An Exonuclease I-Aided Turn-Off Fluorescent Strategy for Alkaline Phosphatase Assay Based on Terminal Protection and Copper Nanoparticles

As an important DNA 3′-phosphatase, alkaline phosphatase can repair damaged DNA caused by replication and recombination. It is essential to measure the level of alkaline phosphatase to indicate some potential diseases, such as cancer, related to alkaline phosphatase. Here, we designed a simple and fast method to detect alkaline phosphatase quantitively. When alkaline phosphatase is present, the resulting poly T-DNA with a 3′-hydroxyl end was cleaved by exonuclease I, prohibiting the formation of fluorescent copper nanoparticles. However, the fluorescent copper nanoparticles can be monitored with the absence of alkaline phosphatase. Hence, we can detect alkaline phosphatase with this turn-off strategy. The proposed method is able to quantify the concentration of alkaline phosphatase with the LOD of 0.0098 U/L. Furthermore, we utilized this method to measure the effects of inhibitor Na3VO4 on alkaline phosphatase. In addition, it was successfully applied to quantify the level of alkaline phosphatase in human serum. The proposed strategy is sensitive, selective, cost effective, and timesaving, having a great potential to detect alkaline phosphatase quantitatively in clinical diagnosis.

Detection of enzymes produced by lactic acid bacteria isolated from traditionally made Serbian cheese and their role in the formation of its specific flavor

Nine species (sixteen isolates) of lactic acid bacteria (LAB) isolated from traditionally made Serbian cheese were evaluated for their enzymatic activities in order to select indigenous strains of technical interest for the manufacture of cheese. These strains were selected based on their previously determined biochemical and physiological characteristics, as well as their antimicrobial activity, and were identified as Lactococcus lactis subsp. lactis (one isolate), Lc. lactis subsp. lactis biovar. diacetylactis (five isolates), Lactobacillus fermentum (two isolates), Lb. plantarum (one isolate), Lb. brevis (one isolate), Enterococcus faecalis (three isolates), E. faecium (one isolate), E. durans (one isolate) and E. hirae (one isolate). The enzymatic activities (acid and alkaline invertases, alkaline phosphatase, alkaline protease, a-amylase) were measured by using the spectrophotometric method. The results indicated that all Lactobacillus isolates showed protease, amylase, and alkaline phosphatase activities, while the activities of acid and alkaline invertases were not observed. The Lactococcus isolates showed protease, acid invertase and alkaline phosphatase activities, except the KGPMF50 isolate, which showed no alkaline phosphatase activity. The tested Enterococcus isolates showed weakly and strain-specific enzymatic activity. The results indicated that the enzymes produced by the investigated strains have a role in the formation of the specific flavor of cheese and that these isolates, especially Lactobacillus isolates, showed the potential for use in the dairy industry or applied biotechnology.

Cytochemical localization of certain hydrolytic enzymes at various stages of development of Achlya flagellata mycelium

The standard coupling azo dyes techniques were used to reveal the activities of acid phosphatase, alkaline phosphatase, esterase and β-galactoidase in the vegetative and reproductive cycle of <i>Achlya flagellata</i>. The end-products of the enzymic reactions, with the exception of E 600 sentisive esterese, which is localized in cytoplasm, occured in cytoplasmic granules. These granules are expected to be spherosomes. Acid phosphatase activity is high in differentiating sporangia, in antheridial hyphae and in degenerating oospheres where hydrolytic processes occur. β-galactosidase is the least active enzyme in the mycelium of <i>Achlya</i>.

Biochemical and electrophoretic analyses of saliva from the predatory reduviid species Rhynocoris marginatus (Fab.).

The saliva of Rhynocoris marginatus consists of amylase, invertase, trehalase, protease, acid phosphatase, alkaline phosphatase, phospholipase, lipase, trypsin, hyaluronidase, and esterase. All enzyme activities were significantly higher in the saliva of female R. marginatus when compared to the saliva of male individuals. The saliva was analyzed by tricine SDS/PAGE, sephadex column chromatography, FT-IR, and MALDI-TOF. The pH of the saliva was slightly alkali. The SDS/PAGE revealed a few proteins with molecular masses greater than 29.5 and 36.2 kDa for male and female predator saliva respectively. The FT-IR spectrum confirmed the acidic, proteinaceous, enzymatic, and aromatic nature of the saliva. The MALDI-TOF-MS revealed the presence of enzymes, proteins, peptides, and other biomolecules. The most prominent peptides were named as RmIT-1 (3.79 kDa), RmIT-2 (9.7 kDa), and RmIT-3 (10.94 kDa) (Rhynocoris marginatus Insect Toxin). Further studies are underway to isolate and identify these biomolecules.

Molecular divergence of locally grown pumpkin (Cucurbita maxima) cultivars through some isozyme tests

Context: Pumpkin (Cucurbita maxima) is highly polymorphic vegetable species and its polymorphism can be analyzed by isozyme molecular marker. Objective: To analyze genetic polymorphism among 10 locally grown pumpkin cultivars by isozyme. Materials and Methods: Fresh leaves of young plant of different cultivars were used for enzyme extraction. Enzyme extracts were prepared by homogenizing of 2 g sample of each cultivar. Prechilled mortar and pestle nestled in ice along with 1% polyvinylpyrrolidone and 2 ml of chilled extraction buffer were used prior to centrifuge. N-PAGE was conducted for different isozymes and stained the gels with specific chemicals for band development. Results: Five isozymes (peroxidase, esterase, acid phosphatase, alkaline phosphatase and malate dehydrogenase) were tested for genetic polymorphism analysis of pumpkin cultivars. Among them esterase, peroxidase and alkaline phosphatase showed polymorphism in different cultivars with 75-, 58.33- and 41.18% respectively. But acid phosphatase and malate dehydrogenase did not show any polymorphism. Esterase and peroxidase produced band quickly than others. Relative mobility of first band of esterase, peroxidase, acid phosphatase, alkaline phosphatase and malate deghdrogenase was 0.063, 0.045, 0.262, 0.07 and 0.093 respectively Conclusion: Out of five isozymes, effective polymorphism was found in esterase and peroxidase test DOI: http://dx.doi.org/10.3329/jbs.v19i0.13006 J. bio-sci. 19 89-93, 2011

Toxicity and Physiological Effect of Essential Oil of Artemisia Annua (Labiatae) on Agriolimax Agrestis L. (Stylommatophora: Limacidae)

Toxicity and Physiological Effect of Essential Oil of Artemisia Annua (Labiatae) on Agriolimax Agrestis L. (Stylommatophora: Limacidae) Essential oil of Artemisia annua L. was investigated to find out its toxicity and physiological aspects on the slug Agriolimax agrestis, in controlled conditions (8±1°C, 75±5 RH and 14:10 LD). The slugs received different concentrations of essential oil treated radish leaves in methanol, while the control received methanol alone. LC10, LC30, LC50 and LC90 values were estimated at 4.67, 5.3, 5.81, 7.25%, respectively. The effect of the essential oil on some important enzymatic components like; cytochrome P450 monnooxygenase, acid phosphatase, alkaline phosphatase, lipase, amylase and protease were significantly increased compared to the control. These results indicate that the plant Artemisia annua L. not only shows toxicity but also shows some irreversible effect on some important biochemical components and deserves further investigation.

Can Homeopathic Arsenic Remedy Combat Arsenic Poisoning in Humans Exposed to Groundwater Arsenic Contamination?: A Preliminary Report on First Human Trial

Groundwater arsenic (As) has affected millions of people globally distributed over 20 countries. In parts of West Bengal (India) and Bangladesh alone, over 100 million people are at risk, but supply of As-free water is grossly inadequate. Attempts to remove As by using orthodox medicines have mostly been unsuccessful. A potentized homeopathic remedy, Arsenicum Album-30, was administered to a group of As affected people and thereafter the As contents in their urine and blood were periodically determined. The activities of various toxicity marker enzymes and compounds in the blood, namely aspartate amino transferase, alanine amino transferase, acid phosphatase, alkaline phosphatase, lipid peroxidation and reduced glutathione, were also periodically monitored up to 3 months. The results are highly encouraging and suggest that the drug can alleviate As poisoning in humans.