Preliminary studies on the antitubercular activity and the mechanism of action of the water extract of garlic (Allium sativum) and its two partially purified proteins (Garlic defensins?)

Water extract of garlic (Allium sativum) inhibited the growth ofMycobacterium tuberculosis H₃₇R_v andM. tuberculosis TRC-C1193 susceptible and resistant to isoniazid respectively. The minimum inhibitory concentration (MIC) was slightly above 80 but less than 160 μg/ml and slightly above 100 but less than 200 μg/ml for the susceptible and resistant strains respectively. Gel filtration in Sephadex G-100 columns showed that two protein fractions (43 & 38 kD) possessed antitubercular activity with much lower MICs of 20–40 μg/ml and 30–60 μg/ml for susceptible strain. Water extract, when added to actively growingM. tuberculosis in their mid log phase prevented their further growth. The water extract of garlic inhibited the incorporation of¹⁴C glycine into whole cells by 81% in 6 hrs. indicating that the primary mechanism of action is by inhibition of protein synthesis.     

Inhibitory and bactericidal activity of the calmodulin antagonist,trifluoperazine, againstMycobacterium avium in vitro and within human monocyte-derived macrophages

The activity of a calmodulin antagonist, trifluoperazine (TFP), was testedin vitro againstMycobacterium avium (ATCC 25291). The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of this compound forM. avium were 20 and 30 μg/ml, respectively. TFP was also found to completely inhibit the growth of 10 isolates ofM. avium (5 patient isolates and 5 environmental isolates) at 30 μg/ml. At near neutral pH (6.8), the MIC of TFP was found to be 20 μg/ml. However, at pH 5.5 (intracellular pH of macrophages), there was a decrease in the inhibitory activity of the compound against this organism. Interestingly, 99.6% ofM. avium within human monocyte-derived macrophages were killed at a drug concentration of 30 μg/ml, which correlates well with the MBC of TFP againstM. avium in vitro. Although the MIC for TFP appears to be higher than that forMycobacterium tuberculosis H₃₇R_v, our studies suggest that calmodulin antagonists might be useful as drugs against infection due toM. avium. It is suggested that administration of TFP in combination with other known drugs may enhance the overall bactericidal effect.     

Effect of some antitubercular drugs on the calmodulin content ofMycobacterium tuberculosis

The effect of the antitubercular drugs isoniazid (10 μg/ml), ethambutol (10 μg/ml), rifampicin (0.5 μg/ml) and streptomycin (1 μg/ml) on the calmodulin like protein (CAMLP) content ofMycobacterium tuberculosis H₃₇R_v andM. tuberculosis H₃₇R_a was investigated. The drugs were added to actively growing cells at their mid log phase of growth (14 days) and after 12 more hours of incubation, CAMLP was estimated. In both the mycobacteria, all the four antitubercular drugs CAMLP.     

Identification of drug-resistantMycobacterium tuberculosis by single strand conformation polymorphism and cleavase fragment length polymorphism

Twenty isolates ofMycobacterium tuberculosis resistant to rifampicin(RIF), isoniazid(INH) and streptomycin(STR) were analysed by Polymerase Chain Reaction (PCR) amplification of rpoB, katG and rrs genes to evaluate comparative diagnostic significance of genetic assays. Mutations were identified by single strand conformation polymorphism (SSCP) and cleavase fragment length polymorphism (CFLP) and were confirmed by DNA sequencing. SSCP of 4 RIF resistant and 14 INH resistant isolates showed an extra peak at the level of 75-bp and 85-bp respectively, while 2 STR resistant isolates showed 2 peaks with 9 bases difference. CFLP showed a different pattern among RIF, INH and STR sensitive and resistant isolates Thus SSCP and CFLP can be used as alternative diagnostic methods for identification of mutations in RIF, INH and STR resistant strains of M.tuberculosis.     

Synthesis,characterization, and in vitro evaluation of gamma radiation-induced PEGylated isoniazid

BackgroundThe search for innovative anti-tubercular agents has received increasing attention in tuberculosis chemotherapy because Mycobacterium tuberculosis infection has steadily increased over the years. This underlines the necessity for new methods of preparation for polymer-drug adducts to treat this important infectious disease. The use of poly(ethylene glycol)(PEG) is an alternative producing anti-tubercular derivatives. However, it is not yet known whether PEGylated isonicotinylhydrazide conjugates obtained by direct links with PEG are useful for therapeutic applications.ResultsHere, we synthesized a PEGylated isoniazid (PEG-g-INH or PEG–INH) by gamma radiation-induced polymerization, for the first time. The new prodrugs were characterized using Raman and UV/Vis spectrometry. The mechanism of PEGylated INH synthesis was proposed. The in vitro evaluation of a PEGylated isonicotinylhydrazide macromolecular prodrug was also carried out. The results indicated that PEG–INH inhibited the bacterial growth above 95% as compared with INH, which showed a lower value (80%) at a concentration of 0.25 μM. Similar trends are observed for 0.1, 1, and 5 μM.ConclusionsIn summary, the research suggests that it is possible to covalently attach the PEG onto INH by the proposed method and to obtain a slow-acting isoniazid derivative with little toxicity in vitro and higher anti-mycobacterial potency than the neat drug.How to cite: González-Torres M, Guzmán-Beltrán S, Mata-Gómez M, et al. Synthesis, characterization, and in vitro evaluation of gamma radiation-induced PEGylated isoniazid. Electron J Biotechnol 2019; 41. https://doi.org/10.1016/j.ejbt.2019.07.005.     

Antimicrobial activity ofTerminalia bellerica

The antimicrobial activity of crude and methanol extract ofTerminalia bellerica dry fruit was tested by disc diffusion method, against 9 human microbial pathogens. Crude aqueous extract of dry fruit at 4 mg concentration showed zone of inhibition ranging from 15.5–28.0 mm.S. aureus was found to be highly susceptible forming highest zone of inhibition, suggesting thatT. bellerica was strongly inhibitory towards this organism. These pathogens were highly sensitive to the methanol extract forming 14.0 to 30.0 mm zone of inhibition suggesting that the methanol extract ofT. bellerica was more effective than crude extract against most of the microbes tested exceptE. coli (enteropathogen) andP. aeruginosa. The minimal inhibitory concentrations (MICs) of crude and methanol extracts were determined by broth dilution technique which ranged from 300 to >2400 μg/ml and 250 μg to >2000 μg/ml respectively, indicating thatT. bellerica was highly effective againstS. aureus with lower MIC values. There were some biochemical alterations induced byT. bellerica. These results indicate thatT. bellerica dry fruit possesses potential broad spectrum antimicrobial activity.     

Diagnostic role of the antibody response to the 38kDa, 16kDa proteins and lipoarabinomannan of mycobacterium tuberculosis

The antibody response to the 38kDa, 16kDa and Lipoarabinomannan (LAM) antigens ofMycobacterium tuberculosis was evaluated using three different ELISAs based on these antigens. The study group included tuberculosis patients (n=52), patients with HIV and TB co-infection (n=10), other chest symptomatics (n=5), HIV infected individuals (n=10), leprosy cases (n=7) and healthy controls (n=75). The results indicate that the 38kDa and LAM based ELISA for IgM/IgG has a low specificity (ranging from 69–85%) and sensitivity (ranging from 55–78%). When three ELISAs are carried out on a single patient the probability of detection of tuberculosis was significantly increased to 95.2% indicating that a single ELISA test is of low sensitivity and that a combination of ELISA’s may be needed to be of any value as a diagnostic test for tuberculosis. Additionally, a western blot assay of the serum antibody response to protein fraction ofM. tuberculosis was analysed in 15 tuberculosis patients and five healthy controls. A multiple antibody response to various M.tuberculosis proteins was observed which varied from patient to patient as compared to controls who showed a single 38–39 kDa protein band positivity. These finding suggest that a western blot assay which determines the antibody response to a set of antigenic components ofM. tuberculosis could be a better serological test for the diagnosis of tuberculosis in our population.     

Detection of enzymes dehydrogenases and proteases inBrugia malayi filarial parasites

Lymphatic filariasis caused mainly by infection fromW. bancrofti andB. malayi remains a major cause of clinical morbidity in tropical and subtropical countries. Analysis ofB. malayi mf, infective larval and adult worm lysates for the activity of enzymes led to the demonstration of activities of three key enzymes of carbohydrate metabolism viz., Malate dehydrogenase (MDH), Malic enzyme (ME) and Glucose-6-phosphate dehydrogenase (G6PDH) in all the three stages of the parasite. The specific activity of all the three dehydrogenases was significantly high in mf lysate compared to their activity in lysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel to their activity inlysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel electrophoresis (PAGE) using 7.5% non-gradient gel showed the presence of two isoforms of each of the three enzymes (MDH, ME & G6PDH) in mf lysate, while only one form of each enzyme was present in L₃ larval and adult worm lysates. Further proteolytic enzyme activity was demonstrated both in microfilarial and infective larval lysates ofB. malayi. While both mf and L₃ larval lysates showed optimal protease activity at alkaline pH of 9.0, the mf lysate showed increased activity also at pH 3.0. The infective larval lysate was markedly inhibited by Tosylamide-L-Phenylalanine chloromethyl ketone (TPCK), a thiol protease inhibitor, while the protease activity in mf lysate was significantly inhibited by both TPCK and a serine protease inhibitor Phenyl Methyl Sulphonyl Flouride (PMSF). In sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using gelatin copolymerized gel, the microfilarial lysate showed 3 protease molecules of 40 kDa, 180 kDa and 200 kDa and the L₃ larval lysate had 6 protease molecules of 18, 25, 37, 49, 70 and 200 kDa size.     

国内发展新型科研机构的举措和动向分析

通过收集各渠道资料,分析近年来有关新型科研机构的政策、文件、调研等一系列活动,指出新型科研机构已成为今后发展的重要方面;并通过对国内新型科研机构的发展举措和动向进行梳理,提出促进新型科研机构发展的相关建议。     

Use of antigen prepared from in vitro culture ofPlasmodium falciparum for the detection of antibodies in sera from patients exposed toPlasmodium vivax

The blood stage parasites fromin vitro cultures ofPlasmodium falciparum were employed for the detection of malaria antibodies in patient sera havingP. vivax infections employing indirect fluorescent antibody (IFA) test. The antibody titres obtained for theP. vivax infected sera ranged from 1∶128 to 1∶1024. Control slides smeared with non-immune sera did not show any fluorescence. On the basis of the results of the present study it maybe concluded that antigen prepared fromin vitro cultures ofP. falciparum cross-reacts withP. vivax antibodies.     

Fixed point properties of the binomial function

Fixed point properties of the binomial function

are developed. It is shown that for any 1 < L < N, T^L_Nhas a unique fixed point p? in (0, 1), and that for large N, the fixed point is L/N. This has application to signal detection schemes commonly used in communication systems. When detecting the presence or absence of a signal with an initial false alarm probability p_FAand an initial detection probability p_D, then T^L_N(p_FA) < p_FAand T^L_N(p_D) > p_Dif, and only if, p_FA < p? < p_D. When this condition is satisfied, as N → ∞, T^L_N(p_FA) → 0 and T^L_N(p_D → 1.     

Chlorella sorokiniana and Chlorella minutissima exhibit antioxidant potentials,inhibit cholinesterases and modulate disaggregation of β-amyloid fibrils

BackgroundMicroalgae are aquatic chlorophyll-containing organisms comprising unicellular microscopic forms, and their biomasses are potential sources of bioactive compounds, biofuels and food-based products. However, the neuroprotective effects of microalgal biomass have not been fully explored. In this study, biomass from two Chlorella species was characterized, and their antioxidant, anticholinesterase and anti-amyloidogenic activities were investigated.ResultsGC–MS analysis of the extracts revealed the presence of some phenols, sterols, steroids, fatty acids and terpenes. Ethanol extract of Chlorella sorokiniana (14.21 mg GAE/g) and dichloromethane extract of Chlorella minutissima (20.65 mg QE/g) had the highest total phenol and flavonoid contents, respectively. All the extracts scavenged 2,2-diphenyl-1-picrylhydrazyl, 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate) and hydroxyl radicals. The highest metal chelating activity of the extracts was observed in the ethanol extracts of C. minutissima (102.60 μg/mL) and C. sorokiniana (107.84 μg/mL). Furthermore, the cholinesterase inhibitory activities of the extracts showed that ethanol extract of C. sorokiniana (13.34 μg/mL) exhibited the highest acetylcholinesterase inhibitory activity, while dichloromethane extract of C. minutissima (11.78 μg/mL) showed the highest butyrylcholinesterase inhibitory activity. Incubation of the β-amyloid protein increased the aggregation of amyloid fibrils after 96 h. However, ethanol extract of C. sorokiniana and C. minutissima inhibited further aggregation of Aβ_1–42 and caused disaggregation of matured protein fibrils compared to the control. This study reveals the modulatory effects of C. sorokiniana and C. minutissima extracts on some mediators of Alzheimer's disease and provides insights into their potential benefits as functional food, nutraceutics or therapeutic agent for the management of this neurodegenerative disease.How to cite: Olasehinde T, Odjadjare EC, Mabinya LV, et al. Chlorella sorokiniana and Chlorella minutissima exhibit antioxidant potentials, inhibit cholinesterases and modulate disaggregation of β-amyloid fibrils. Electron J Biotechnol 2019;40. https://doi.org/10.1016/j.ejbt.2019.03.008     

Isolation ofMycobacterium Tuberculosis 31 kDa antigen protein of diagnostic interest from culture filtrate using anti-ES-31 antibody by affinity chromatography

Proteins secreted into the culture medium byMycobacterium tuberculosis (M. tb) are shown to be source of antigens of immunodiagnostic interest. Anin vitro released 31 kDa antigen ESAS-7F isolated fromM.tb H₃₇Ra culture filtrate by salt precipitation, SDS-PAGE and cation exchange fast protein liquid chromatography (FPLC) was shown earlier to be a diagnostically important antigen fraction. In this report, we describe the isolation of ESAS-7F antigen using monospecific antibody coupled to sepharose CL-4B column. The percentage recovery of ESAS-7F antigen using affinity chromatography was approximately 8% of the total ES antigen proteins compared to 0.05% obtained by conventional purification steps using salt precipitation, SDS-PAGE and FPLC. Similar seroreactivity was observed by the antigen isolated by both the methods in indirect ELISA. Affinity chromatography helped in an increased recovery of ESAS-7F antigen and obviates the need for time consuming conventional purification steps.     

Polyphenolic extracts of walnut (Juglans regia) green husk containing juglone inhibit the growth of HL-60 cells and induce apoptosis

BackgroundJuglone is a naphthoquinone currently obtained by chemical synthesis with biological activities including antitumor activity. Additionally, juglone is present in the green husk of walnut, which suggests evaluating the effect of GH extracts on carcinogenic cell lines.ResultsWalnut green husk ethanolic extract was obtained as 169.1 mg _juglone/100 g _{Green Husk} and antioxidant activity (ORAC) of 44,920 μmol Trolox Equivalent/100 g DW _{Green Husk}. At 1 μM juglone in HL-60 cell culture, green husk extract showed an antiproliferative effect, but pure juglone did not; under these conditions, normal fibroblast cells were not affected. A dose-dependent effect on mitochondrial membrane potential loss was observed. Apoptosis of HL-60 was detected at 10 μM juglone. Despite high ORAC values, neither purified juglone nor the extract showed protective effects on HL-60 cells under oxidative conditions.ConclusionsGreen husk extract generates an antiproliferative effect in HL-60 cells, which is related to an induction of the early stages of apoptosis and a loss of mitochondrial membrane potential. The normal cells were not affected when juglone is present at concentrations of 1 μM, while at higher concentrations, there is loss of viability of both cancerous and healthy cells.How to cite: Soto-Maldonado C, Vergara-Castro M, Jara-Quezada J, et al. Polyphenolic extracts of walnut (Juglans regia) green husk containing juglone inhibit the growth of HL-60 cells and induce apoptosis. Electron J Biotechnol 2019;39. https://doi.org/10.1016/j.ejbt.2019.02.001.     

Antitubercular activity of garlic (allium sativum) extract on combination with conventional antitubercular drugs in tubercular lymphadenitis

Based on our demonstration earlier that ethanol extract, water extract and a compound purified from garlic possessedin vitro antitubercular activity against drug resistant and susceptibleMycobacterium tuberculosis, we tried the effect of garlic extract in 30 patients of tubercular lymphadenitis. For ethical considerations, two groups of patients, 30 each, were given antitubercular therapy (ATT) consisting of isoniazid, rifampicin, ethambutol and pyrazinamide for 30 days. For the next 15 days (31 to 45 days) group 1 patients received 3–6 garlic pearls per day in addition to ATT while group 2 patients received ATT only. From 46th day onwards both the groups received ATT only for 6–8 months. Antitubercular activity of the serum samples collected on 45th day was assessed by its effect on the growth ofM. tuberculois. The serum of group 1 patients showed significantly much higher antitubercular activity than that of group 2 patients. Further, there was relief of dyspeptic symptoms caused by ATT therapy in patients of group 1 with garlic plus ATT therapy but no change in group 2 patients with ATT only. Liver function and hematological tests were normal in both the groups after 6 months of therapy. Garlic extracts or compounds have a good potential as antitubercular(s) drug if given as a supplement to ATT.     

Immunoproteomic analysis of secretory proteins ofAspergillus fumigatus with specific IGE immunoreactivity

Allergenic/antigenic proteins are known to induce Type I and Type III hypersensitivity reactions leading to allergic bronchopulmonary aspergillosis (ABPA) in immunocompetent host. The common structural features or intrinsic properties of the allergens/antigens leading to allergenicity in a host are not well understood. In the current report, comparative analysis of proteins on two dimensional gel electrophoresis (2D-3) and specific IgE immunoblots ofA. fumigatus secretory proteins (1^st, 2^nd and 3^rd week culture filtrate proteins) was carried out. We observed a total of 159 proteins in 1^st, 2^nd and 3^rd week culture filtrates ofA. fumigatus. Specific IgE immunoreactivity was observed in 75 proteins with different intensity. Third week culture filtrate showed maximum number of proteins, 142, and specific IgE immunoreactive proteins, 65. MALDI-TOF analysis resulted in putative identification of two allergens as hypothetical protein YBL057c fromSaccharomyces cereviseae and unnamed protein product fromDebaryomyces hansenii (similar to IPF14568 ofCandida albicans). Identification of a repertoire of specific IgE immunoreactive proteins will facilitate the studies on structure-function relationship of these proteins relevant for diagnosis and pathogenesis.     

Prostaglandin,cortisol and catecholamines in angina pectoris

Plasma prostaglandin E₂ (PGE₂)-like activity, cortisol and catecholamines were estimated in twenty stable angina pectoris and thirty control cases. PGE₂ like activity was significantly decreased (0.7 ng/ml ± 0.44) in angina patients compared to control (1.6 ng/ml ± 0.7). The plasma catecholamines were only marginally elevated in angina patients (242.8 ng/100 ml ± 14.2) compared to healthy individuals (236.2 ng/100 ml ± 5.5). The changes in plasma cortisol levels were not significant in angina (18.4 μg%± 6.5) in comparison to control (16.9 μg% ± 3.2).     

Bionomics of Mansonioides mosquitoes in relation to community structure of hydrophytes/breeding habitats in Cherthala,Kerala

Three species of Mansonioides vectors viz.,Ma. annulifera, Ma. uniformis andMa. indiana were found in Cherthala taluk, Kerala which is one of the endemic areas due toB. malayi. The immatures of Mansonioides thrive mainly in association with macrophytic hydrophytes such asP. stratiotes, S. molesta andE. crassipes in perennial habitats (ponds, channels/ canals etc.,) andI. miliaceae in seasonal habitats (fallow lands etc.) Breeding potential was higher (130.19) in clean ponds withP. stratiotes, compared to that of polluted ones (40.69). However, the polluted habitats infested with the same host plants were found to be the most productive forMa. annulifera, with an average daily adult emergence rate of 601/100 sq.m.). The clean habitats played a major role in the contribution ofMa. uniformis, whereS. molesta in the perennial habitats and I. miliaceae in the seasonal fallow lands were the favourable plants contributing a daily output of 12.5/100 sq.m and 221.81/100 sq.m. respectively.E. crassipes infested polluted habitats formed the major source forMa. indiana, the emergence rate being 13.89/100 sq.m. The perennial habitats supported mainly the breeding ofMa. annulifera (70.82%), whereas the seasonal habitats contributed the major chunk ofMa. uniformis (92.54%) andMa. indiana (71.43%). The bionomics of Mansonioides mosquitoes are thus shown to be greatly influenced by the community structure of hydrophytes and also the nature of breeding habitats.     

Strategy of oxygen transfer coefficient control on the l-erythrulose fermentation by newly isolated Gluconobacter kondonii

BackgroundThe effect of diverse oxygen transfer coefficient on the l-erythrulose production from meso-erythritol by a newly isolated strain, Gluconobacter kondonii CGMCC8391 was investigated. In order to elucidate the effects of volumetric mass transfer coefficient (k_La) on the fermentations, baffled and unbaffled flask cultures, and fed-batch cultures were developed in present work.ResultsWith the increase of the k_La value in the fed-batch culture, l-erythrulose concentration, productivity and yield were significantly improved, while cell growth was not the best in the high k_La. Thus, a two-stage oxygen supply control strategy was proposed, aimed at achieving high concentration and high productivity of l-erythrulose. During the first 12 h, k_La was controlled at 40.28 h^-1 to obtain high value for cell growth, subsequently k_La was controlled at 86.31 h^-1 to allow for high l-erythrulose accumulation.ConclusionsUnder optimal conditions, the l-erythrulose concentration, productivity, yield and DCW reached 207.9 ± 7.78 g/L, 6.50 g/L/h, 0.94 g/g, 2.68 ± 0.17 g/L, respectively. At the end of fermentation, the l-erythrulose concentration and productivity were higher than those in the previous similar reports.     

Preliminary studies on the detection ofMycobacterium avium-intracellulare complex using DNA probe from a clinical isolate

Genomic DNA from a clinical isolate ofMycobacterium avium-intracellulare complex was purified and cloned in PBR 322 at the tetracycline resistance site using Bam HI restriction enzyme. A 16 kb cloned fragment was purified, radiolabeled and used as a probe. Genomic DNA isolated from nineteen MAC strains, threeM. tuberculosis strains and oneM. kansasii strain were digested with Eco RI restriction enzyme, Southern blotted and hybridized with the 16 kb cloned and labeled fragment. Twelve MAC strains showed positive hybridization although five strains gave faint signals. Positive hybridization was noted in two out of the threeM. tuberculosis strains, possibly due to shared DNA homology. No signal was received from the singleM. kansasii strain used in this study.