The bactericidal effects of Lactobacillus acidophilus,garcinol and Protykin® compared to clarithromycin,on Helicobacter pylori

Chronic infection with Helicobacter pylori causes peptic ulcers, gastric cancer and lymphoma. We evaluated the inhibitory effects of the probiotic Lactobacillus acidophilus DDS-1J, the antibiotic clarithromycin and the natural antioxidants garcinol and Protykin^® (containing 50% trans-resveratrol) on Helicobacter pylori strain ATCC 49503. The findings of this study indicate that Lactobacillus acidophilus DDS-1J exerts a growth inhibitory effect on H. pylori at a ratio of 1:1 or higher in vitro. In the case of clarithromycin, garcinol and resveratrol, the bactericidal effect is time and concentration dependent. Clarithromycin completely inhibited growth at 62.5 g/ml at 6 h and at 31.5 g/ml at 12 h. For garcinol the highest concentration needed for complete inhibition was 31.5 g/ml at 6 h and 3.9 g/ml after 12 h incubation. For resveratrol, significant inhibition was noted at 1000 g/ml at 12 h only. The bactericidal effect of garcinol was reduced by the addition of resveratrol at all concentrations 125 g/ml at 6 and 12 h. We conclude from this study that Lactobacillus acidophilus DDS-1J inhibits H. pylori at 1:1 and higher ratios. Also, between the two antioxidants, garcinol is much more potent than resveratrol as a bactericidal agent against H. pylori, and that resveratrol may antagonize this effect. Finally, our study showed equivalent or better bactericidal activity of garcinol compared to clarithromycin against H. pylori at 6 and 12 h incubation, indicating a potential role for this antioxidant in treatment for H. pylori infection.     

Dose- and time-dependent effects of a novel (−)-hydroxycitric acid extract on body weight,hepatic and testicular lipid peroxidation,DNA fragmentation and histopathological data over a period of 90 days

(–)-Hydroxycitric acid (HCA), a natural extract from the dried fruit rind of Garcinia cambogia (family Guttiferae), is a popular supplement for weight management. The dried fruit rind has been used for centuries as a condiment in Southeastern Asia to make food more filling and satisfying. A significant number of studies highlight the efficacy of Super CitriMax (HCA-SX, a novel 60% calcium-potassium salt of HCA derived from Garcinia cambogia) in weight management. These studies also demonstrate that HCA-SX promotes fat oxidation, inhibits ATP-citrate lyase (a building block for fat synthesis), and lowers the level of leptin in obese subjects. Acute oral, acute dermal, primary dermal irritation and primary eye irritation toxicity studies have demonstrated the safety of HCA-SX. However, no long-term safety of HCA-SX or any other (–)-hydroxycitric acid extract has been previously assessed. In this study, we have evaluated the dose- and time-dependent effects of HCA-SX in Sprague-Dawley rats on body weight, hepatic and testicular lipid peroxidation, DNA fragmentation, liver and testis weight, expressed as such and as a % of body weight and brain weight, and histopathological changes over a period of 90 days. The animals were treated with 0, 0.2, 2.0 and 5.0% HCA-SX as feed intake and the animals were sacrificed on 30, 60 or 90 days of treatment. The feed and water intake were assessed and correlated with the reduction in body weight. HCA-SX supplementation demonstrated a reduction in body weight in both male and female rats over a period of 90 days as compared to the corresponding control animals. An advancing age-induced marginal increase in hepatic lipid peroxidation was observed in both male and female rats as compared to the corresponding control animals. However, no such difference in hepatic DNA fragmentation and testicular lipid peroxidation and DNA fragmentation was observed. Furthermore, liver and testis weight, expressed as such and as a percentage of body weight and brain weight, at 30, 60 and 90 days of treatment, exhibited no significant difference between the four groups. Taken together, these results indicate that treatment of HCA-SX over a period of 90 days results in a reduction in body weight, but did not cause any changes in hepatic and testicular lipid peroxidation, DNA fragmentation, or histopathological changes.     

Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS^-/-) are protected against ALI. In both wild-type and eNOS^-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS^-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS^-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS^-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS^-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr³⁴. Finally, we found that the reduction in NOS uncoupling in eNOS^-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.     

Amyloid Properties of the Mouse Egg Zona Pellucida

The zona pellucida (ZP) surrounding the oocyte is an extracellular fibrillar matrix that plays critical roles during fertilization including species-specific gamete recognition and protection from polyspermy. The mouse ZP is composed of three proteins, ZP1, ZP2, and ZP3, all of which have a ZP polymerization domain that directs protein fibril formation and assembly into the three-dimensional ZP matrix. Egg coats surrounding oocytes in nonmammalian vertebrates and in invertebrates are also fibrillar matrices and are composed of ZP domain-containing proteins suggesting the basic structure and function of the ZP/egg coat is highly conserved. However, sequence similarity between ZP domains is low across species and thus the mechanism for the conservation of ZP/egg coat structure and its function is not known. Using approaches classically used to identify amyloid including conformation-dependent antibodies and dyes, X-ray diffraction, and negative stain electron microscopy, our studies suggest the mouse ZP is a functional amyloid. Amyloids are cross-β sheet fibrillar structures that, while typically associated with neurodegenerative and prion diseases in mammals, can also carry out functional roles in normal cells without resulting pathology. An analysis of the ZP domain from mouse ZP3 and ZP3 homologs from five additional taxa using the algorithm AmylPred 2 to identify amyloidogenic sites, revealed in all taxa a remarkable conservation of regions that were predicted to form amyloid. This included a conserved amyloidogenic region that localized to a stretch of hydrophobic amino acids previously shown in mouse ZP3 to be essential for fibril assembly. Similarly, a domain in the yeast protein α-agglutinin/Sag 1p, that possesses ZP domain-like features and which is essential for mating, also had sites that were predicted to be amyloidogenic including a hydrophobic stretch that appeared analogous to the critical site in mouse ZP3. Together, these studies suggest that amyloidogenesis may be a conserved mechanism for ZP structure and function across billions of years of evolution.     

Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair

Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.     

Expression,purification and substrate specificities of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2

3-Nitotoluene dioxygenase (3-NTDO) is the first enzyme in the degradation pathway of 3-nitrotoluene (3-NT) by Diaphorobacter sp. strain DS2. The complete gene sequences of 3-NTDO were PCR amplified from genomic DNA of Diaphorobacter sp., cloned, sequenced and expressed. The 3-NTDO gene revealed a multi component structure having a reductase, a ferredoxin and two oxygenase subunits. Clones expressing the different subunits were constructed in pET21a expression vector system and overexpressed in E. coli BL21(DE3) host. Each subunit was individually purified separately to homogeneity. The active recombinant enzyme was reconstituted in vitro by mixing all three purified subunits. The reconstituted recombinant enzyme could catalyse biotransformations on a variety of organic aromatics.     

Broad-host-range plasmid-mediated metabolic perturbations in Pseudomonas fluorescens 13525

Genetic engineering of fluorescent pseudomonads for various industrially, agriculturally and environmentally important bioprocesses often involves the use of suitable plasmids. Plasmid-mediated alterations in host physiology and metabolism are poorly understood for this group of organisms. Thus, we investigated the metabolic perturbations in Pseudomonas fluorescens 13525 due to the independent and combined presence of broad-host-range plasmids, pBBR1MCS-2 (copy number 30) and pUCPM18 derived pAB4 and pAB8 (copy number 14-16). Presence of pAB4 and pAB8 not only significantly increased the growth rate and glucose utilization of P. fluorescens 13525, but also increased glucose dehydrogenase activity and gluconic acid production indicating enhanced direct oxidative pathway for glucose catabolism. Additionally, increased secretion of pyruvic, acetic, and citric acids caused faster media acidification in presence of pAB4 and pAB8. Simultaneous presence of pAB4/pAB8 in Pf (pAB48) and pAB4/pBBR1MCS-2 in Pf (pAB4BBR1MCS-2) reduced their respective copy numbers to nearly half. Pf (pAB48) demonstrated further increase in direct oxidation pathway without altering growth and glucose depletion rates, as compared with single transformants. Conversely, pBBR1MCS-2 plasmid did not greatly alter P. fluorescens 13525 metabolism when present independently but masked the effects imposed by pAB4 when present in its combination. In conclusion, P. fluorescens 13525 redesigns its metabolism in response to the presence of plasmids irrespective of their nature, by enhancing anaplerosis with a simultaneous reduction in catabolism as indicated by increased pyruvate carboxylase and decreased citrate synthase activities, respectively. Such information will be helpful for vector designing during genetic engineering of fluorescent pseudomonads.