Levels of enzymes in leukaemic mice treated withAeromonas L-asparaginase

L-asparaginase isolated in our laboratory fromAeromonas has been found to be antileukaemic. In the present study changes in the levels of serum enzymes in leukaemic mice and under treatment withAeromonas L-asparaginase has been compared. A significant increase in the levels of serum lactate dehydrogenase with tumour growth and a decrease during therapy was observed. A significant decrease in alanine transaminase activity during tumour growth and an increase during treatment was noticed. Increased levels of aspartate transaminase and alkaline phosphatase was observed during enzyme therapy. Total acid phosphatase was found to be increased during tumour growth and decreased considerably during treatment.     

Effect of<Emphasis Type="Italic">Vitex negundo</Emphasis> leaf extract on the free radicals scavengers in complete Freund's adjuvant induced arthritic rats

The effect of the oral administration ofVitex negundo leaf extract on the levels of enzymic and non-enzymic antioxidants were studied in the adjuvant induced arthritic (AIA) rats The levels of antioxidant enzymes such as SOD, CAT, GPx, G6PD, GSH and Vit-C were estimated in various groups of the experimental rats. It was observed that the antioxidant enzyme levels in the AIA were significantly low when compared to normal rats. A significant decrease in enzymic antioxidant—SOD, CAT, GPx, G6PD and non-enzymic antioxidant—GSH, Vit-C were observed in the liver of AIA rats compared to the normal rats. These results suggest that the leaf extract ofVitex negundo possesses antioxidant activity.     

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.     

Secondary immunization inhibits drug induced apoptosis<Emphasis Type="Italic">in vivo</Emphasis>

Immunization with a proper dose of an antigenic stimulus leads to cell proliferation and antibody response of circulating lymphocytes. We have previously observed that Secondary immunized spleenocytes resist ceramide-mediated apoptosisin vitro. Our present study is aimed at investigating thein vivo effect of immunization on apoptosis. Mice were subjected to either Primary or Secondary dose with Tetanus Toxoid. Unimmunized spleenocytes served as controls. Unimmunized, Primary and Secondary immunized mice were later exposed to chemotherapeutic drugs such as Etoposide/Methotrexate/Vincristine to induce apoptosis. Apoptosis was studied by using the Feulgen reaction on 5μ thin parafin sections of spleen. It was observed that Secondary immunized mice showed a lower percentage of apoptosis as compared to Primary or Unimmunized mice that was subjected to either of the chemotherapeutic drugs. It was thus concluded that Secondary immunization inhibits the process of chemotherapeutic drug induced apoptosis in vivo.     

Altered antioxidant enzyme profile in wound healing

The effects of the alcoholic extract of the flowers ofIxora coccinea were studied on some of the antioxidant enzymes in dead space wounds created in rats. Increases in the tensile strength of the wound and in the level of lysyl oxidase, the crucial enzyme for collagen maturation, were observed indicating a definite prohealing action. In addition, a highly significant increase in the levels of catalase, glutathione peroxidase and glutathione reductase was observed in the drug-treated group. Thus, the gain in tensile strength may be attributed not only to the better cross-linking but also to the antioxidant properties of the drug.     

Induction of IgE antibody response by the green seed extract of<Emphasis Type="Italic">Vigna sinensis</Emphasis> in mice

Sensitization to ingested foods is a known fact and several food allergens have been characterized. It has been observed in our survey that the people complained of allergic symptoms after consumption of the vegetableVigna sinensis. In this study, the experiments were carried to investigate the IgE antibody response against the green seed extract of vigna sinensis in mice and found that the primary, secondary and tertiary immunization with or without adjuvant by different doses induced a significant production of IgE antibodies. The presence of IgE antibodies in the mice sera were determined by passive cutaneous anaphylaxis and enzyme linked immunosorbent assay. It was also confirmed that these allergens were found to be heat resistant and shared a common epitope(s) with the other legume foods, as evidenced by the cross-reactive studies.     

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.     

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.     

Immune response to n-terminal and c-terminal deletion mutants of Aspergillus fumigatus major allergen ASP F 3

The ubiquitous fungus Aspergillus fumigatus causes allergic rhinitis, asthma, sinusitis and allergic bronchopulmonary aspergillosis. A number of major allergens from A. fumigatus are purified, but their structure-function role in the pathogenesis of disease is not known. Such information is essential for devising alternative therapy of fungal allergic diseases. In the present study, N-terminal and C-terminal deletion mutants ofAsp f 3 were constructed and their immunopathological responses studied in a mice model of allergy. Three mutants viz,Asp f 3 (aa 33–168), (aa 1–142), and (aa 23–142) were made by deleting certain amino acids from epitopic regions of full lengthAsp f 3, a major allergen of A. furnigatus. TheAsp f 3 and three mutated proteins were expressed in pET vector. The C-terminal deletion mutantAsp f 3 (aa 1–142) induced elevated IFN-γ but low levels of IL-4 by spleen cells. This mutant also showed significant downregulation of peripheral blood eosinophils and lung inflammation in immunized mice. The N-terminal deletion mutantAsp f 3 (aa 33–168) also exhibited an immuno-suppressive effect in terms of IgE production and induction of Th2 cytokine. The results indicate thatrAsp f 3 and its deletion mutants induced distinct immune-inflammatory responses in mice on challenge with these proteins. The non-IgE binding deletion mutants ofAsp f 3 (aa 1–142 and aa 33–168) could deviate Th2 immune response with a concomitant reduction in airway inflammation and infiltration of inflammatory cells.     

Antihyperglycemic and antilipidperoxidative effects of<Emphasis Type="Italic">Tephrosia purpurea</Emphasis> seed extract in streptozotocin induced diabetic rats

The aim of the present study was to evaluate the antihyperglycemic and antilipidperoxidative effects of ethanolic seed extract ofTephrosia purpurea (TpEt) in streptozotocin induced diabetic rats. Hyperglycemia associated with an altered hexokinase and glucose 6 phosphatase activities, elevated lipid peroxidation, disturbed enzymatic and non-enzymatic antioxidants status were observed in streptozotocin induced diabetic rats. Oral administration of “TpEt” at a dose of 300mg/kg bw showed significant antihyperglcemic and antilipidperoxidative effects as well as increased the activities of enzymatic antioxidants and levels of non enzymatic antioxidants. We also noticed that the antihyperglycemic effect of plant drug (TpEt) was comparable to that of the reference drug glibenclamide. Our results clearly indicate that “TpEt” has potent antihyperglycemic and antilipidperoxidative effects in streptozotocin induced diabetic rats and therefore further studies are warranted to isolate and characterize the bioactive antidiabetic principles from “TpEt”.     

Prevalence of multiple antibiotic resistance and R-plasmid inEscherichia coli isolates of hospital sewage of Aligarh city in India

R-plasmids that transfer antibiotic resistance are common in the non-pathogenicEscherichia coli of the gastro-intestinal tract of human beings and domestic animals, which inturn may enter into sewage. Therefore we have isolated 30Escherichia coli isolates from hospital sewage of Aligarh city. These isolates were tested for their resistance and sensitivity against 10 antibiotics. 90% isolates showed resistance against ampicillin and sulphamethizole. Of the total 30E. coli isolates 86.6% were resistant to erythromycin and rifampicin but none of them was resistant to kanamycin and streptomycin. Plasmids (mol. wt. 16.5 mega daltons) were isolated from five differentE. coli strains which harboured only a single plasmid and were characterized on the basis of antibiogram. Moreover, the transformation experiments were also performed to confirm the resistant character on the plasmid. We conclude that multiple drug resistance among most of theE. coli isolates is plasmid borne.     

Effect of Lecithin and silymarin on D-galactosamine induced toxicity in isolated hepatocytes and rats

To investigate Lecithin for its hepatoprotective activity against D-galactosamine (D-GalN) induced toxicity in freshly isolated rat hepatocytes and animal models. Freshly isolated rat hepatocytes were exposed to Dgalactosamine (30 mM) along with/without lecithin (100 μg/ml) and the levels of selected liver enzymes were measured. Thirty six Wistar strain albino rats were used for the in vivo investigations. Lecithin 50 and 100 mg/kg.b.wt were administered for one week by oral route. Liver damage was induced by intra peritoneal administration of 400 mg/kg b.wt D-galactosamine. The antihepatotoxic effect of lecithin was observed in freshly isolated rat hepatocytes at concentration 100 μg/ml and was found to be similar to that of the standard silymarin used. Its in vivo hepatoprotective effect at 100 mg/kg b.wt was comparable with that of the standard silymarin at 100 mg/kg body weight. Lecithin was able to normalise the biochemical levels which were altered due to D-galactosamine intoxication in freshly isolated rat hepatocytes and also in animal models.     

Use of specific IgE to fractions of gynandropsis gynandra pollen inin vitro diagnostic test

The study was aimed at presence of specific IgE antibody levelsinvitro to the identified antigen. Based on positive skin test with Gynandropsis gynandra and elevated levels of total IgE (>325 IU/ml) 104 patients were selected. Healthy, asymptomatic individuals (25) with low total IgE (<325 IU/ml) were included as controls. The mean OD values by ELISA for specific IgE were 0.67±0.21, 0.57±0.18 and 0.56±0.18 with whole pollen antigen, 46-37 kD fraction and 36-32 kD fraction, respectively. The specificity and sensitivity between skin test positivity with whole pollen antigen verses fraction with mol.wt 46-37 kD was 90% and 90% and for fraction with mol.wt 36-32 kD was found to be 81.1% and 89.4%. The clusters with molecular weights 46-37 kD and 36-32 kD may be useful inin vitro diagnostic test. Fractions within these clusters need to be identified for a higher specificity.     

Acute and chronic toxicity studies on partially purified hypoglycemic preparation from water extract of bark ofFicus bengalensis

Acute and chronic toxicity studies were conducted to assess toxicity of a partially purified preparation from the water extract of the bark ofFicus bengalensis, which was demonstrated in our earlier studies to have significant hypoglycemic and hypocholesteroiemic effect on alloxan induced, mild and severe diabetes in rabbits. LD₅₀ of this preparation was found to be ∼1 gm/kg in rats when given orally. For chronic toxicity studies 3 doses of aqueous preparation were given to 3 groups of rats. First group received 5 times ED₅₀ (50 mg/kg), second group 10 times ED₅₀ (100 mg/kg) and the third group 15 times ED₅₀ (150 mg/kg) for 3 months. Fourth group which served as control was given water. After three months, blood was collected for studying biochemical and hematological parameters. Blood glucose, serum cholesterol, liver and kidney function tests, haemoglobin, total and differential leukocyte count were determined. Animals were sacrificed and histopathological examination of liver, heart and kidneys was carried out. Results of the study showed that partially purified preparation fromFicus bengalensis is not toxic by all the above mentioned parameters.     

Hypocholesterolemic and hepatoprotective effects of flaxseed chutney: Evidence from animal studies

Rats fed with hypercholesterolemic diet showed a significant increase in serum total—cholesterol, liver homogenate total-cholesterol, HDL-cholesterol and changed LDL-cholesterol, and HDL/LDL ratio in comparison to control. Flaxseedchutney (FC) supplemented diet (15%, w/w) was found to be more effective in restoring lipid profile changes in rats fed with cholesterol, (1.0%). The activities of serum marker enzymes glutamate oxaloacetate transminase (GOT), glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP) were elevated significantly in carbon tetrachloride induced rats. Administration of flaxseedchutney (15%, w/w) resulted in depletion of serum marker enzymes and exhibited recoupment thus showing significant hepatoprotective effect. It was observed that flaxseedchutney supplemented diet could lower the serum cholesterol and as a potential source of antioxidants it could exert protection against hepatotoxic damage induced by carbon tetrachloride (CCl₄) in rats.     

Myeloperoxidase in Chronic Kidney Disease

Numerous lines of evidence implicate a role of myeloperoxidase (MPO) in the pathogenesis of cardiovascular disease (CVD). It is a well accepted fact that patients with chronic kidney disease (CKD) are at an increased risk for CVD. MPO is a pro-oxidant enzyme which could be involved in the increased susceptibility of these patients to CVD. Hence, the levels of plasma MPO was determined in healthy controls as well as in patients with CKD [stratified with the level of their kidney failure as CKD stages II–V (end stage renal disease)]. Plasma MPO was assayed by a spectrophotometric method. Serum urea and creatinine were estimated on a clinical chemistry analyzer using standard laboratory procedures. The mean plasma MPO levels were significantly lower with advancing stages of renal failure (P < 0.001). There was a positive correlation between MPO and GFR (r = +0.89, P < 0.001) and a negative correlation with urea (r = −0.85, P < 0.001) and creatinine (r = −0.82, P < 0.001). While an inverse association was observed between plasma MPO and urea in CKD patients, such an association was not observed in control subjects (P = 0.43). In conclusion, the decline in plasma MPO levels may be due to the inhibitory effect of uraemic toxins on the enzyme.     

Enhanced production of glutaminase-free l-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines

BackgroundThe increasing rate of breast cancer globally requires extraordinary efforts to discover new effective sources of chemotherapy with fewer side effects. Glutaminase-free l-asparaginase is a vital chemotherapeutic agent for various tumor malignancies. Microorganisms from extreme sources, such as marine bacteria, might have high l-asparaginase productivity and efficiency with exceptional antitumor action toward breast cancer cell lines.Resultsl-Asparaginase-producing bacteria, Bacillus velezensis isolated from marine sediments, were identified by 16S rRNA sequencing. l-Asparaginase production by immobilized cells was 61.04% higher than that by free cells fermentation. The significant productivity of enzyme occurred at 72 h, pH 6.5, 37°C, 100 rpm. Optimum carbon and nitrogen sources for enzyme production were glucose and NH₄Cl, respectively. l-Asparaginase was free from glutaminase activity, which was crucial medically in terms of their severe side effects. The molecular weight of the purified enzyme is 39.7 KDa by SDS-PAGE analysis and was ideally active at pH 7.5 and 37°C. Notwithstanding, the highest stability of the enzyme was found at pH 8.5 and 70°C for 1 h. The enzyme kinetic parameters displayed V_max at 41.49 μmol/mL/min and a K_m of 3.6 × 10⁻⁵ M, which serve as a proof of the affinity to its substrate. The anticancer activity of the enzyme against breast adenocarcinoma cell lines demonstrated significant activity toward MDA-MB-231 cells when compared with MCF-7 cells with IC₅₀ values of 12.6 ± 1.2 μg/mL and 17.3 ± 2.8 μg/mL, respectively.ConclusionThis study provides the first potential of glutaminase-free l-asparaginase production from the marine bacterium Bacillus velezensis as a prospect anticancer pharmaceutical agent for two different breast cancer cell lines.How to cite: Mostafa Y, Alrumman S, Alamri S, et al. Enhanced production of glutaminase-free L-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines. Electron J Biotechnol 2019;42. https://doi.org/10.1016/j.ejbt.2019.10.001.     

Enzymatic changes in liver in Calcium oxalate stone forming rats treated with sodium pentosan polysulphate

The effect of sodium pentosan polysulphate (SPP) in calcium oxalate stone forming rats was studied in relation to enzymatic changes in liver. A significant increase in liver glycollate oxidase (GAO) activity was observed in stone forming rats fed sodium glycollate. SPP treatment lowered the enzyme acitivity in both stone formers and 30 days drug treated control rats. Moderate elevation in LDH activity was seen in the calculogenic group and SPP had minimal effect. The lowering of alkaline and acid phosphatase activities in stone formers was normalised with drug administration. Increases in total, Na⁺, K⁺-and Ca²⁺-ATPase levels in the calculogenic rats was lowered considerably with SPP treatment. Inorganic pyrophosphatase and aminotransferases were slightly reduced in glycollate-fed rats. SPP administration further lowered the pyrophosphatase level. The decrease in liver GAO during SPP administration with a consequent reduction in kidney oxalate may prove beneficial in preventing recurrence.     

Interaction ofLabeo rohita lectin (LRP) with mycobacterial surface oligosaccharide

A C-type mannose specific lectin (LRP) isolated from plasma of fresh water fishLabeo rohita showed 500 times higher specificity towards cell surface oligosaccharide (LAM) ofMycobacterium tuberculosis (H37Rv). Using biotinylated LRP, binding between lectin and LAM was demonstrated by ELISA and it was observed that even 3ng of oligosaccharides might be detected using only 1μg of biotinylated lectin.     

Hypoglycemic, hypolipidemic and antioxidant properties of combination ofCurcumin fromCurcuma longa, Linn, and partially purified product fromAbroma augusta, Linn. in streptozotocin induced diabetes

Dietary spice components ofCurcuma longa andAbroma augusta have been screened for their protective effect against reactive oxygen species induced lipid peroxidation. They have been found to be efficient antioxidant when administered in combination. The purpose of the study was to investigate the effect of oral administration (300mg/Kg) of the aqueous extract of turmeric whose active ingredient isCurcumin andAbromine powder as a hypoglycemic agent mixed with diet. The effect of this aqueous extract on blood glucose, lipid peroxidation (LPO) and the antioxidant defense system in rat tissues like liver, lung, kidney and brain was studied for 8 weeks in streptozotocin induced diabetic rats. The administration of an aqueous extract of turmeric and abromine powder resulted in a significant reduction in blood glucose and an increase in total haemoglobin. The aqueous extract also resulted in decreased free radical formation in the tissues studied. The decrease in thiobarbituric acid reactive substances (TBARS) and increase in reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) clearly showed the antioxidant property of the mixture. It is suggested that these changes initially counteract the oxidative stress in diabetes however, a gradual decrease in the antioxidative process may be one of the factors which results in chronic diabetes. These results indicate that the mixture of the two plants have shown antidiabetic activity and also reduced oxidative stress in diabetes. A combination ofAbroma augusta and Curcuma longa also restored the other general parameters in diabetic animals. The results were statistically analyzed and indicated that combination of herbal extracts showed better efficacy as compared to individual herbal plant extracts used.