New unsymmetric dinuclear Cu(II)Cu(II) complexes and their relevance to copper(II) containing metalloenzymes and DNA cleavage

The new homodinuclear complexes, [Cu(2)(II)(HLdtb)(mu-OCH(3))](ClO(4))(2) (1) and [Cu(2)(II)(Ldtb)(mu-OCH(3))](BPh(4)) (2), with the unsymmetrical N(5)O(2) donor ligand (H(2)Ldtb) - {2-[N,N-Bis(2-pyridylmethyl)aminomethyl]-6-[N',N'-(3,5-di-tert-butylbenzyl-2-hydroxy)(2-pyridylmethyl)]aminomethyl}-4-methylphenol have been synthesized and characterized in the solid state by X-ray crystallography.In both cases the structure reveals that the complexes have a common {Cu(II)(mu-phenoxo)(mu-OCH(3))Cu(II)} structural unit.Magnetic susceptibility studies of 1 and 2 reveal J values of -38.3 cm(-1) and -2.02 cm(-1), respectively, and that the degree of antiferromagnetic coupling is strongly dependent on the coordination geometries of the copper centers within the dinuclear {Cu(II)(mu-OCH(3))(mu-phenolate)Cu(II)} structural unit.Solution studies in dichloromethane, using UV-Visible spectroscopy and electrochemistry, indicate that under these experimental conditions the first coordination spheres of the Cu(II) centers are maintained as observed in the solid state structures, and that both forms can be brought into equilibrium ([Cu(2)(HLdtb)(mu-OCH(3))](2+)=[Cu(2)(Ldtb)(mu-OCH(3))](+)+H(+)) by adjusting the pH with Et(3)N (Ldtb(2-) is the deprotonated form of the ligand).On the other hand, potentiometric titration studies of 1 in an ethanol/water mixture (70:30 V/V; I=0.1M KCl) show three titrable protons, indicating the dissociation of the bridging CH(3)O(-) group.The catecholase activity of 1 and 2 in methanol/water buffer (30:1 V/V) demonstrates that the deprotonated form is the active species in the oxidation of 3,5-di-tert-butylcatechol and that the reaction follows Michaelis-Menten behavior with k(cat)=5.33 x 10(-3)s(-1) and K(M)=3.96 x 10(-3)M. Interestingly, 2 can be electrochemically oxidized with E(1/2)=0.27 V vs.Fc(+)/Fc (Fc(+)/Fc is the redox pair ferrocinium/ferrocene), a redox potential which is believed to be related to the formation of a phenoxyl radical.Since these complexes are redox active species, we analyzed their activity toward the nucleic acid DNA, a macromolecule prone to oxidative damage.Interestingly these complexes promoted DNA cleavage following an oxygen dependent pathway.     

Photosynthesis research in Italy: a review

This historical review was compiled and edited by Giorgio Forti, whereas the other authors of the different sections are listed alphabetically after his name, below the title of the paper; they are also listed in the individual sections. This review deals with the research on photosynthesis performed in several Italian laboratories during the last 50 years; it includes research done, in collaboration, at several international laboratories, particularly USA, UK, Switzerland, Hungary, Germany, France, Finland, Denmark, and Austria. Wherever pertinent, references are provided, especially to other historical papers in Govindjee et al. [Govindjee, Beatty JT, Gest H, Allen JF (eds) (2005) Discoveries in Photosynthesis. Springer, Dordrecht]. This paper covers the physical and chemical events starting with the absorption of a quantum of light by a pigment molecule to the conversion of the radiation energy into the stable chemical forms of the reducing power and of ATP. It describes the work done on the structure, function and regulation of the photosynthetic apparatus in higher plants, unicellular algae and␣in photosynthetic bacteria. Phenomena such as photoinhibition and the protection from it are also included. Research in biophysics of photosynthesis in Padova (Italy) is discussed by G.M. Giacometti and G.␣Giacometti (2006).     

A versatile and sensitive tritium-based radioassay for measuring hydrogenase activity in aquatic sediments

We present a method for the measurement of hydrogenase (H(2)ase) activity in aquatic sediments. The assay is based on the H(2)ase-mediated isotopic exchange between dissolved molecular hydrogen (H(2)) and water. A slurry of sediment material is incubated with a tritiated hydrogen (HT) headspace in a glass syringe on a rotary shaker. The method includes a procedure for preparing HT from radiolabeled sodium borohydride, which is a useful alternative to purchasing HT directly. A method for measuring HT specific activity based on liquid scintillation counting is also presented. Validation tests were run using live and frozen cultures of Clostridium pasteurianum and Desulfovibrio vulgaris, and freshly collected marine sediments. Adherence to Michaelis-Menten kinetics was demonstrated. An interassay coefficient of variation of 15% was determined using frozen C. pasteurianum cultures as reference material. Serial dilutions of cultures and sediments showed that measured H(2)ase activity scales with cell concentration, and indicate that the method can detect C. pasteurianum cell concentrations of between 300 and 3000 cells/ml. This technique allows measurement of H(2)ase activity in a variety of environmental samples, and will be particularly useful in the study of deep marine sediments with low microbial activity.     

Regulation of aldosterone production from zona glomerulosa cells by ANG II and cAMP: evidence for PKA-independent activation of CaMK by cAMP

Aldosterone production in zona glomerulosa (ZG) cells of adrenal glands is regulated by various extracellular stimuli (K(+), ANG II, ACTH) that all converge on two major intracellular signaling pathways: an increase in cAMP production and calcium (Ca(2+)) mobilization. However, molecular events downstream of the increase in intracellular cAMP and Ca(2+) content are controversial and far from being completely resolved. Here, we found that Ca(2+)/calmodulin-dependent protein kinases (CaMKs) play a predominant role in the regulation of aldosterone production stimulated by ANG II, ACTH, and cAMP. The specific CaMK inhibitor KN93 strongly reduced ANG II-, ACTH-, and cAMP-stimulated aldosterone production. In in vitro kinase assays and intact cells, we could show that cAMP-induced activation of CaMK, using the adenylate cyclase activator forskolin or the cAMP-analog Sp-5,6-DCI-cBIMPS (cBIMPS), was not mediated by PKA. Activation of the recently identified cAMP target protein Epac (exchange protein directly activated by cAMP) by 8-pCPT-2'-O-Me-cAMP had no effect on CaMK activity and aldosterone production. Furthermore, we provide evidence that cAMP effects in ZG cells do not involve Ca(2+) or MAPK signaling. Our results suggest that ZG cells, in addition to PKA and Epac/Rap proteins, contain other as yet unidentified cAMP mediator(s) involved in regulating CaMK activity and aldosterone secretion.     

Effects of a 50 Hz magnetic field on Dictyostelium discoideum (Protista)

Some studies have demonstrated that a few biological systems are affected by weak, extremely low frequency (ELF) electromagnetic fields (EMFs), lower than 10 mT. However, to date there is scanty evidence of this effect on Protists in the literature. Due to their peculiarity as single-cell eukaryotic organisms, Protists respond directly to environmental stimuli, thus appearing as very suitable experimental systems. Recently, we showed the presence of propionylcholinesterase (PrChE) activity in single-cell amoebae of Dictyostelium discoideum. This enzyme activity was assumed to be involved in cell-cell and cell-environment interactions, as its inhibition affects cell aggregation and differentiation. In this work, we have exposed single-cell amoebae of D. discoideum to an ELF-EMF of about 200 microT, 50 Hz, for 3 h or 24 h at 21 degrees C. A delay in the early phase of the differentiation was observed in 3 h exposed cells, and a significant decrease in the fission rate appeared in 24 h exposed cells. The PrChE activity was significantly lower in 3 h exposed cells than in the controls, whereas 24 h exposed cells exhibited an increase in this enzyme activity. However, such effects appeared to be transient, as the fission rate and PrChE activity values returned to the respective control values after a 24 h stay under standard conditions.     

New functions of XPC in the protection of human skin cells from oxidative damage

Xeroderma pigmentosum (XP) C is involved in the recognition of a variety of bulky DNA-distorting lesions in nucleotide excision repair. Here, we show that XPC plays an unexpected and multifaceted role in cell protection from oxidative DNA damage. XP-C primary keratinocytes and fibroblasts are hypersensitive to the killing effects of DNA-oxidizing agents and this effect is reverted by expression of wild-type XPC. Upon oxidant exposure, XP-C primary keratinocytes and fibroblasts accumulate 8,5'-cyclopurine 2'-deoxynucleosides in their DNA, indicating that XPC is involved in their removal. In the absence of XPC, a decrease in the repair rate of 8-hydroxyguanine (8-OH-Gua) is also observed. We demonstrate that XPC-HR23B complex acts as cofactor in base excision repair of 8-OH-Gua, by stimulating the activity of its specific DNA glycosylase OGG1. In vitro experiments suggest that the mechanism involved is a combination of increased loading and turnover of OGG1 by XPC-HR23B complex. The accumulation of endogenous oxidative DNA damage might contribute to increased skin cancer risk and account for internal cancers reported for XP-C patients.     

The Drosophila inhibitor of apoptosis protein DIAP2 functions in innate immunity and is essential to resist gram-negative bacterial infection

The founding member of the inhibitor of apoptosis protein (IAP) family was originally identified as a cell death inhibitor. However, recent evidence suggests that IAPs are multifunctional signaling devices that influence diverse biological processes. To investigate the in vivo function of Drosophila melanogaster IAP2, we have generated diap2 null alleles. diap2 mutant animals develop normally and are fully viable, suggesting that diap2 is dispensable for proper development. However, these animals were acutely sensitive to infection by gram-negative bacteria. In Drosophila, infection by gram-negative bacteria triggers the innate immune response by activating the immune deficiency (imd) signaling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the pathway of mammalian tumor necrosis factor receptor 1 (TNFR1). diap2 mutant flies failed to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumbed to bacterial infection. Our genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1, and Relish. Therefore, DIAP2 functions in the host immune response to gram-negative bacteria. In contrast, we find that the Drosophila TNFR-associated factor (Traf) family member Traf2 is dispensable in resistance to gram-negative bacterial infection. Taken together, our genetic data identify DIAP2 as an essential component of the Imd signaling cascade, protecting the organism from infiltrating microbes.     

Queen-worker differences in spermatheca reservoir of phylogenetically basal ants

Ant queens mate when young and store sperm in their spermatheca to fertilize eggs for several years until their death. In contrast, workers in most species never mate. We have compared the histological organization of spermathecae in 25 poneromorph species exhibiting various degrees of queen-worker dimorphism. The spermathecae of both castes in all species are similar in having a reservoir connected by a sperm duct to the ovary, and a paired gland opening into this duct. The reservoir of queens typically has a columnar epithelium in the hilar region (near the opening of the sperm duct), whereas the epithelium in the distal region is cuboidal. Abundant mitochondria together with apical microvilli and basal invaginations indicate an osmoregulatory function. In contrast, the reservoir epithelium of workers is flattened throughout and lacks these transport characteristics. This single difference shows the importance of a columnar epithelium in the reservoir for sperm storage. However, our data have not revealed inter-specific variations in the development of the hilar region linked with higher fecundity. We have found no consistent differences in associated structures, such as the spermatheca gland or sperm ducts, or in the musculature between queens and workers.This work was funded by IWT, FWO, KULeuven OT and JSPS.