Effect of protein modification by malondialdehyde on the interaction between the oxygen-evolving complex 33 kDa protein and photosystem II core proteins

Previously we observed that the oxygen-evolving complex 33 kDa protein (OEC33) which stabilizes the Mn cluster in photosystem II (PSII), was modified with malondialdehyde (MDA), an end-product of peroxidized polyunsaturated fatty acids, and the modification increased in heat-stressed plants (Yamauchi et al. 2008). In this study, we examined whether the modification of OEC33 with MDA affects its binding to the PSII complex and causes inactivation of the oxygen-evolving complex. Purified OEC33 and PSII membranes that had been removed of extrinsic proteins of the oxygen-evolving complex (PSII∆OEE) of spinach (Spinacia oleracea) were separately treated with MDA. The binding was diminished when both OEC33 and PSII∆OEE were modified, but when only OEC33 or PSII∆OEE was treated, the binding was not impaired. In the experiment using thylakoid membranes, release of OEC33 from PSII and corresponding loss of oxygen-evolving activity were observed when thylakoid membranes were treated with MDA at 40°C but not at 25°C. In spinach leaves treated at 40°C under light, maximal efficiency of PSII photochemistry (F _v/F _m ratio of chlorophyll fluorescence) and oxygen-evolving activity decreased. Simultaneously, MDA contents in heat-stressed leaves increased, and OEC33 and PSII core proteins including 47 and 43 kDa chlorophyll-binding proteins were modified with MDA. In contrast, these changes were to a lesser extent at 40°C in the dark. These results suggest that MDA modification of PSII proteins causes release of OEC33 from PSII and it is promoted in heat and oxidative conditions.     

Virulence determinants in vancomycin-resistant <Emphasis Type="Italic">Enterococcus faecium van</Emphasis>A isolated from different sources at University Hospital of Londrina,Paraná, Brazil

Enterococcus faecium, especially those showing multidrug resistance, has emerged as a significant cause of healthcare-associated infections worldwide. However, relatively little is known about the virulence and pathogenesis of this species. The aim of this study was to determine the occurrence of four putative virulence determinants of E. faecium and to correlate them with phenotypic traits. Using forty E. faecium vanA-type isolates from hospitalized patients and their environmental vicinity, we determined the following: the antimicrobial susceptibility profile, occurrence of the genes cylA, efaA, esp, and gelE, hemolytic and gelatinase activities, capacity to form biofilm and in vitro adhesion to epithelial cells. All isolates were shown to be resistant to vancomycin and teicoplanin, as well as to two or more other antimicrobials. All isolates harbored at least one putative virulence marker, and the prevalence was as follows: esp, 87.5%; efaA, 82.5%; gelE, 70%; and cylA, 65%. The presence of 4 genes was observed in 32.5% isolates. The presence of the efaA was associated with the presence of esp, regardless of the source of the isolates. A positive association with the presence of cylA and hemolytic activity in the sheep blood agar assay was observed. No association was found for gelE and gelatinase production in the agar plate assay, for efaA and LLC-MK2 cell adhesion, and for esp and biofilm formation on polystyrene surface. These results show the presence of putative virulence genes in multiple antimicrobial resistant E. faecium isolates from different sources in a hospital setting.     

Synthesis and evaluation of novel phosphate ester analogs as neutral sphingomyelinase inhibitors

A novel sphingomyelin inhibitor RY221B-a, which contains a bipyridyl moiety as a metal coordination site was designed based upon the mechanism of phosphate ester hydrolysis. RY221B-a was synthesized from N-Boc-sphingosine in three steps via selective etherification using stannyl acetal. Synthesized RY221B-a exhibited relatively-strong inhibitory activity against Bc-SMase (IC₅₀ = 1.2 μM).     

Up-regulation of circadian clock gene Period 2 in the prostate mesenchymal cells during flutamide-induced apoptosis

Androgen regulates the proper development and physiological function of the prostate. Here, we investigated the modulation of androgen and androgen receptor (AR) antagonist on circadian oscillations of a clock core gene Period 2 (Per2) in rat prostate mesenchymal cells (PMCs). Circadian oscillations were analyzed with the real-time monitoring system of gene expression using transgenic rats introduced with mouse Per2 promoter fused to a destabilized luciferase (Per2-dLuc) reporter gene. Analyses of circadian oscillations, immunofluorescence, and androgen response element (ARE)-luciferase reporter assay revealed that circadian clocks are operative and the AR protein is functional in PMCs in vitro. Androgen such as testosterone (T) and dihydrotestosterone (DHT) did not cause any changes in circadian Per2-dLuc oscillations of confluent cells. Conversely, flutamide (FL) up-regulated the amplitude of circadian Per2-dLuc oscillations in a dose-dependent manner, whereas T antagonized the action of FL. The PER2 protein was markedly accumulated by FL treatment and localized in both the nucleus and cytoplasm during the first peak period of circadian Per2-dLuc oscillations. Simultaneously, FL treatment increased apoptotic cell death. Collectively, the present study demonstrates that a clock gene Per2 is up-regulated in PMCs during FL-induced apoptotic cell death. Thus, circadian oscillations of Per2 gene expression may be closely linked to the cellular states of PMCs such as apoptotic cell death.     

Oxidative stress in mature rat testis and its developmental changes

Active oxygen causes various problems including male infertility through the oxidation of DNA, proteins, and lipids. In the present study, we examined the immunohistochemical localization of molecules involved in oxidative stress including 8-hydroxy-2-deoxyguanosine (8-OHdG), superoxide dismutase (SOD), and protein disulfide isomerase (PDI) in mature and developing rat testes. In mature rat testes, 8-OHdG was detected in leptotene, zygotene, and early pachytene spermatocytes, while its expression was weak in late pachytene stage spermatocytes. On the other hand, SOD was detected in late pachytene spermatocytes but not in early pachytene and former spermatocytes, suggesting the efficient removal of active oxygen by SOD in late pachytene spermatocytes. In developing rat testes, 8-OHdG expression peaked at 4 weeks when spermatocytes started to differentiate to the late pachytene stage, while SOD started to be expressed at 4 weeks after birth. These findings suggest that the defense system against oxidative stress by SOD is developed in late pachytene stage spermatocytes at 4 weeks after birth. The present findings aid our understanding of the defensive mechanism against oxidative stress in developing and mature testes.     

Effects of buspirone on posthypoxic ventilatory behavior in the C57BL/6J and A/J mouse strains.

Buspirone, a partial agonist of the serotonergic 5-HT1A receptor, improves breathing irregularities in humans with Rett syndrome or brain stem injury. The purpose of this study was to examine whether buspirone alters posthypoxic ventilatory behavior in C57BL/6J (B6) and A/J mouse strains. Measurements of ventilatory behavior were collected from unanesthetized adult male mice (n=6 for each strain) using the plethysmographic method. Mice were given intraperitoneal injections of vehicle or several doses of buspirone and exposed to 2 min of hypoxia (10% O2) followed by rapid reoxygenation (100% O2). Twenty minutes later, mice were tested for hypercapnic response (8% CO(2)-92% O2). On a separate day, mice were injected with the 5-HT1A receptor antagonist 4-iodo-N-{2-[4-(methoxyphenyl)-1-piperazinyl] ethyl}-N-2-pyridinylbenzamide (p-MPPI) before the injection of buspirone, and measurements were repeated. In separate studies, arterial blood-gas analysis was performed for each strain (n=12 in B6 and 10 in A/J) with buspirone or vehicle. In both strains, buspirone stimulated ventilation at rest. In the B6 mice, the hypoxic response was unchanged, but the response to hypercapnia was reduced with buspirone (5 mg/kg; P<0.05). With reoxygenation, vehicle-treated B6 exhibited periodic breathing and greater variation in ventilation compared with A/J (P<0.01). In B6 animals, >or=3 mg/kg of buspirone reduced variation and prevented the occurrence of posthypoxic periodic breathing. Both effects were reversed by p-MPPI. Treatment effect of buspirone was not explained by a difference in resting arterial blood gases. We conclude that buspirone improves posthypoxic ventilatory irregularities in the B6 mouse through its agonist effects on the 5-HT1A receptor.     

Observation of an isotope-sensitive low-frequency Raman band specific to metmyoglobin

A resonance Raman band involving significantly the iron(III)-histidine stretching (upsilonFe-His) character is identified for metmyoglobin (metMb) through isotope sensitivity of its low-frequency resonance Raman bands, but the identification was not successful for methemoglobin (metHb) and its isolated alpha and beta subunits. A band at 218 cm-1 of natural abundance metMb exhibited a low-frequency shift for 15N-His-labeled metMb (-1.4 cm-1 shift), while the strong porphyrin bands at 248 and 271 cm-1 did not shift significantly. The frequency of the 218-cm-1 band of metMb decreased by 1.6 cm-1 in D2O, probably due to Ndelta-deuteration of the proximal His, in a similar manner to that of the upsilonFe-His band of deoxyMb in D2O. This 218-cm-1 band shifted slightly to a lower frequency in H2(18)O, whereas it did little upon 54Fe isotopic substitution (<0.3 cm-1), presumably because of the six-coordinate structure. The lack of the 54Fe-isotope shift shows that the 218-cm-1 band is specific to metMb and not due to the deoxy species. The intensity of this band decreased for hydroxymetMb and was indiscernible for cyanometMb. For metHb and its alpha and beta subunits, however, the frequencies of the band around 220 cm-1 were not D2O sensitive. These results suggest an assignment of the band around 220 cm-1 to a pyrrole tilting mode, which significantly contains the Fe-His stretching character for metMb but scarcely for metHb and its subunits. The differences in the isotope sensitivity of this band in different proteins are considered to reflect the heme distortion from the planarity and the Fe-His geometry specific to individual proteins.     

Synthesis and characterization of chromium(III) octaphenylporphyrin complexes with various axial ligands: An insight into porphyrin distortion

Influence of axial ligands, MeCN, H₂O, py, and piperidine (pip), on distortion of (2,3,5,10,12,13,15,20-octaphenylporphinato)chromium(III), [Cr(OPP)]⁺, was investigated by X-ray crystallography and UV-vis and ESR spectroscopies. In crystal structures of [Cr(OPP)(MeCN)(H₂O)]ClO₄ · MeCN and [Cr(OPP)(H₂O)₂]ClO₄ · 3THF, the OPP²⁻ ligand had a planar structure. On the other hand, crystal structures of [Cr(OPP)(pip)₂]ClO₄ · 2CH₂Cl₂ and [Cr(OPP)(py)₂]ClO₄ exhibited a waved structure and a saddle-shaped structure with ruffling, respectively. In the UV-vis spectrum of [Cr(OPP)(py)₂]ClO₄ in CH₂Cl₂, the large red shift of the Soret band was observed. Furthermore, the small D value of 0.10 cm⁻¹ was obtained for [Cr(OPP)(py)₂]ClO₄ from the ESR spectrum in frozen 1,2-dichloroethane solution at 5 K. These results suggest that the OPP²⁻ ligand is distorted both in solid and in solution, and that the axial ligand would exert some effects on the porphyrin distortion.     

Different patterns of 5alpha-reductase expression, cellular distribution, and testosterone metabolism in human follicular dermal papilla cells

Androgens regulate hair growth, and 5α-reductase (5αR) plays a pivotal role in the action of androgens on target organs. To clarify the molecular mechanisms responsible for controlling hair growth, the present study presents evidence that the human follicular dermal papilla cells (DPCs) from either beard (bDPCs) or scalp hair (sDPCs) possess endogenous 5αR activity. Real-time RT-PCR revealed that the highest level of 5αR1 mRNA was found in bDPCs, followed by sDPCs, and a low but detectable level of 5αR1 mRNA was observed in fibroblasts. Minimally detectable levels of 5αR2 mRNA were found in all three cell types. A weak band at 26 kDa corresponding to the human 5αR1 protein was detected by Western blot in both DPCs, but not in fibroblasts. Immuonofluorescence analysis confirmed that 5αR1 was localized to the cytoplasm rather than in the nuclei in both DPCs Furthermore, a 5αR assay using [¹⁴C]testosterone labeling in intact cells revealed that testosterone was transformed primarily into androstenedione, and in small amounts, into DHT. Our results demonstrate that the 5αR activities of either bDPCs or sDPCs are stronger than that of dermal fibroblasts, despite the fact that the major steroidogenic activity is attributed to 17β-HSD rather than 5αR among the three cell types. The 5αR1 inhibitor MK386 exhibited a more potent inhibitory effect on 5αR activity than finasteride (5αR2 inhibitor) in bDPCs.