking down dual oxidase 2 by selective RNA targeting (siRNA) reduced epidermal growth factor receptor phosphorylation, and MUC3 and MUC5AC gene expression. Extracellular reactive oxygen species produced by dual oxidase 2, upon stimulation by epidermal growth factor, stabilize epidermal growth factor receptor phosphorylation and activate extracellular signal-regulated kinases 1/2–protein kinase C which induce MUC5AC and MUC3. Extracellular reactive oxygen species produced by dual oxidase 2 that are known to directly kill bacteria, also contribute to the maintenance of the epidermal growth factor-amplification loop, which induces mucins. These data suggest a new function of dual oxidase 2 protein in the luminal protection of the gastrointestinal tract through the induction of mucin expression by growth factors.     

Genetic factors in individual radiation sensitivity

Cancer risk and radiation sensitivity are often associated with alterations in DNA repair, cell cycle, or apoptotic pathways. Interindividual variability in mutagen or radiation sensitivity and in cancer susceptibility may also be traced back to polymorphisms of genes affecting e.g. DNA repair capacity. We studied possible associations between 70 polymorphisms of 12 DNA repair genes with basal and initial DNA damage and with repair thereof. We investigated DNA damage induced by ionizing radiation in lymphocytes isolated from 177 young lung cancer patients and 169 cancer-free controls. We also sought replication of our findings in an independent sample of 175 families (in total 798 individuals). DNA damage was assessed by the Olive tail moment (OTM) of the comet assay. DNA repair capacity (DRC) was determined for 10, 30 and, 60 min of repair.Genes involved in the single-strand-repair pathway (SSR; like XRCC1 and MSH2) as well as genes involved in the double-strand-repair pathway (DSR; like RAD50, XRCC4, MRE11 and ATM) were found to be associated with DNA damage. The most significant association was observed for marker rs3213334 (p = 0.005) of XRCC1 with basal DNA damage (B), in both cases and controls. A clear additive effect on the logarithm of OTM was identified for the marker rs1001581 of the same LD-block (p = 0.039): B_CC = −1.06 (95%-CI: −1.16 to −0.96), B_CT = −1.02 (95%-CI: −1.11 to −0.93) and B_TT = −0.85 (95%-CI: −1.01 to −0.68). In both cases and controls, we observed significantly higher DNA basal damage (p = 0.007) for carriers of the genotype AA of marker rs2237060 of RAD50 (involved in DSR). However, this could not be replicated in the sample of families (p = 0.781). An alteration to DRC after 30 min of repair with respect to cases was observed as borderline significant for marker rs611646 of ATM (involved in DSR; p = 0.055), but was the most significant finding in the sample of families (p = 0.009).Our data indicate that gene variation impacts measurably on DNA damage and repair, suggesting at least a partial contribution to radiation sensitivity and lung cancer susceptibility.     

Pentraxin 3 is an anti-inflammatory protein associated with lipid-induced interleukin 10 in vitro

Pentraxin 3 (PTX3) is an acute phase protein expressed in response to pro-inflammatory stimuli during atherosclerosis. However, recent findings suggest that PTX3 is a counter-regulatory protein which enhances the anti-inflammatory response.ObjectiveTherefore, the capacity of PTX3 to alter the inflammatory milieu following in vitro stimulation of PBMCs with the pro-inflammatory lipid, palmitate, was examined.MethodsPBMCs from 17 healthy male donors were isolated and cultured under four separate conditions; 200 μmol/L palmitate, a physiologically relevant concentration of PTX3, in combination (pal + PTX3), and an unstimulated time-course control.ResultsPalmitate-induced production of the counter-regulatory protein PTX3 was positively associated with the production of the anti-inflammatory cytokine interleukin 10 (IL-10) following in vitro stimulation of human PBMCs. Furthermore, stimulation of PBMCs in vitro with 500 pg/mL PTX3 elicited a significantly greater increase in IL-10 production compared to the palmitate stimulated conditions. However, PTX3 stimulation did not result in the production of the pro-inflammatory cytokines IL-1β, IL-6, and tumor necrosis factor alpha, and when combined with palmitate, did not alter the pro-inflammatory milieu from PBMCs in this study.ConclusionThese findings provide evidence supporting the role of PTX3 as a mediator of the anti-inflammatory response in physiologically relevant conditions, and suggests that PTX3 counter regulates the development of atherosclerosis by enhancing the production of IL-10.     

Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroA_A.sp. A phylogenetic analysis revealed that AroA_A.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The K_M for PEP and IC₅₀ [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroA_A.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC₅₀ [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroA_A.sp. The specific activity of the wild-type AroA_A.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.     

Portulaca oleracea reduces triglyceridemia,cholesterolemia, and improves lecithin: cholesterol acyltransferase activity in rats fed enriched-cholesterol diet

PurposeThe effects of Portulaca oleracea (Po) lyophilized aqueous extract were determined on the serum high-density lipoproteins (HDL₂ and HDL₃) amounts and composition, as well as on lecithin: cholesterol acyltansferase (LCAT) activity.MethodsMale Wistar rats (n = 12) were fed on 1% cholesterol-enriched diet for 10 days. After this phase, hypercholesterolemic rats (HC) were divided into two groups fed the same diet supplemented or not with Portulaca oleracea (Po-HC) (0.5%) for four weeks.ResultsSerum total cholesterol (TC) and triacylglycerols (TG), and liver TG values were respectively 1.6-, 1.8-, and 1.6-fold lower in Po-HC than in HC group. Cholesterol concentrations in LDL-HDL₁, HDL₂, and HDL₃ were respectively 1.8, 1.4-, and 2.4-fold decreased in Po-HC group. HDL₂ and HDL₃ amounts, which were the sum of apolipoproteins (apos), TG, cholesteryl esters (CE), unesterified cholesterol (UC), and phospholipids (PL) contents, were respectively 4.5-fold higher and 1.2-fold lower with Po treatment. Indeed, enhanced LCAT activity (1.2-fold), its cofactor-activator apo A-I (2-fold) and its reaction product HDL₂-CE (2.1-fold) were observed, whereas HDL₃-PL (enzyme substrate) and HDL₃-UC (acyl group acceptor) were 1.2- and 2.4-fold lower.ConclusionPortulaca oleracea reduces triglyceridemia, cholesterolemia, and improves reverse cholesterol transport in rat fed enriched-cholesterol diet, contributing to anti-atherogenic effects.     

Reference database of the gait cycle for young healthy Tunisian adults

BackgroundQuantified gait analysis is a rising technology used increasingly to assess motor disorders. Normal reference data are required in order to evaluate patients, but there are no reference data available for the Tunisian healthy population.AimTo assess the features of normal Tunisian gait pattern, and examine the intrinsic reliability of spatio-temporal, kinematic and kinetic parameters within a new specific reference database.MethodsEighteen healthy active-young adults (age: 23.30 ± 2.54 years, height: 1.78 ± 0.04 m and, weight: 70.00 ± 4.80 kg) have participated to five trials of step gait where the dominant lower limb were recorded. Two over the five trials were randomly selected to be further analyzed. Twenty-three spatio-temporal, kinematic and kinetic parameters determined from 3-dimensional gait analysis. The intrinsic reliability was examined for each variable and our results were compared with those available in the literature.ResultsTwelve over 23 parameters have an excellent intrinsic reliability (P > 0.05, ICC > 0.9 and SEM < 5% of the grand mean). There are similarities with other studies (P < 0.05) but we noticed the existence of some specificity (the height of hip extension peak and the low cadence of gait) that could characterize the Tunisian population.ConclusionA specific reference database of the gait cycle has been established for healthy Tunisian active-young adults and excellent inter-trial reliability may be observed for different variables.     

Antileishmanial activity of essential oil from Chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in BALB/c mice

Chenopodium ambrosioides have been used during centuries by native people to treat parasitic diseases.Aims of the studyTo compare the in vivo anti-leishmanial activity of the essential oil (EO) from C. ambrosioides and its major components (ascaridole, carvacrol and caryophyllene oxide).Materials and methodsAnti-leishmanial effect was evaluated in BALB/c mice infected with Leishmania amazonensis and treated with the EO, main compounds and artificial mix of pure components by intralesional route at 30 mg/kg every 4 days during 14 days. Diseases progression and parasite burden in infected tissues were determined.ResultsEO prevented lesion development compared (p < 0.05) with untreated animals and treated with vehicle. In addition, the efficacy of EO was also statistically superior (p < 0.05) compared with the glucantime-treated animals. No potential effects were observed with pure components treatment. Mix of pure compounds cause death of animals after 3 days of treatment.ConclusionsOur results demonstrate the superiority of EO against experimental cutaneous leishmaniasis caused by L. amazonensis.     

Effects of ophiopogonin D and spicatoside A derived from Liriope Tuber on secretion and production of mucin from airway epithelial cells

In the present study, we investigated whether aqueous extract of Liriope Tuber, ophiopogonin D and spicatoside A derived from Liriope Tuber affect basal or phorbol ester (phorbol 12-myristate 13-acetate, PMA)-induced airway mucin production and secretion from airway epithelial cells. Confluent NCI-H292 cells were treated with each agent for 24 h (basal production) or pretreated with each agent for 30 min and then stimulated with PMA for 24 h (PMA-induced production and secretion), respectively. MUC5AC airway mucin production and secretion were measured by ELISA. The results were as follows: (1) aqueous extract of Liriope Tuber stimulated basal mucin production and did not inhibit but increased PMA-induced mucin production; (2) ophiopogonin D and spicatoside A stimulated basal mucin production and did not inhibit but increased PMA-induced mucin production; (3) two compounds increased PMA-induced mucin secretion. These results suggest that ophiopogonin D and spicatoside A can increase mucin production and secretion, by directly acting on airway epithelial cells and, at least in part, explain the traditional use of aqueous extract of Liriope Tuber as expectorants in diverse inflammatory pulmonary diseases.     

Resveratrol and curcumin synergistically induces apoptosis in cigarette smoke condensate transformed breast epithelial cells through a p21Waf1/Cip1 mediated inhibition of Hh-Gli signaling

Combination therapy using two or more small molecule inhibitors of aberrant signaling cascade in aggressive breast cancers is a promising therapeutic strategy over traditional monotherapeutic approaches. Here, we have studied the synergistic mechanism of resveratrol and curcumin induced apoptosis using in vitro (cigarette smoke condensate mediated transformed breast epithelial cell, MCF-10A-Tr) and in vivo (tumor xenograft mice) model system. Resveratrol exposure increased the intracellular uptake of curcumin in a dose dependent manner and caused apoptosis in MCF-10A-Tr cells. Approximately, ten fold lower IC₅₀ value was noted in cells treated with the combination of resveratrol (3 μM) and curcumin (3 μM) in comparison to 30 μM of resveratrol or curcumin alone. Resveratrol + curcumin combination caused apoptosis by increasing Bax/Bcl-xL ratio, Cytochrome C release, cleaved product of PARP and caspase 3 in cells. Interestingly, this combination unaltered the protein expressions of WNT-TCF and Notch signaling components, β-catenin and cleaved notch-1 val1744, respectively. Furthermore, the combination also significantly decreased the intermediates of Hedgehog-Gli cascade including SMO, SHH, Gli-1, c-MYC, Cyclin-D1, etc. and increased the level of p21^Waf/Cip1 in vitro and in vivo. A significant reduction of Gli- promoter activity was noted in combinational drug treated cells in comparison to individual drug treatment. Un-alteration of the expressions of the above proteins and Gli1 promoter activity in p21^Waf/Cip1 knockout cells suggests this combination caused apoptosis through p21^Waf/Cip1. Thus, our findings revealed resveratrol and curcumin synergistically caused apoptosis in cigarette smoke induced breast cancer cells through p2  ^Waf/Cip1 mediated inhibition of Hedgehog-Gli cascade.     

Effects of praeruptorin C on blood pressure and expression of phospholamban in spontaneously hypertensive rats

BackgroundThe traditional Chinese medicine Praeruptorin c (Pra-c) has many physiological and pharmacological effects, including antagonistic effects on blood pressure and calcium levels, maintenance of cellular calcium homeostasis, and improved cardiac systolic and diastolic function. It is potentially a novel and versatile drug for the treatment and prevention of cardiovascular diseases.ObjectiveTo explore the possible impact of Pra-c on blood pressure in SHR and its mechanism of action.Materials and methodsTwenty SHR were randomly divided into a Pra-c group [Pra-c was administered intragastrically, 20 mg kg⁻¹ d⁻¹, n = 10] or an untreated control group (n = 10), containing 10 age-matched SD rats. Each group of rats was followed for 8 weeks. Before and during the treatment, tail artery systolic blood pressure was measured using a tail-cuff every 2 weeks. After 8 weeks, the rats were sacrificed and RNA was extracted from homogenates of cardiac tissue. Tissue from the left ventricle was fixed, sectioned and H&E stained to assess possible changes in myocardial cell structure and morphology. Semi-quantitative RT-PCR was used to assess changes in phospholamban gene expression in treated and untreated rats.ResultsSHR treated with Pra-c for 8 weeks had a lower systolic pressure than untreated SHR (p < 0.05), two measures of cardiac damage, the heart mass index and left ventricle mass index (HMI and LVMI, respectively) were improved, and the level of PLB mRNA expression was lower in the untreated SHR group (p < 0.05).Discussion and conclusionWith continuous hypertension, SHR gradually formed or developed cardiac hypertrophy and fibrosis. Pra-c had a clear effect on blood pressure in SHR, and reversed SHR ventricular remodeling by upregulating the gene expression of sarcoplasmic reticulum PLB.     

Norepinephrine stimulation of alpha1D-adrenoceptor promotes proliferation of pulmonary artery smooth muscle cells via ERK-1/2 signaling

It has been shown that the sympathetic nervous system is activated in pulmonary arterial hypertension (PAH). Norepinephrine (NE) levels are increased by chemoreflex-dependent sympathetic overactivation and involved in pulmonary vascular remodeling. However, the underlying mechanisms of the remodeling induced by NE are poorly understood. In this study, we found that, in vivo, the expression of tyrosine hydroxylase and the concentration of plasma NE were increased in PAH rats compared with normal rats. Increases in ventricular hypertrophy and medial width of the pulmonary arteries were reversed by prazosin, α₁-adrenoceptor (α₁-AR) antagonists, in PAH rats. Elevated expression of α_1D-AR was detected in PAH rats. In addition, prazosin reduced the increasing expression of PCNA, CyclinA and CyclinE induced by hypoxia. In vitro, MTT assay, flow cytometry, Western blotting and immunofluorescence were performed to investigate the effects of NE on proliferation of pulmonary artery smooth muscle cells (PASMCs). We revealed that NE promoted PASMCs viability, increased the expression of PCNA, CyclinA and CyclinE, made more cells from G₀/G₁ phase to G₂/M + S phase and enhanced the microtubule formation. Above NE-induced changes could be suppressed by BMY 7378, an inhibitor of α_1D-AR. Furthermore, ERK-1/2 pathway was activated by NE. U0126, a specific inhibitor for ERK-1/2, attenuated the NE-induced proliferation of PASMCs under normoxia and hypoxia. Taken together, our results suggest that NE which stimulates α_1D-AR promotes proliferation of PASMCs and the effect is, at least in part, mediated via the ERK-1/2 pathway.     

Synthesis and antiproliferative activity of two diastereomeric lignan amides serving as dimeric caffeic acid-l-DOPA hybrids

Two new diastereomeric lignan amides (4 and 5) serving as dimeric caffeic acid-l-DOPA hybrids were synthesized. The synthesis involved the FeCl₃-mediated phenol oxidative coupling of methyl caffeate to afford trans-diester 1a as a mixture of enantiomers, protection of the catechol units, regioselective saponification, coupling with a suitably protected l-DOPA derivative, separation of the two diastereomers thus obtained by flash column chromatography and finally global chemoselective deprotection of the catechol units. The effect of hybrids 4 and 5 and related compounds on the proliferation of two breast cancer cell lines with different metastatic potential and estrogen receptor status (MDA-MB-231 and MCF-7) and of one epithelial lung cancer cell line, namely A-549, was evaluated for concentrations ranging from 1 to 256 μM and periods of treatment of 24, 48 and 72 h. Both hybrids showed interesting and almost equipotent antiproliferative activities (IC₅₀ 64–70 μM) for the MDA-MB-231 cell line after 24–48 h of treatment, but they were more selective and much more potent (IC₅₀ 4–16 μM) for the MCF-7 cells after 48 h of treatment. The highest activity for both hybrids and both breast cancer lines was observed after 72 h of treatment (IC₅₀ 1–2 μM), probably as the result of slow hydrolysis of their methyl ester functions.     

Kososan,a standardized traditional Japanese herbal medicine,reverses sleep disturbance in socially isolated mice via GABAA-benzodiazepine receptor complex activation

PurposeKososan (KSS), a traditional Japanese medicine with a distinct aroma, is clinically used to treat affective disorders but its antidepressant-like effect has not been thoroughly investigated. In this study, we investigated the effects of inhaled and orally administered KSS on sleep disturbances in socially isolated mice.MethodsFour-weeks-old male ddy mice were housed either in social isolation or in groups for 4–6 weeks before the experiment. KSS was orally administered (0.5 or 1.0 g/kg) or inhaled (0.5, 1.0, or 2.5 g/0.125 m³) 60 min before pentobarbital administration. Stress levels in mice were evaluated by the duration of pentobarbital-induced sleeping time.ResultsSleeping time was shorter in socially-isolated mice than in group-housed mice. Oral and inhaled KSS prolonged sleeping time in stressed mice, but had no effect on sleeping time of group-housed mice. Prolonged sleeping time after oral KSS was significantly inhibited (p < 0.05) by bicuculline (3 mg/kg, i.p.), a GABA_A antagonist, but not by flumazenil (3 mg/kg, i.p.), a selective benzodiazepine antagonist. Prolonged sleeping time after KSS inhalation was significantly inhibited (p < 0.05) by flumazenil but not by bicuculline.ConclusionsOur findings suggest that KSS activates GABA_A-benzodiazepine receptor complex and reverses shortened pentobarbital-induced sleep caused by social isolation.     

2-Arachidonoylglycerol modulates human endothelial cell/leukocyte interactions by controlling selectin expression through CB1 and CB2 receptors

Accumulated evidence points to a key role for endocannabinoids in cell migration, and here we sought to characterize the role of these substances in early events that modulate communication between endothelial cells and leukocytes. We found that 2-arachidonoylglycerol (2-AG) was able to initiate and complete the leukocyte adhesion cascade, by modulating the expression of selectins. A short exposure of primary human umbilical vein endothelial cells (HUVECs) to 2-AG was sufficient to prime them towards an activated state: within 1 h of treatment, endothelial cells showed time-dependent plasma membrane expression of P- and E-selectins, which both trigger the initial steps (i.e., capture and rolling) of leukocyte adhesion. The effect of 2-AG was mediated by CB₁ and CB₂ receptors and was long lasting, because endothelial cells incubated with 2-AG for 1 h released the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α) for up to 24 h. Consistently, TNF-α-containing medium was able to promote leukocyte recruitment: human Jurkat T cells grown in conditioned medium derived from 2-AG-treated HUVECs showed enhanced L-selectin and P-selectin glycoprotein ligand-1 (PSGL1) expression, as well as increased efficiency of adhesion and trans-migration. In conclusion, our in vitro data indicate that 2-AG, by acting on endothelial cells, might indirectly promote leukocyte recruitment, thus representing a potential therapeutic target for treatment of diseases where impaired endothelium/leukocyte interactions take place.     

Home environment and cord blood levels of lead,arsenic, and zinc on neurodevelopment of 24 months children living in Chitwan Valley,Nepal

In a birth cohort living in Chitwan Valley, lowland Nepal, we have previously reported inverse associations between in utero levels of lead (Pb), arsenic (As) and neurodevelopment at birth measured by the Brazelton Neonatal Behavioral Assessment Scale, third edition (NBAS III). In the present paper, a follow-up of the same cohort was made on 24-month-old infants regarding the neurodevelopmental effects of these metals, taking the postnatal environment into account. In total, the same100 mother-infant pairs as the previous study, whose Pb, As, and Zn concentrations in cord blood were known, were recruited. Postnatal raising environment was evaluated using the Home Observation for Measurement of Environment (HOME) scale. Neurodevelopment of children at 24 months of age (n = 74) was assessed using the Bayley Scale of Infant Development, Second Edition (BSID II). Multivariable regression adjusting for covariates was performed to determine the associations of in utero levels of toxic and essential elements and the home environment with neurodevelopment scores. Unlike the NBAS III conducted for newborns, none of the BSID II cluster scores in 24-month-old infants were associated with cord blood levels of Pb, As, and Zn. The total HOME score was positively associated with the mental development scale (MDI) score (coefficient = 0.67, at 95% CI = 0.03 to 1.31). In this cohort, a detrimental effect of in utero Pb and As on neurodevelopmental indicators observed at birth disappeared at 24 months, while an association between neurodevelopment and home environment continued.     

Effect of cyanobacterial extracellular products on high-frequency in vitro induction and elongation of Gossypium hirsutum L organs through shoot apex explants

The challenging task of bringing high efficiency transformed plants attracts lot of attention in recent times. In search for this, there have been many attempts made using, different techniques like tissue culture and plant breeding methods. Here we report a suitable alternative facile route, where cyanobacterial extracellular products are utilized as growth regulators and its performance validated on Gossypium hirsutum L. MS medium is tested with cyanobacterial extracellular products of Nostoc ellipsosporum, Dolichospermum flos-aquae and Oscillatoria acuminata .Our best results show that the addition of O. acuminata extracellular product with plant growth hormones gives the excellent induction and elongation in cotton. In addition to this, the multiple shoot was obtained on MS medium fortified with 1.0 mg L^?1 BA with 8% O. acuminata and 1.5 mg L^?1 TDZ with 12% O. acuminata. High frequency of shoot elongation supplemented with MS medium, iP 2.5 mg L^?1 and 16% O. acuminata and root production MS medium fortified with 12% O. acuminata best responsible for regeneration in cotton plants. The rooted plants were hardened and transferred to soil with 90% survival rate.     

A high efficiency gene disruption strategy using a positive–negative split selection marker and electroporation for Fusarium oxysporum

The Fusarium oxysporum species complex consists of fungal pathogens that cause serial vascular wilt disease on more than 100 cultivated species throughout the world. Gene function analysis is rapidly becoming more and more important as the whole-genome sequences of various F. oxysporum strains are being completed. Gene-disruption techniques are a common molecular tool for studying gene function, yet are often a limiting step in gene function identification. In this study we have developed a F. oxysporum high-efficiency gene-disruption strategy based on split-marker homologous recombination cassettes with dual selection and electroporation transformation. The method was efficiently used to delete three RNA-dependent RNA polymerase (RdRP) genes. The gene-disruption cassettes of three genes can be constructed simultaneously within a short time using this technique. The optimal condition for electroporation is 10 μF capacitance, 300 Ω resistance, 4 kV/cm field strength, with 1 μg of DNA (gene-disruption cassettes). Under these optimal conditions, we were able to obtain 95 transformants per μg DNA. And after positive–negative selection, the transformants were efficiently screened by PCR, screening efficiency averaged 85%: 90% (RdRP₁), 85% (RdRP₂) and 77% (RdRP₃). This gene-disruption strategy should pave the way for high throughout genetic analysis in F. oxysporum.