Study of cell killing and morphology on S180 by ultrasound activating hematoporphyrin derivatives

Sonodynamic therapy (SDT) is one of antitumor strategies that kill tumor cells through the synergistic effects caused by the combined use of HpD and US[1]. SDT is based on the following principle. Ultrasound used in SDT can penetrate the deep tissue and activate HpD, which accu- mulated in tumor cell, and produce highly active oxygen species[2] such as singlet oxygen (1O2), which can destroy the structure of tumor cells. So far the studies on the SDT have focused mainly on the mechan…     

Pyrogallol inhibits the growth of lung cancer Calu-6 cells via caspase-dependent apoptosis

Pyrogallol (PG) is a polyphenol compound and a known O₂⁻ generator. We evaluated the effects of PG on the growth and apoptosis of human pulmonary adenocarcinoma Calu-6 cells. PG decreased the viability of Calu-6 cells in a dose- and time-dependent manner. The induction of apoptosis by PG was accompanied by the loss of mitochondrial membrane potential (ΔΨ_m), cytochrome c release from mitochondria and activation of caspase-3 and caspase-8. All tested caspase inhibitors, especially the pan-caspase inhibitor (Z-VAD), markedly rescued Calu-6 cells from PG-induced cell death. Rescue was accompanied by inhibition of caspase-3 activation and PARP cleavage. Treatment with Z-VAD also prevented the loss of mitochondrial membrane potential (ΔΨ_m). In conclusion, PG inhibits the growth of Calu-6 cells via caspase-dependent apoptosis.     

Functional expression of amine oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> (AO-I) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.     

Internal charge transfer in cytochrome c oxidase at a limited proton supply: Proton pumping ceases at high pH

Background

In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O₂ is linked to proton uptake from solution to form H₂O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane.

Methods

In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O₂. These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles.

Results

The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O₂ reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH.

Conclusions

The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the “wrong” side of the membrane) and that at this pH the net proton-uptake stoichiometry is ∼ 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme.

General significance

These results provide new insights into the function of cytochrome c oxidase.     

Does anthocyanin degradation play a significant role in determining pigment concentration in plants?

In contrast to the detailed knowledge available on anthocyanin synthesis, very little is known about its stability and catabolism in plants. Here we review evidence supporting in planta turnover and degradation of anthocyanins. Transient anthocyanin accumulation and disappearance during plant development or changes in environmental conditions suggest that anthocyanin degradation is controlled and induced when beneficial to the plant. Several enzymes have been isolated that degrade anthocyanins in postharvest fruit that may be candidates for in vivo degradation. Three enzyme groups that control degradation rates of anthocyanins in fruit extracts and juices are polyphenol oxidases, peroxidases and β-glucosidases. Evidence supporting the involvement of peroxidases and β-glucosidases in in vivo anthocyanin degradation in Brunfelsia flowers is presented. Understanding the in vivo anthocyanin degradation process has potential for enabling increased pigmentation and prevention of color degradation in crops.     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.     

Phylogenetic analysis of New World screwworm fly, Cochliomyia hominivorax, suggests genetic isolation of some Caribbean island populations following colonization from South America

Abstract.  Larval infestations of the New World screwworm (NWS) fly, Cochliomyia hominivorax , cause considerable economic losses through the direct mortality and reduced production of livestock. Since the 1950s, NWS populations in North and Central America have been the target of virtually continuous eradication attempts by sterile insect technique (SIT). Nevertheless, in some areas, such as Jamaica, SIT-based control programmes have failed. Reasons for the failure of SIT-based programmes in some locations are unknown, but it is hypothesized that failure may be related to the mating incompatibility between sterile and wild flies or to the existence of sexually incompatible cryptic species. Accordingly, the current research investigates intraspecific phylogenetic relationships and associated biogeographic patterns between NWS populations from the Caribbean and South America, which represent those populations involved in, or earmarked for, forthcoming SIT programmes. Uniquely, this study also includes analyses of two North American samples, collected in Texas in 1933 and 1953 prior to initiation of the SIT-based eradication programme. The study utilizes three nucleotide datasets: elongation factor-1α (nuclear); cytochrome oxidase subunit 1 (mitochondrial), and 12S rRNA (mitochondrial). Phylogenetic analysis of these data, representing populations from across the Caribbean, South America and Texas, indicates sub-structuring of fly populations on several of the larger Caribbean islands, suggesting a period of isolation and/or founder effects following colonization from South America; significantly, our findings do not support a North American origin for Cuban flies. The importance of these findings in the light of proposed SIT programmes in the region is discussed.     

Amperometric biosensor based on enzymes immobilized in hybrid mesoporous membranes for the determination of acetylcholine

Acetylcholine sensor is successfully prepared by using immobilized enzymes, i.e., acetylcholinesterase and choline oxidase within separate hybrid mesoporous silica membranes with 12 nm pore diameter (F127M). The measurement was based on the detection of hydrogen peroxide produced by two sequential enzyme reactions. The determination range and the response time are 6.0–800 μM and within approximately 3 min, respectively. The sensor is very stable compared to free enzymes and 80% of the initial response was maintained even after storage for 80 days. These results show that two enzymes are successfully immobilized and well stabilized, and at the same time, two sequential enzyme reactions efficiently proceed within the separate hybrid mesoporous membranes. Further, we studied the possible detection of organophosphorus pesticides in terms of the inhibition of acetylcholinesterase activity, i.e., the decrease of current response, and demonstrated that the nanomolar concentrations of pesticide (DZN-oxon) can be detected with our sensor.     

Functionally undefined gene, yggE, alleviates oxidative stress generated by monoamine oxidase in recombinant Escherichia coli

Real-time PCR analysis showed that yggE gene was about two and three times up-regulated in Escherichia coli cells exposed to UVA irradiation and thermal elevation, respectively, suggesting that this gene is responsive to physiological stress. The yggE gene was introduced into E. coli BL21 cells, together with a monoamine oxidase (MAO) gene as a model source for oxidative stress generation. The distribution of independently isolated transformants (two dozen isolates) was examined in terms of MAO activity and cell vitality. In the case of control strain expressing MAO alone, the largest number of transformants existed in the low range of MAO activity less than 2 units mg⁻¹ and the number significantly decreased at increased MAO activity. On the other hand, the distribution of MAO/YggE-coexpressing transformants shifted to higher MAO activity with frequent appearance in the activity range of 4–8 units mg⁻¹. The yggE gene product therefore has a possible function for alleviating the stress generated in the cells.     

Arylalkylamine vanadium salts as new anti-diabetic compounds

Vanadium compounds show insulin-like effects in vivo and in vitro. Several clinical studies have shown the efficacy of vanadium compounds in type 2 diabetic subjects. However, a major concern is safety, which calls for the development of more potent vanadium compounds. For that reason different laboratories develop strategies to decrease the therapeutic dose of vanadate. One of these strategies use substrates of semicarbazide-sensitive amine oxidase (SSAO)/vascular adhesion protein-1 (VAP-1), a bifunctional protein with amine oxidase activity and adhesive properties implicated in lymphocyte homing at inflammation sites. Substrates of SSAO combined with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 glucose transporter recruitment to the plasma membrane in 3T3-L1 adipocytes and in rat adipocytes. This combination also shows anti-diabetic effects in various animal models of type 1 and type 2 diabetes. Benzylamine/vanadate administration generates peroxovanadium locally in pancreatic islets, which stimulates insulin secretion, and also produces peroxovanadium in adipose tissue, thereby activating glucose metabolism in adipocytes and in neighboring muscle. This opens up the possibility of using the SSAO/VAP-1 activity as a local generator of protein tyrosine phosphatase inhibitors in anti-diabetic therapy. More recently a novel class of arylalkylaminevanadium salts have shown potent insulin-mimetic effects downstream of the insulin receptor. Administration of these compounds lowers glycemia and normalizes the plasma lipid profile in type 1 and type 2 models of diabetes. The combination of different approaches to decrease vanadium doses, among them chelating agents and SSAO substrates, should permit to develop safe and efficient vanadium based agents safe for diabetes treatment.