Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Charge separation and energy transfer in the photosystem II core complex studied by femtosecond midinfrared spectroscopy

The core of photosystem II (PSII) of green plants contains the reaction center (RC) proteins D1D2-cytb559 and two core antennas CP43 and CP47. We have used time-resolved visible pump/midinfrared probe spectroscopy in the region between 1600 and 1800 cm(-1) to study the energy transfer and charge separation events within PSII cores. The absorption difference spectra in the region of the keto and ester chlorophyll modes show spectral evolution with time constants of 3 ps, 27 ps, 200 ps, and 2 ns. Comparison of infrared (IR) difference spectra obtained for the isolated antennas CP43 and CP47 and the D1D2-RC with those measured for the PSII core allowed us to identify the features specific for each of the PSII core components. From the presence of the CP43 and CP47 specific features in the spectra up to time delays of 20-30 ps, we conclude that the main part of the energy transfer from the antennas to the RC occurs on this timescale. Direct excitation of the pigments in the RC evolution associated difference spectra to radical pair formation of PD1+PheoD1- on the same timescale as multi-excitation annihilation and excited state equilibration within the antennas CP43 and CP47, which occur within approximately 1-3 ps. The formation of the earlier radical pair ChlD1+PheoD1-, as identified in isolated D1D2 complexes with time-resolved mid-IR spectroscopy is not observed in the current data, probably because of its relatively low concentration. Relaxation of the state PD1+PheoD1-, caused by a drop in free energy, occurs in 200 ps in closed cores. We conclude that the kinetic model proposed earlier for the energy and electron transfer dynamics within the D1D2-RC, plus two slowly energy-transferring antennas C43 and CP47 explain the complex excited state and charge separation dynamics in the PSII core very well. We further show that the time-resolved IR-difference spectrum of PD1+PheoD1- as observed in PSII cores is virtually identical to that observed in the isolated D1D2-RC complex of PSII, demonstrating that the local structure of the primary reactants has remained intact in the isolated D1D2 complex.     

Enantioselective metabolism of primaquine by human CYP2D6

Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated. Six diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (Hss LDH) or plasmodial lactate dehydrogenase (Pf LDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro. All tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of Pf LDH; specificity between the residues involved in H-bonds of the Pf LDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of Pf LDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive. The presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than Pf LDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).     

Identification of the first steps in charge separation in bacterial photosynthetic reaction centers of Rhodobacter sphaeroides by ultrafast mid-infrared spectroscopy: electron transfer and protein dynamics

Time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy in the region between 1600 and 1800 cm⁻¹ was used to investigate electron transfer, radical pair relaxation, and protein relaxation at room temperature in the Rhodobacter sphaeroides reaction center (RC). Wild-type RCs both with and without the quinone electron acceptor Q_A, were excited at 600 nm (nonselective excitation), 800 nm (direct excitation of the monomeric bacteriochlorophyll (BChl) cofactors), and 860 nm (direct excitation of the dimer of primary donor (P) BChls (P_L/P_M)). The region between 1600 and 1800 cm⁻¹ encompasses absorption changes associated with carbonyl (CO) stretch vibrational modes of the cofactors and protein. After photoexcitation of the RC the primary electron donor P excited singlet state (P*) decayed on a timescale of 3.7 ps to the state (where B_L is the accessory BChl electron acceptor). This is the first report of the mid-IR absorption spectrum of ; the difference spectrum indicates that the 9-keto CO stretch of B_L is located around 1670-1680 cm⁻¹. After subsequent electron transfer to the bacteriopheophytin H_L in ∼1 ps, the state was formed. A sequential analysis and simultaneous target analysis of the data showed a relaxation of the radical pair on the ∼20 ps timescale, accompanied by a change in the relative ratio of the and bands and by a minor change in the band amplitude at 1640 cm⁻¹ that may be tentatively ascribed to the response of an amide CO to the radical pair formation. We conclude that the drop in free energy associated with the relaxation of , is due to an increased localization of the electron hole on the P_L half of the dimer and a further consequence is a reduction in the electrical field causing the Stark shift of one or more amide CO oscillators.     

Reduced susceptibility to eccentric exercise-induced muscle damage in resistance-trained men is not linked to resistance training-related neural adaptations

The purpose of this study was to examine the acute effects of maximal concentric vs. eccentric exercise on the isometric strength of the elbow flexor, as well as the biceps brachii muscle electromyographic (EMG) responses in resistance-trained (RT) vs. untrained (UT) men. Thirteen RT men (age: 24 ± 4 years; height: 180.2 ± 7.7 cm; body weight: 92.2 ± 16.9 kg) and twelve UT men (age: 23 ± 4 years; height: 179.2 ± 5.0 cm; body weight: 81.5 ± 8.6 kg) performed six sets of ten maximal concentric isokinetic (CON) or eccentric isokinetic (ECC) elbow flexion exercise in two separate visits. Before and after the exercise interventions, maximal voluntary contractions (MVCs) were performed for testing isometric strength. In addition, bipolar surface EMG signals were detected from the biceps brachii muscle during the strength testing. Both CON and ECC caused isometric strength to decrease, regardless of the training status. However, ECC caused greater isometric strength decline than CON did for the UT group (p = 0.006), but not for the RT group. Both EMG amplitude and mean frequency significantly decreased and increased, respectively, regardless of the training status and exercise intervention. Resistance-trained men are less susceptible to eccentric exercise-induced muscle damage, but this advantage is not likely linked to the chronic resistance training-induced neural adaptations.