Antisecretory,Gastroprotective, Antioxidant and Anti-Helicobcter Pylori Activity of Zerumbone from Zingiber Zerumbet (L.) Smith

Background

Zingiber zerumbet Smith is a perennial herb, broadly distributed in many tropical areas. In Malaysia, it’s locally known among the Malay people as “lempoyang” and its rhizomes, particularly, is widely used in traditional medicine for the treatment of peptic ulcer disease beyond other gastric disorders.

Aim of the study

The aim of the current study is to evaluate the gastroprotective effect of zerumbone, the main bioactive compound of Zingiber zerumbet rhizome, against ethanol-induced gastric ulcer model in rats.

Materials and Methods

Rats were pre-treated with zerumbone and subsequently exposed to acute gastric ulcer induced by absolute ethanol administration. Following treatment, gastric juice acidity, ulcer index, mucus content, histological analysis (HE and PAS), immunohistochemical localization for HSP-70, prostaglandin E2 synthesis (PGE₂), non-protein sulfhydryl gastric content (NP-SH), reduced glutathione level (GSH), and malondialdehyde level (MDA) were evaluated in ethanol-induced ulcer in vivo. Ferric reducing antioxidant power assay (FRAP) and anti-H. pylori activity were investigated in vitro.

Results

The results showed that the intragastric administration of zerumbone protected the gastric mucosa from the aggressive effect of ethanol-induced gastric ulcer, coincided with reduced submucosal edema and leukocyte infiltration. This observed gastroprotective effect of zerumbone was accompanied with a significant (p <0.05) effect of the compound to restore the lowered NP-SH and GSH levels, and to reduce the elevated MDA level into the gastric homogenate. Moreover, the compound induced HSP-70 up-regulation into the gastric tissue. Furthermore, zerumbone significantly (p <0.05) enhanced mucus production, showed intense PAS stain and maintained PG content near to the normal level. The compound exhibited antisecretory activity and an interesting minimum inhibitory concentration (MIC) against H. pylori strain.

Conclusion

The results of the present study revealed that zerumbone promotes ulcer protection, which might be attributed to the maintenance of mucus integrity, antioxidant activity, and HSP-70 induction. Zerumbone also exhibited antibacterial action against H. pylori.     

A Reevaluation of the Voluntary Medical Male Circumcision Scale-Up Plan in Zimbabwe

Background

The voluntary medical male circumcision (VMMC) program in Zimbabwe aims to circumcise 80% of males aged 13–29 by 2017. We assessed the impact of actual VMMC scale-up to date and evaluated the impact of potential alterations to the program to enhance program efficiency, through prioritization of subpopulations.

Methods and Findings

We implemented a recently developed analytical approach: the age-structured mathematical (ASM) model and accompanying three-level conceptual framework to assess the impact of VMMC as an intervention. By September 2014, 364,185 males were circumcised, an initiative that is estimated to avert 40,301 HIV infections by 2025. Through age-group prioritization, the number of VMMCs needed to avert one infection (effectiveness) ranged between ten (20–24 age-group) and 53 (45–49 age-group). The cost per infection averted ranged between $811 (20–24 age-group) and $5,518 (45–49 age-group). By 2025, the largest reductions in HIV incidence rate (up to 27%) were achieved by prioritizing 10–14, 15–19, or 20–24 year old. The greatest program efficiency was achieved by prioritizing 15–24, 15–29, or 15–34 year old. Prioritizing males 13–29 year old was programmatically efficient, but slightly inferior to the 15–24, 15–29, or 15–34 age groups. Through geographic prioritization, effectiveness varied from 9–12 VMMCs per infection averted across provinces. Through risk-group prioritization, effectiveness ranged from one (highest sexual risk-group) to 60 (lowest sexual risk-group) VMMCs per infection averted.

Conclusion

The current VMMC program plan in Zimbabwe is targeting an efficient and impactful age bracket (13–29 year old), but program efficiency can be improved by prioritizing a subset of males for demand creation and service availability. The greatest program efficiency can be attained by prioritizing young sexually active males and males whose sexual behavior puts them at higher risk for acquiring HIV.     

The Effect of Thermophoresis on Unsteady Oldroyd-B Nanofluid Flow over Stretching Surface

There are currently only a few theoretical studies on convective heat transfer in polymer nanocomposites. In this paper, the unsteady incompressible flow of a polymer nanocomposite represented by an Oldroyd-B nanofluid along a stretching sheet is investigated. Recent studies have assumed that the nanoparticle fraction can be actively controlled on the boundary, similar to the temperature. However, in practice, such control presents significant challenges and in this study the nanoparticle flux at the boundary surface is assumed to be zero. We have used a relatively novel numerical scheme; the spectral relaxation method to solve the momentum, heat and mass transport equations. The accuracy of the solutions has been determined by benchmarking the results against the quasilinearisation method. We have conducted a parametric study to determine the influence of the fluid parameters on the heat and mass transfer coefficients.     

Cyclic AMP signaling contributes to neural plasticity and hyperexcitability in AH sensory neurons following intestinal Trichinella spiralis-induced inflammation

Trichinella spiralis infection causes hyperexcitability in enteric after-hyperpolarising (AH) sensory neurons that is mimicked by neural, immune or inflammatory mediators known to stimulate adenylyl cyclase (AC)/cyclic 3',5'-adenosine monophosphate (cAMP) signaling. The hypothesis was tested that ongoing modulation and sustained amplification in the AC/cAMP/phosphorylated cAMP related element binding protrein (pCREB) signaling pathway contributes to hyperexcitability and neuronal plasticity in gut sensory neurons after nematode infection. Electrophysiological, immunological, molecular biological or immunochemical studies were done in T. spiralis-infected guinea-pigs (8000 larvae or saline) after acute-inflammation (7 days) or 35 days p.i., after intestinal clearance. Acute-inflammation caused AH-cell hyperexcitability and elevated mucosal and neural tissue levels of myeloperoxidase, mast cell tryptase, prostaglandin E2, leukotrine B4, lipid peroxidation, nitric oxide and gelatinase; lower level inflammation persisted 35 days p.i. Acute exposure to blockers of AC, histamine, cyclooxygenase or leukotriene pathways suppressed AH-cell hyperexcitability in a reversible manner. Basal cAMP responses or those evoked by forskolin (FSK), Ro-20-1724, histamine or substance P in isolated myenteric ganglia were augmented after T. spiralis infection; up-regulation also occurred in AC expression and AC-immunoreactivity in calbindin (AH) neurons. The cAMP-dependent slow excitatory synaptic transmission-like responses to histamine (mast cell mediator) or substance P (neurotransmitter) acting via G-protein coupled receptors (GPCR) in AH neurons were augmented by up to 2.5-fold after T. spiralis infection. FSK, histamine, substance P or T. spiralis acute infection caused a 5- to 30-fold increase in cAMP-dependent nuclear CREB phosphorylation in isolated ganglia or calbindin (AH) neurons. AC and CREB phosphorylation remained elevated 35 days p.i.. Ongoing immune activation, AC up-regulation, enhanced phosphodiesterase IV activity and facilitation of the GPCR-AC/cAMP/pCREB signaling pathway contributes to T. spiralis-induced neuronal plasticity and AH-cell hyperexcitability. This may be relevant in gut nematode infections and inflammatory bowel diseases, and is a potential therapeutic target.     

Synthesis, liposomal preparation, and in vitro toxicity of two novel dodecaborate cluster lipids for boron neutron capture therapy

A new class of lipids, containing the closo-dodecaborate cluster, has been synthesized. Two lipids, S-(N, N-(2-dimyristoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-14) and S-(N, N-(2-dipalmitoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-16) are described. Both of them have a double-tailed lipophilic part and a headgroup carrying two negative charges. Differential scanning calorimetry shows that B-6-14 and B-6-16 bilayers have main phase transition temperatures of 18.8 and 37.9 degrees C, respectively. Above the transition temperature of 18.8 degrees C, B-6-14 can form liposomal vesicles, representing the first boron-containing lipid with this capability. Upon cooling below the transition temperature, stiff bilayers are formed. When incorporated into liposomal formulations with equimolar amounts of distearoyl phosphatidylcholine (DSPC) and cholesterol, stable liposomes are obtained. The zeta-potential measurements indicate that both B-6-14- and B-6-16-containing vesicles are negatively charged, with the most negative potential described of any liposome so far. The liposomes are of high potential value as transporters of boron to tumor cells in treatments based on boron neutron capture therapy (BNCT). Liposomes prepared from B-6-14 were slightly less toxic in V79 Chinese hamster cells (IC50 5.6 mM) than unformulated Na2B12H11SH (IC50 3.9 mM), while liposomes prepared from B-6-16 were not toxic even at 30 mM.     

2-Cysteine Peroxiredoxins and Thylakoid Ascorbate Peroxidase Create a Water-Water Cycle That Is Essential to Protect the Photosynthetic Apparatus under High Light Stress Conditions

Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H₂O₂)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H₂O₂, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H₂O₂- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX.Plants are frequently exposed to different abiotic stresses, including high light (HL), UV irradiation, heat, cold, and drought. A component common to these stresses is the rapid formation of reactive oxygen species (ROS) as the result of metabolic dysbalances. A major ROS produced under moderate light (ML) and, in particular, HL photooxidative stress conditions was shown to be singlet oxygen, ¹O₂, that is produced in illuminated chloroplasts predominantly at the PSII (Triantaphylidès et al., 2008). Most of the singlet oxygen is quenched by carotenoids and tocopherols or reacts with galactolipids in thylakoid membranes, yielding galactolipid hydroperoxides (Zoeller et al., 2012; Farmer and Mueller, 2013). In addition, superoxide radicals, O₂·⁻, are produced predominantly at the PSI and rapidly dismutate to hydrogen peroxide (H₂O₂) either spontaneously or because of being catalyzed by superoxide dismutase. Hence, lipid peroxides and H₂O₂ are produced close to the photosystems and may damage thylakoid proteins. In this context, 2-Cys peroxiredoxin (PRX) enzymes have been implicated in the reductive detoxification of lipid peroxides and H₂O₂ (König et al., 2002).During photosynthesis, light energy absorbed by PSII is used to split water molecules, and the electrons are channeled from PSII through PSI to ferredoxin (Fd). As a result, electrons flow from water to Fd. The main electron sink reaction is the Fd NADP oxidoreductase-catalyzed production of NADPH that functions as an electron donor to reduce carbon dioxide to sugars. Under HL conditions, excessive excitation energy is dissipated into heat, which was indicated by nonphotochemical quenching of chlorophyll fluorescence. In addition, excessive photosynthetic electrons can be donated from PSI to O₂, yielding O₂·⁻ (Miyake, 2010). This process, the Mehler reaction, creates an alternative electron sink and electron flow. Superoxide anion radicals, O₂·⁻, can be dismutated to O₂ and H₂O₂ by a thylakoid-attached copper/zinc superoxide dismutase (Cu/ZnSOD; Rizhsky et al., 2003). H₂O₂ can then be reduced to water by peroxidases. As a result, O₂ molecules originating from the water-splitting process at PSII are reduced to water by electrons originating from PSI. This process is termed the water-water cycle (WWC) that is thought to protect the photosynthetic apparatus from excessive light and alleviate photoinhibition.In the classical WWC, the Mehler-ascorbate peroxidase (MAP) pathway, ascorbate peroxidases (APXs) have been considered as key enzymes in the reductive detoxification of H₂O₂ in chloroplasts (Kangasjärvi et al., 2008). APXs reduce H₂O₂ to water and oxidize ascorbate to monodehydroascorbate radicals. NADPH functions as an electron donor to regenerate ascorbate by monodehydroascorbate radical reductase. There are two functional APX homologs in plastids: a 33-kD stromal ascorbate peroxidase (sAPX) and a 38-kD thylakoid ascorbate peroxidase (tAPX). The latter tAPX is thought to reside close to the site of H₂O₂ generation at PSI. Surprisingly, knockout-tAPX mutants as well as double mutants lacking both the tAPX and the sAPX exhibited no visible symptoms of stress after long-term (1–14 d) HL (1.000 µmol photons m⁻² s⁻¹) exposure (Giacomelli et al., 2007; Kangasjärvi et al., 2008; Maruta et al., 2010). Moreover, the photosynthetic efficiency of PSII (as judged by the maximum photochemical efficiency of PSII in the dark-adapted state [F_v/F_m]), H₂O₂ production, antioxidant levels (ascorbate, glutathione, and tocopherols), protein oxidation, and anthocyanin accumulation were similar between light-stressed mutant and wild-type plants. Hence, other H₂O₂ detoxification mechanisms can efficiently compensate for the lack of the sAPX and tAPX detoxification system.In addition to APX, glutathione peroxidases and PRXs may reduce H₂O₂ to water. It has been postulated that, in the chloroplast, two highly homologous thylakoid-associated 2-Cys peroxiredoxins (2CPs), 2CPA and 2CPB, can create an alternative ascorbate-independent WWC (Dietz et al., 2006). In support of this concept, HL stress-acclimated tapx sapx double-mutant plants showed increased levels of 2-Cys PRX compared with wild-type plants (Kangasjärvi et al., 2008). Because the two plastidial 2CPA and 2CPB dynamically interact with the stromal side of thylakoid membranes and are capable of reducing peroxides, 2-Cys PRX enzymes may be involved in both H₂O₂ detoxification and reduction of lipid peroxides in thylakoids (König et al., 2002).The reaction mechanism of 2-Cys PRX is highly conserved and involves a Cys residue, which becomes transiently oxidized to sulphenic acid (termed the peroxidatic Cys residue), thereby reducing H₂O₂ to water. The sulphenic acid is subsequently attacked by a second Cys residue, termed resolving Cys residue, yielding an intermolecular disulfide bridge and water (Dietz, 2011).At high peroxide concentrations, the peroxidase function of 2-Cys PRX becomes inactivated through overoxidation, and excess H₂O₂ may function as a redox signal (Puerto-Galán et al., 2013). It has been postulated that 2-Cys PRXs function as a floodgate that allows H₂O₂ signaling only under oxidative stress conditions (Wood et al., 2003; Dietz, 2011; Puerto-Galán et al., 2013). In addition to its function as peroxidase, 2-Cys PRX may also serve as proximity-based thiol oxidases and chaperones (König et al., 2013).The genome of Arabidopsis (Arabidopsis thaliana) contains two 2CP genes. To study 2-Cys PRX function, transgenic plants with reduced 2-Cys PRX levels were generated by antisense suppression (Baier et al., 2000) as well as crossing of transfer DNA (T-DNA) insertion mutants (Pulido et al., 2010). The T-DNA insertion double mutant was shown to contain less than 5% of the wild-type content of 2CPA and no 2CPB. Hence, full knockout lines lacking both 2-Cys PRXs have not yet been established. Under standard growth conditions, 2-Cys PRX double mutants (similar to plastid APX-deficient plants) also did not show a photooxidative stress phenotype that might be because of compensation by alternative H₂O₂ reduction systems (Pulido et al., 2010). Because of the lack of a clear phenotype of the 2-Cys PRX double-knockdown mutant under ML conditions, the physiological functions of 2CPA and 2CPB remain to be elucidated.The main aim of this study was to identify the physiological function of 2CPA and 2CPB under HL stress conditions, when the WWC is of particular importance in protecting the photosynthetic apparatus from photooxidative damage. We investigated mutants completely deficient in 2-Cys PRX (2cpa 2cpb) or tAPX (tapx) and in addition, 2cpa 2cpb tapx triple knockout plants to study the extent of the functional overlap between these enzymes. Results suggest that 2-Cys PRXs are involved in a 2-Cys PRX-dependent WWC that seems to be more important in protecting the photosynthetic apparatus than the tAPX-dependent WWC, the MAP cycle.     

Broad-scale latitudinal patterns of genetic diversity among native European and introduced house sparrow (Passer domesticus) populations

Introduced species offer unique opportunities to study evolution in new environments, and some provide opportunities for understanding the mechanisms underlying macroecological patterns. We sought to determine how introduction history impacted genetic diversity and differentiation of the house sparrow (Passer domesticus), one of the most broadly distributed bird species. We screened eight microsatellite loci in 316 individuals from 16 locations in the native and introduced ranges. Significant population structure occurred between native than introduced house sparrows. Introduced house sparrows were distinguished into one North American group and a highly differentiated Kenyan group. Genetic differentiation estimates identified a high magnitude of differentiation between Kenya and all other populations, but demonstrated that European and North American samples were differentiated too. Our results support previous claims that introduced North American populations likely had few source populations, and indicate house sparrows established populations after introduction. Genetic diversity also differed among native, introduced North American, and Kenyan populations with Kenyan birds being least diverse. In some cases, house sparrow populations appeared to maintain or recover genetic diversity relatively rapidly after range expansion (<50 years; Mexico and Panama), but in others (Kenya) the effect of introduction persisted over the same period. In both native and introduced populations, genetic diversity exhibited large-scale geographic patterns, increasing towards the equator. Such patterns of genetic diversity are concordant with two previously described models of genetic diversity, the latitudinal model and the species diversity model.     

The cysteine protease α-clostripain is not essential for the pathogenesis of Clostridium perfringens-mediated myonecrosis

Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process.     

Effect of resveratrol and beta-sitosterol in combination on reactive oxygen species and prostaglandin release by PC-3 cells

The objective of this project was to identify some possible mechanisms by which two common phytochemicals, resveratrol and beta-sitosterol, inhibit the growth of human prostate cancer PC-3 cells. These mechanisms include the effect of the phytochemicals on apoptosis, cell cycle progression, prostaglandin synthesis and the production of reactive oxygen species (ROS). Prostaglandins have been known to play a role in regulating cell growth and apoptosis. PC-3 cells were supplemented with 50 microM resveratrol or 16 microM beta-sitosterol alone or in combination for up to 5 days. Phytochemical supplementation resulted in inhibition in cell growth. beta-Sitosterol was more potent than resveratrol and the combination of the two resulted in greater inhibition than supplementation with either alone. Long-term supplementation with resveratrol or beta-sitosterol elevated basal prostaglandin release but beta-sitosterol was much more potent than resveratrol in this regard. beta-Sitosterol was more effective than resveratrol in inducing apoptosis and the combination had an intermediate effect after 1 day of supplementation. Cells supplemented with resveratrol were arrested at the G1 phase and at the G2/M phase in the case of beta-sitosterol while the combination resulted in cell arrest at the two phases of the cell cycle. beta-Sitosterol increased ROS production while resveratrol decreased ROS production. The combination of the two phytochemicals resulted in an intermediate level of ROS. The observed changes in prostaglandin levels and ROS production by these two phytochemicals may suggest their mediation in the growth inhibition. The reduction in ROS level and increase by resveratrol supplementation in PC-3 cells reflects the antioxidant properties of resveratrol. It was concluded that these phytochemicals may induce the inhibition of tumor growth by stimulating apoptosis and arresting cells at different locations in the cell cycle and the mechanism may involve alterations in ROS and prostaglandin production.