Chemical analysis and antioxidant activity in vitro of a β-D-glucan isolated from Dictyophora indusiata

In this study, a water-soluble polysaccharide (PPS) from Dictyophora indusiata was purified and investigated through a combination of gel chromatography (Sephadex G-200), infrared (IR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS). The results indicated that PPS has a backbone of β-conformation, mainly consist of glucose (98.58%). And the antioxidant activities of PPS were investigated in vitro including reducing power, hydroxyl assay, superoxide radical assay and DPPH scavenge activity. The results showed that PPS has antioxidant activities in all these assay systems, suggesting it may be explored as a novel natural antioxidant with potential therapeutic properties in mammalian systems.     

Cholinesterase inhibitory activity of tinosporide and 8-hydroxytinosporide isolated from Tinospora cordifolia: In vitro and in silico studies targeting management of Alzheimer’s disease

Tinosporide and 8-hydroxytinosporide isolated from Tinospora cordifolia were evaluated for acetylcholinesterase (AChE) and butylcholinesterase (BuChE) inhibitory activities. The structure of the compound was confirmed by spectroscopic analysis, whereas cholinesterase inhibition was investigated by Ellman method using donepezil as standard drug and the data were presented as IC₅₀ (μg/ml ± SEM). Furthermore, donepezil, tinosporide and 8-hydroxytinosporide were executed for docking analysis. The results from the isolated compounds TC-16R confirmed as tinosporide promisingly inhibited AChE with IC₅₀ value of 13.45 ± 0.144, whereas TC-19R confirmed as 8-hydroxytinosporide moderately inhibited AChE with IC₅₀ value of 46.71 ± 0.511. In case of BuChE inhibition, the IC₅₀ values were found to be 408.50 ± 17.197 and 317.26 ± 6.918 for tinosporide and 8-hydroxytinosporide, respectively. The in silico studies revealed that the ligand tinosporide fit with the binding sites and inhibited AChE. Overall, the study findings suggested that tinosporide would be a complementary noble molecule of donepezil which is correlated with its pharmacological activity through in vitro studies, while 8-hydroxytinosporide modestly inhibited BuChE and the results are very close to the standard donepezil.     

Influence of manufacturing processes on in vitro properties of the probiotic strain Lactobacillus rhamnosus Lcr35®

Probiotics are administered as complex manufactured products and yet most studies on probiotic bacterial strains have been performed with native culture strains. Little is known about the influence of industrial processes on the properties of the microorganisms. In this study, we comparatively assessed the characteristics of the probiotic bacterial strain Lactobacillus rhamnosus (Lcr35(?)) together with four of its commercial formulations, including three intestinal formulas (BACILOR with Lcr Restituo(?) packet and capsule and FLOREA Lcr Lenio(?)) and one vaginal formula (GYNOPHILUS Lcr Regenerans(?)). Lcr35(?) grown from the intestinal formulas displayed increased resistance to acidic pH and bile stress, especially FLOREA (Lcr Lenio(?)), which showed a 4.5log higher number of viable bacteria compared to the results obtained with the control native Lcr35(?) strain. Adhesion to intestinal cells was significantly higher with Lcr Restituo(?) packet and Lcr Restituo(?) capsule vs Lcr35(?). Bacteria from the vaginal formulation GYNOPHILUS had increased ability to metabolize glycogen thereby increasing lactic acid production. In vitro growth inhibition of the pathogen Candida albicans was significantly higher with bacteria from the vaginal formulation (4.5 log difference) and in the presence of vaginal epithelial cells than with the native strain. Our results show that the manufacturing process influences strain properties and should therefore be adapted according to the strain and the therapeutic indication.     

Prevalence of antimicrobial resistance among bacterial pathogens isolated from cattle in different European countries: 2002–2004

Background

Acute phase proteins haptoglobin (Hp), serum amyloid A (SAA) and lipopolysaccharide binding protein (LBP) have suggested to be suitable inflammatory markers for bovine mastitis. The aim of the study was to investigate acute phase markers along with clinical parameters in two consecutive intramammary challenges with Escherichia coli and to evaluate the possible carry-over effect when same animals are used in an experimental model.

Methods

Mastitis was induced with a dose of 1500 cfu of E. coli in one quarter of six cows and inoculation repeated in another quarter after an interval of 14 days. Concentrations of acute phase proteins haptoglobin (Hp), serum amyloid A (SAA) and lipopolysaccharide binding protein (LBP) were determined in serum and milk.

Results

In both challenges all cows became infected and developed clinical mastitis within 12 hours of inoculation. Clinical disease and acute phase response was generally milder in the second challenge. Concentrations of SAA in milk started to increase 12 hours after inoculation and peaked at 60 hours after the first challenge and at 44 hours after the second challenge. Concentrations of SAA in serum increased more slowly and peaked at the same times as in milk; concentrations in serum were about one third of those in milk. Hp started to increase in milk similarly and peaked at 36–44 hours. In serum, the concentration of Hp peaked at 60–68 hours and was twice as high as in milk. LBP concentrations in milk and serum started to increase after 12 hours and peaked at 36 hours, being higher in milk. The concentrations of acute phase proteins in serum and milk in the E. coli infection model were much higher than those recorded in experiments using Gram-positive pathogens, indicating the severe inflammation induced by E. coli.

Conclusion

Acute phase proteins would be useful parameters as mastitis indicators and to assess the severity of mastitis. If repeated experimental intramammary induction of the same animals with E. coli is used in cross-over studies, the interval between challenges should be longer than 2 weeks, due to the carry-over effect from the first infection.     

In vitro evaluation of single- and multi-strain probiotics: Inter-species inhibition between probiotic strains, and inhibition of pathogens

Many studies comparing the effects of single- and multi-strain probiotics on pathogen inhibition compare treatments with different concentrations. They also do not examine the possibility of inhibition between probiotic strains with a mixture. We tested the ability of 14 single-species probiotics to inhibit each other using a cross-streak assay, and agar spot test. We then tested the ability of 15 single-species probiotics and 5 probiotic mixtures to inhibit Clostridium difficile, Escherichia coli and S. typhimurium, using the agar spot test. Testing was done with mixtures created in two ways: one group contained component species incubated together, the other group of mixtures was made using component species which had been incubated separately, equalised to equal optical density, and then mixed in equal volumes. Inhibition was observed for all combinations of probiotics, suggesting that when used as such there may be inhibition between probiotics, potentially reducing efficacy of the mixture. Significant inter-species variation was seen against each pathogen. When single species were tested against mixtures, the multi-species preparations displayed significantly (p < 0.05 or less) greater inhibition of pathogens in 12 out of 24 cases. Despite evidence that probiotic species will inhibit each other when incubated together in vitro, in many cases a probiotic mixture was more effective at inhibiting pathogens than its component species when tested at approximately equal concentrations of biomass. This suggests that using a probiotic mixture might be more effective at reducing gastrointestinal infections, and that creating a mixture using species with different effects against different pathogens may have a broader spectrum of action that a single provided by a single strain.     

Antimicrobial activity of enterocin EJ97 against 'Bacillus macroides/Bacillus maroccanus' isolated from zucchini purée

AIMS: Activity of the bacteriocin EJ97 produced by Enterococcus faecalis EJ97 against strains of 'Bacillus macroides/B. maroccanus' isolated from spoiled zucchini purée was investigated. METHODS AND RESULTS: The influence of several factors like bacteriocin concentration, incubation temperature, pH, growth medium and chemical perservatives on bacteriocin activity was investigated. Enterocin EJ97 [2 arbitrary units (AU) per millilitre] had a marked bactericidal effect on strain INRA P53-2 after 4 h of incubation at 37 degrees C, 24 h at 15 degrees C or 48 h at 4 degrees C. Activity was markedly reduced at pH values of 5.0 and 9.0, but was potentiated by sodium nitrite, sodium benzoate, sodium lactate and sodium tripolyphosphate. Inhibition of strain INRA P53-2 in a commercial vegetable purée required a 10-fold higher bacteriocin concentration. Strain EJ97 was able to grow and produce bacteriocin on vegetable purée, but no inhibition of strain INRA P53-2 was detected. CONCLUSIONS: The concentration-dependent bactericidal activity of enterocin EJ97 against strain INRA P53-2 was higher at 37 degrees C and neutral pH, and was potentiated by chemical preservatives. Although enterocin EJ97 was less active in vegetable purée, the concentrations providing bactericidal activity in this food matrix are practical for commercial use. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterocin EJ97 may have a potential for use in the prevention of food spoilage caused by 'B. macroides/B. maroccanus'.     

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood γδT cells isolated from glioblastoma patients

γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies. Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of γδT cells in cancer patients. Received: 23 January 1998 / Accepted: 20 May 1998     

In vitro replication studies of carboxymethylated DNA lesions with Saccharomyces cerevisiae polymerase η

Humans are exposed to N-nitroso compounds (NOCs) both endogenously and exogenously from a number of environmental sources, and NOCs are both mutagenic and carcinogenic. After metabolic activation, some NOCs can induce carboxymethylation of nucleobases through a diazoacetate intermediate, which could give rise to p53 mutations similar to those seen in human gastrointestinal cancers. It was previously found that the growth of polymerase η-deficient human cells was inhibited by treatment with azaserine, a DNA carboxymethylation agent, suggesting the importance of this polymerase in bypassing the azaserine-induced carboxymethylated DNA lesions. In this study, we examined how carboxymethylated DNA lesions, which included N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA), N(4)-carboxymethyl-2'-deoxycytidine (N(4)-CMdC), N3-carboxymethylthymidine (N3-CMdT), and O(4)-carboxymethylthymidine (O(4)-CMdT), perturbed the efficiency and fidelity of DNA replication mediated by Saccharomyces cerevisiae polymerase η (pol η). Our results from steady-state kinetic assay showed that pol η could readily bypass and extend past N(6)-CMdA and incorporated the correct nucleotides opposite the lesion and its neighboring 5'-nucleoside with high efficiency. By contrast, the polymerase could bypass N(4)-CMdC inefficiently, with substantial misincorporation of dCMP followed by dAMP, though pol η could extend past the lesion with high fidelity and efficiency when dGMP was incorporated opposite the lesion. On the other hand, yeast pol η experienced great difficulty in bypassing O(4)-CMdT and N3-CMdT, and the polymerase inserted preferentially the incorrect dGMP opposite these two DNA lesions; the extension step, nevertheless, occurred with high fidelity and efficiency when the correct dAMP was opposite the lesion, as opposed to the preferentially incorporated incorrect dGMP. These results suggest that these lesions may contribute significantly to diazoacetate-induced mutations and those in the p53 gene observed in human gastrointestinal tumors.     

Antimicrobial activity of human β-defensin 4 analogs: Insights into the role of disulfide linkages in modulating activity

Human β-defensins (HBDs) are cationic antimicrobial peptides that are components of the innate immune system. They are characterized by three disulfide bridges. However, the number of cationic residues as well as the presence of lysine and arginine residues vary. In HBD4, the cationic residues occur predominantly in the N-terminal segment, unlike in HBD1–3. We have examined the antimicrobial activity of peptides spanning the N- and C-terminal segments of HBD4. We have introduced one, two and three disulfide bridges in the peptides corresponding to the N-terminal segments. Peptides corresponding to the N-terminal segment had identical sequences and variation was only in the number and spacing of cysteines and disulfide bridges. Antimicrobial activity to varying extents was observed for all the peptides. When two disulfide bridges were present, decrease in antimicrobial potency as well as sensitivity of activity to salt was observed. Enhanced antimicrobial activity was observed when three disulfide bridges were present. The antimicrobial potency was similar to HBD4 except against Escherichia coli and was attenuated in the presence of salt. While the presence of three disulfide bridges did not constrain the peptide to a rigid β-sheet, the activity was considerably more as compared to the peptides with one or two disulfide bridges. The peptides enter bacterial and fungal cells rapidly without membrane permeabilization and appear to exert their activity inside the cells rather than at the membrane.     

Kinetic studies on the stimulation of Na+−H+ exchange activity in renal brush border membranes isolated from thyroid hormone-treated rats

Summary Na⁺–H⁺ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na⁺], treatment altered the initial rate of Na⁺ uptake by increasingV _m (hyperthyroid, 18.9±1.1 nmol Na⁺ · mg^–1 · 2 sec^–1; normal, 8.9±0.3 nmol Na⁺ · mg^–1 · 2 sec^–1), and not the apparent affinityK _Na ⁺ (hyperthyroid, 7.3±1.7mm; normal, 6.5±0.9mm). When examined as a function of [H⁺] and at a subsaturating [Na⁺] (1mm), hyperthyroidism resulted in the proportional increase in Na⁺ uptake at every intravesicular pH measured. A positive cooperative effect on Na⁺ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change then (hyperthyroid, 1.2±0.06; normal, 1.2±0.07) or the [H⁺]_0.5 (hyperthyroid, 0.39±0.08 m; normal, 0.44±0.07 m) but increased the apparentV _m (hyperthyroid, 1.68±0.14 nmol Na⁺ · mg^–1 · 2 sec^–1; normal 0.96±0.10 nmol Na⁺ · mg^–1 · 2 sec^–1). The uptake of Na⁺ in exchange for H⁺ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H⁺ translocation or debinding was rate limiting for Na⁺–H⁺ exchange since Na⁺–Na⁺ exchange activity was greater than Na⁺–H⁺ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na⁺–Na⁺ and Na⁺–H⁺ exchange. We conclude that hyperthyroidism leads to either an increase in the number of functional exchangers in the membrane or exactly proportional increases in the rate-limiting steps for Na⁺–Na⁺ and Na⁺–H⁺ exchange activity.     

Synthesis,in vitro α-glucosidase inhibitory activity and molecular docking studies of new thiazole derivatives

Current study based on the synthesis of new thiazole derivatives via “one pot” multicomponent reaction, evaluation of their in vitro α-glucosidase inhibitory activities, and in silico studies. All synthetic compounds were fully characterized by ¹H NMR, ¹³C NMR and EIMS. CHN analysis was also performed. These newly synthesized compounds showed activities in the range of IC₅₀ = 9.06 ± 0.10–82.50 ± 1.70 μM as compared to standard acarbose (IC₅₀ = 38.25 ± 0.12 μM). It is worth mentioning that most of the compounds such as 1 (IC₅₀ = 23.60 ± 0.39 μM), 2 (IC₅₀ = 22.70 ± 0.60 μM), 3 (IC₅₀ = 22.40 ± 0.32 μM), 4 (IC₅₀ = 26.5 ± 0.40 μM), 6 (IC₅₀ = 34.60 ± 0.60 μM), 7 (IC₅₀ = 26.20 ± 0.43 μM), 8 (IC₅₀ = 14.06 ± 0.18 μM), 9 (IC₅₀ = 17.60 ± 0.28 μM), 10 (IC₅₀ = 27.16 ± 0.41 μM), 11 (IC₅₀ = 19.16 ± 0.19 μM), 12 (IC₅₀ = 9.06 ± 0.10 μM), 13 (IC₅₀ = 12.80 ± 0.21 μM), 14 (IC₅₀ = 11.94 ± 0.18 μM), 15 (IC₅₀ = 16.90 ± 0.20 μM), 16 (IC₅₀ = 12.60 ± 0.14 μM), 17 (IC₅₀ = 16.30 ± 0.29 μM), and 18 (IC₅₀ = 32.60 ± 0.61 μM) exhibited potent inhibitory potential. Molecular docking study was performed in order to understand the molecular interactions between the molecule and enzyme. Newly identified α-glucosidase inhibitors except few were found to be completely non-toxic.     

N⁶-Alkyladenosines: Synthesis and evaluation of in vitro anticancer activity

A series of adenosine analogues differently substituted in N⁶-position were synthesized to continue our studies on the relationships between structure and biological activity of iPA. The structures of the compounds were confirmed by standard studies of ¹H NMR, MS and elemental analysis. These molecules were then evaluated for their anti-proliferative activity on bladder cancer cells. We found that some of these compounds possess anti-proliferative activity but have no effect on cell invasion and metalloprotease activity.     

Small leucine-rich proteoglycans in atherosclerotic lesions: novel targets of chronic statin treatment?

Small leucine‐rich proteoglycans (SLRPs), such as decorin and biglycan, regulate the assembly and turnover of collagenous matrix. The aim of the study was to analyse the effect of chronic rosuvastatin treatment on decorin, biglycan and the collagen matrix in ApoE‐deficient mice. Twenty‐week‐old male ApoE‐deficient mice received normal chow or 20 mg rosuvastatin/kg × day for 32 weeks. Subsequently, matrix composition was analysed by histochemistry and immunostaining at the aortic root and in innominate arteries of ApoE deficient mice as well as in human carotid endarterectomy specimens. Immunoblotting of proteoglycans was performed from aortic extracts of ApoE‐deficient mice. Immunohistochemistry and immunoblotting revealed strongly increased decorin and biglycan deposition in atherosclerotic plaques at the aortic root and in innominate arteries. In contrast, versican and perlecan expression was not changed by rosu‐vastatin. Furthermore, matrix metalloproteinase 2 and gelatinolytic activity were decreased in response to rosuvastatin and a condensed collagen‐rich matrix was formed. In carotid endarterectomy specimens of statin‐treated patients increased decorin and biglycan accumulation was detected as well. Drug treatment did not change low‐density lipoprotein (LDL) plasma levels in ApoE‐deficient mice and did not significantly affect lipid retention at the aortic root level as demonstrated by oil‐red O staining and immunohistochemistry of LDL. Long‐term treatment with rosuvastatin caused pronounced remodelling of atherosclerotic plaque matrix characterized specifically by enrichment with SLRPs and formation of a condensed collagen matrix. Therefore, decorin and biglycan might represent novel targets of statin treatment that contribute to a stable plaque phenotype.     

Use of VIRAZOLE® to eradicate odontoglossum ringspot virus from in vitro cultures of Cymbidium Sw.

Odontoglossum Ringspot Virus has been eradicated from Cymbidium Sw. through chemotherapy based on incorporation of ribavirin (VIRAZOLE^®) into the in vitro culture medium of protocorms. Applications of the virustatic agent for several consecutive subcultures freed protocorms of the virus. Acclimated plantlets regenerated from those protocorms are healthy as determinated by enzyme-linked immunosorbent assay (ELISA). No resurgence of virus occurred over a period of 5 years. Besides, trueness to type was total at flowering level and the batch grown was perfectly homogeneous.To secure fast and effective eradication of the virus during the consecutive subcultures of protocorms with ribavirin, three factors proved to be of prime importance: accurate isolation of new growths from initial tissues, VIRAZOLE^® concentration and frequency of transplanting in new media.     

Cardioprotective potential of N,α-l-rhamnopyranosyl vincosamide,an indole alkaloid,isolated from the leaves of Moringa oleifera in isoproterenol induced cardiotoxic rats: In vivo and in vitro studies

Hitherto unknown protective effect of N,α-l-rhamnopyranosyl vincosamide (VR), isolated from Moringa oleifera leaves in isoproterenol (ISO)-induced cardiac toxicity was evaluated in rats. Oral administration of VR at 40 mg/kg for 7 days markedly reduced the ISO-induced increase in the levels of serum cardiac markers such as troponin-T, creatine kinase-MB, lactate dehydrogenase and glutamate pyruvate transaminase as well as cardiac lipid peroxidation with a parallel increase in the cellular antioxidants suggesting its cardio-protective and free radical scavenging potential, which was latter confirmed by in vitro study. Rats treated with test compound also improved the ISO-induced abnormal changes in ECG as well as in cardiac histology. A reduction in myocardial necrosis was further evidenced by the tri-phenyl tetrazolium chloride (TTC) stain in isolated test drug pretreated rats.These findings suggest the cardio-protective potential of the isolated alkaloid and possibly the beneficial action is mediated through its free radical scavenging property.     

In vitro assessment of probiotic properties of <Emphasis Type="Italic">Bacillus</Emphasis> isolated from naturally fermented congee from Inner Mongolia of China

A total of thirty-three strains of Bacillus were isolated from sixteen samples of naturally fermented congee in Inner Mongolia of China and identified by 16S rDNA sequence analysis. Probiotic properties including acid, bile tolerance and artificial gastrointestinal juice resistance as well as inhibition on pathogenic bacteria were used for screening of Bacillus. After the preliminary selection, four strains including Bacillus licheniformis IMAUB1002, Bacillus subtilis IMAUB1011, Bacillus amyloliquefaciens IMAUB1014 and Bacillus amyloliquefaciens IMAUB1034 showed high tolerance to simulated gastric juice at pH 2.0 for 3 h with survival rate all above 92%. And then through gastrointestinal transit, survival rates of these four strains were above 90%. Furthermore, Bacillus licheniformis IMAUB1002 performed well in tolerance to bile salt (0.6%) and inhibitory activity to five food-borne pathogens among four strains of Bacillus. The results suggested that Bacillus licheniformis IMAUB1002 should be considered as a potential probiotics. Further study will be focused on evaluation of these porbiotics properties in vivo and clarification of its other functional properties so as to use it in functional foods production in future.     

Antimicrobial resistance among Salmonella serovars isolated from different sources in Brazil during 1978–1983

A total of 748 Salmonella strains (97 serovars) isolated from human (291), animal (119), environmental (141), food (102) and animal feed (95) sources were examined for resistance to 9 antimicrobial agents. Most of the human isolates were from hospitalized patients (282). An overall resistance rate of 98.8% was determined with 100% for human and environmental isolates. Resistance to sulfadiazine (87.7%) was most common, followed by streptomycin (61.2%), ampicillin (39%) and trimethoprim-sulphamethoxazole (37.9%). Fifty one different resistance patterns were identified with Su (164 strains), Su-Sm (122) and Su-Sm-Tc-Cm-Km-Ap-Nx-Gm-Tm (95) predominating, the latter occurring only in human isolates. Multiple resistance was most frequently found among human isolates, particularly in S. derby and S. typhimurium strains. The relationship between antibiotic resistance, serovar and source of isolation of the Salmonella strains is discussed.