Nano-sized manganese-calcium cluster in photosystem II

Cyanobacteria, algae, and plants are the manufacturers that release O₂ via water oxidation during photosynthesis. Since fossil resources are running out, researchers are now actively trying to use the natural catalytic center of water oxidation found in the photosystem II (PS II) reaction center of oxygenic photosynthetic organisms to synthesize a biomimetic supercatalyst for water oxidation. Success in this area of research will transcend the current bottleneck for the development of energy-conversion schemes based on sunlight. In this review, we go over the structure and function of the water-oxidizing complex (WOC) found in Nature by focusing on the recent advances made by the international research community dedicated to achieve the goal of artificial water splitting based on the WOC of PS II.     

Axenic cultivation of Entamoeba dispar in newly designed yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium

Yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium that allows axenic cultivation of Entamoeba dispar was designed based on casein-free yeast extract-iron-serum (YI-S) medium, and the usefulness of the medium was assessed. The main differences from YI-S medium are replacement of glucose by gluconic acid, addition of dihydroxyacetone and D-galacturonic acid monohydrate, and sterilization by filtration. This medium promoted the axenic growth of 5 strains of E. dispar (2 strains of nonhuman primate isolates and 3 strains of human isolates). In addition, to clarify the biological basis for the growth of E. dispar in this medium, analyses of relevant enzymes on the glycolytic pathway of the amoebae as well as of the protozoans that are the best culture supplement for amoebae are being performed.     

Regulation of cAMP homeostasis by the efflux protein MRP4 in cardiac myocytes

Recent studies indicate that members of the multidrug-resistance protein (MRP) family belonging to ATP binding cassette type C (ABCC) membrane proteins extrude cyclic nucleotides from various cell types. This study aimed to determine whether MRP proteins regulate cardiac cAMP homeostasis. Here, we demonstrate that MRP4 is the predominant isoform present at the plasma membrane of cardiacmyocytes and that it mediates the efflux of cAMP in these cells. MRP4-deficient mice displayed enhanced cardiac myocyte cAMP formation, contractility, and cardiac hypertrophy at 9 mo of age, an effect that was compensated transiently by increased phosphodiesterase expression at young age. These findings suggest that cAMP extrusion via MRP4 acts together with phosphodiesterases to control cAMP levels in cardiac myocytes.     

Human mutation in the anti-apoptotic heat shock protein 20 abrogates its cardioprotective effects

The small heat shock protein Hsp20 protects cardiomyocytes against apoptosis, and phosphorylation at its Ser16 site enhances its cardioprotection. To determine whether genetic variants exist in human Hsp20, which may modify these beneficial effects, we sequenced the coding region of the Hsp20 gene in 1347 patients suffering from dilated cardiomyopathy and 744 subjects with no heart disease. We identified a C59T substitution in the human Hsp20 gene in one patient and three individuals without heart disease. All subjects were heterozygous for this mutation, which changes a fully conserved proline residue into leucine at position 20 (P20L), resulting in secondary structural alterations. To examine the potential functional significance of the P20L-Hsp20 human variant, adult rat cardiomyocytes were infected with Ad.GFP (where Ad is adenovirus and GFP is green fluorescent protein), Ad.WT-Hsp20 (where WT is wild-type), and Ad.P20L-Hsp20 and subjected to simulated ischemia/reperfusion injury. Expression of WT-Hsp20 resulted in significant attenuation of apoptosis compared with the GFP control. However, the P20L-Hsp20 mutant showed no protection against apoptosis, assessed by Hoechst staining and DNA fragmentation. The loss of cardioprotection by the mutant Hsp20 was associated with its diminished phosphorylation at Ser16 compared with WT-Hsp20. Furthermore, maximal stimulation of cardiomyocytes with isoproterenol or protein kinase A-mediated phosphorylation in vitro confirmed the impaired ability of the mutant Hsp20 to become phosphorylated at Ser16. In conclusion, we have identified a P20L substitution in human Hsp20, which is associated with diminished phosphorylation at Ser16 and complete abrogation of the Hsp20 cardioprotective effects which may adversely affect the ability of human carriers to cope with cellular stress.     

Regulation of postsynaptic structure and protein localization by the Rho-type guanine nucleotide exchange factor dPix.

Mutations in dpix were recovered from a large-scale screen in Drosophila for genes that control synaptic structure. dpix encodes dPix, a Rho-type guanine nucleotide exchange factor (RtGEF) homologous to mammalian Pix. Here we show that dPix plays a major role in regulating postsynaptic structure and protein localization at the Drosophila glutamatergic neuromuscular junction. dpix mutations lead to decreased synaptic levels of the PDZ protein Dlg, the cell adhesion molecule Fas II, and the glutamate receptor subunit GluRIIA, and to a complete reduction of the serine/threonine kinase Pak and the subsynaptic reticulum. The electrophysiology of these mutant synapses is nearly normal. Many, but not all, dpix defects are mediated through dPak, a member of the family of Cdc42/Rac1-activated kinases. Thus, a Rho-type GEF and Rho-type effector kinase regulate postsynaptic structure.     

Serine phosphorylation-dependent coregulation of topoisomerase I by the p14ARF tumor suppressor

p14ARF (ARF) and topoisomerase I play central roles in cancer and have recently been shown to interact. The interaction activates topoisomerase I, an important target for camptothecin-like chemotherapeutic drugs, but the regulation of the interaction is poorly understood. We have used the H358 and H23 lung cancer cell lines and purified recombinant human topoisomerase I to demonstrate that the ARF/topoisomerase I interaction is regulated by topoisomerase I serine phosphorylation, a modification that regulates topoisomerase I activity. Both cell lines express wild-type ARF and topoisomerase I proteins at equivalent levels, but H23 topoisomerase I, unlike that of H358 cells, is largely devoid of serine phosphorylation, has low activity, and complexes poorly with ARF. The ability of H23 topoisomerase I to complex with ARF can be restored by treatment with the serine kinase, casein kinase II. Consistent with these observations, we show that the response of H23 cells to camptothecin treatment is unaffected by changes in intracellular levels of ARF. However, in H358 and PC-3 cells, which express a serine phosphorylated topoisomerase I that complexes with ARF, ectopic overexpression of ARF causes sensitization to camptothecin, and siRNA-mediated down-regulation of endogenous ARF causes desensitization to camptothecin. These biological responses correlate with increased and decreased levels, respectively, of ARF/topoisomerase I complex and DNA-bound topoisomerase I. Thus, ARF is a serine phosphorylation-dependent coregulator of topoisomerase I in vivo, and it regulates cellular sensitivity to camptothecin by interacting with topoisomerase I. Certain cancer associated defects affecting ARF/topoisomerase I complex formation could contribute to cellular resistance to camptothecin.     

Preferential platination of an activated cellular promoter by cis-diamminedichloroplatinum.

This study examines how accessibility to cisplatin on various genomic regions in T47D breast cancer cells, including the retinoic acid receptor beta gene promoter and coding region and the dihydrofolate reductase gene promoter and coding region, is affected by treatment of the cells with 9-cis retinoic acid, a treatment that activates the retinoic acid receptor beta gene promoter in these cells. A PCR-based assay was used to measure cisplatin adduct density based on the inhibition of PCR amplification of templates from cisplatin treated versus untreated cells. Treatment of cells with 9-cis retinoic acid enhanced accessibility to cisplatin on the retinoic acid receptor beta gene promoter region, but not on the coding regions of that gene nor on the dihydrofolate reductase gene promoter or coding regions, where accessibilities to cisplatin remained 2-4 times lower than on the activated retinoic acid receptor beta gene promoter. Examination of smaller regions within this promoter region showed a repression of platination in the 500 bp region surrounding the TATA box in cells prior to 9-cis retinoic acid treatment, which was abolished following promoter activation. Differences in sequence composition between the various regions could not fully account for differences in platination, suggesting that structural features such as bends in retinoic acid receptor beta gene promoter DNA following gene activation, create energetically favorable sites for platination, and contribute to the cytotoxicity of the drug.     

Evaluation of antigenotoxicity effects of umbelliprenin on human peripheral lymphocytes exposed to oxidative stress

The protective properties of a prenylated coumarin, umbelliprenin (UMB), on the human lymphocytes DNA lesions were tested. Lymphocytes were isolated from blood samples taken from healthy volunteers. DNA breaks and resistance to H₂O₂-induced damage were measured using a single-cell microgel electrophoresis technique under alkaline conditions (comet assay). Human lymphocytes were incubated in UMB (10, 25, 50, 100, 200, and 400 μM) alone or a combination of different concentrations of UMB (10, 25, 50, 100, 200, and 400 μM) and 25 μM H₂O₂. Untreated cells, ascorbic acid (AA; 25, 50, 100, 200, and 400 μM) and H₂O₂ (25 μM) were considered as negative control, positive control, and the standard antioxidant agent for our study, respectively. Single cells were analyzed with “TriTek Cometscore version 1.5” software. The DNA damage was expressed as percent tail DNA. UMB exhibited a concentration-dependent increase in protection activity against DNA damage induced by 25 μM H₂O₂ (from 67.28% to 39.17%). The antigenotoxic activity of AA, in the range 0–50 μM, was greater than that of UMB. However, no significant difference (p > 0.05) in the protective activity was found between UMB and AA at concentrations of approximately higher than 50 μM.     

Amperometric detection of insulin at renewable sol-gel derived carbon ceramic electrode modified with nickel powder and potassium octacyanomolybdate(IV)

A renewable three-dimensional chemically modified carbon ceramic electrode (CCE) containing nickel powder and K4[Mo(CN)8] was constructed by sol-gel technique. The electrochemical properties and stability of modified electrode was evaluated by cyclic voltammetry in pH range 4-10. The redox couple of [Mo(CN)8] (4-/3-) was shown both as a solute in electrolyte solution and as a component of a carbon based conducting composite electrode. The apparent electron transfer rate constant (ks) and transfer coefficient (alpha) were determined by cyclic voltammetry and they were about 17.1 and 0.57 s(-1), respectively. The catalytic activity of the modified CCE toward insulin oxidation was investigated at pH range of 3-8 by cyclic votammetry. The modified electrode showed excellent electrocatalytic activity toward insulin electroxidation at physiological pH value. The modified electrode was used for insulin detection chronoamperometrically at pH 7. Under optimized condition in amperometry method, the concentration calibration range, detection limit and sensitivity were 0.5-500 nM, 0.45 nM and 6140 nA/microM, respectively. Flow injection amperometric determination of insulin at pH 7.4, at this modified electrode yielded a calibration curve with the following characteristics, linear dynamic range 100-500 pM; sensitivity 8.1 nA/nM and detection limit 40 pM (based on S/N = 3). The inherent stability at wide pH range, high sensitivity, low detection limit, low cost and ease of preparation are of advantageous of this insulin sensor. This sensor indicates great promise for monitory insulin in chromatographic effluents.