The NMR structure of FliK, the trigger for the switch of substrate specificity in the flagellar type III secretion apparatus

The flagellar cytoplasmic protein FliK controls hook elongation by two successive events: by determining hook length and by stopping the supply of hook protein. These two distinct roles are assigned to different parts of FliK: the N-terminal half (FliK_N) determines length and the C-terminal half (FliK_C) switches secretion from the hook protein to the filament protein. The interaction of FliK_C with FlhB, the switchable secretion gate, triggers the switch. By NMR spectroscopy, we demonstrated that FliK is largely unstructured and determined the structure of a compact domain in FliK_C. The compact domain, denoted the FliK_C core domain, consists of two α-helices, a β-sheet with two parallel and two antiparallel strands, and several exposed loops. Based on the functional data obtained by a series of deletion mutants of the FliK_C core domain, we constructed a model of the complex between the FliK_C core domain and FlhB_C. The model suggested that one of the FliK_C loops has a high probability of interacting with the C-terminal domain of FlhB (FlhB_C) as the FliK molecule enters the secretion gate. We suggest that the autocleaved NPTH sequence in FlhB contacts loop 2 of FliK_C to trigger the switching event. This contact is sterically prevented when NPTH is not cleaved. Thus, the structure of FliK provides insight into the mechanism by which this bifunctional protein triggers a switch in the export of substrates.     

Correspondences in the Behavior of the Electroretinogram and of the Potentials Evoked at the Visual Cortex

Electrical potentials from the eye (ERG) and from the contralateral visual cortex were recorded in response to flashes of white and of colored light of various intensities and durations. The evoked potentials were found to parallel the behavior of the ERG in several significant respects. Selective changes in the ERG brought about by increasing the light intensity and by light adaptation led to parallel selective changes in the cortical responses. The dual waves (b₁, b₂) of the ERG were found to have counterparts in two cortical waves (c₁, c₂) which, in respect to changes in light intensity and to light adaptation, behaved analogously to the two retinal components. The responses evoked at high intensity showed only the diphasic c₁-potential. As stimulus intensity was lowered the c₁-wave decreased in magnitude and a delayed c₂-component appeared. The c₂-potential increased in amplitude as light intensity of the flash was further reduced. Eventually the c₂-wave, too, decreased as stimulus reduction continued. There was no wave length specificity in regard to either the duplex b-waves or duplex cortical waves. Both appeared at all wave lengths from 454 mµ to 630 mµ. The two cortical waves evoked by brief flashes of colored light showed all the behavior to changes in stimulus intensity and to light adaptation that occurred with white light.     

Kinetics of electron transfer between the primary and the secondary electron acceptor in reaction centers from Rhodopseudomonas sphaeroides

Photoreduction of the two ubiquinone molecules, UQ₁ and UQ₂, bound to purified reaction center from Rhodopseudomonas sphaeroides induces different absorption band shifts of bacteriochlorophyll and bacteriopheophytin molecules depending on which ubiquinone is photoreduced. This allows us to study electron transfer between UQ₁ and UQ₂ directly by absorption spectrometry. The results support a model in which electrons are transferred one by one from UQ₁ to UQ₂ with a half-time of 200 μs, and two by two from fully reduced UQ₂ to the secondary acceptor pool.     

The effect of exogenous phosphate deficiency on the activity of acid phosphatase of the root of two maize genotypes

In two maize genotypes, the effect of exogenous phosphate deficiency on acid phosphatase activity of the apical part of primary root was followed in dry and imbided grains. Higher acid phosphatase activity was found in the genotype LG 5. The enzyme activity increased after 18 h grain imbibition in the two genotypes. After 48 h germination no differences were found between the genotypes. After 24 h cultivation of root segments in a solution of 0.25 mM CaSO₄.2H₂O,0.1 mM MgSO₄.7H₂O, and further after 3,6 and 24 h cultivation in solutions with and without phosphate genotypic differences were found in acid phosphatase activity as well as in dry mass production. An increase in enzymatic activity due to exogenous phosphate deficiency was registered in the two genotypes only after 24 h cultivation.     

An FTIR study on the structure of the oxygen-evolving Mn-cluster of Photosystem II in different spin forms of the S2 state

The S₂ state of the oxygen-evolving Mn-cluster of Photosystem II (PS II) is known to have different forms that exhibit the g =2 multiline and g = 4.1 EPR signals. These two spin forms are interconvertible at > 200 K and the relative amplitudes of the two signals are dependent on the species of cryoprotectant and alcohol contained in the medium. Also, it was recently found that the mutiline form can be converted to the g = 4.1 form by absorption of near-infrared light by the Mn-cluster itself at around 150 K [Boussac et al. (1996) Biochemistry 35: 6984–6989]. We have used light-induced Fourier transform infrared (FTIR) difference spectroscopy to study the structural difference in these two S₂ forms. FTIR difference spectra for S₂/S₁ as well as for S₂Q_A ^-/S₁Q_A measured at cryogenic temperatures using PS II membranes in the presence of various cryoprotectants, and monohydric alcohols did not show any specific differences except for intensities of amide I bands, which were larger when ethylene glycol or glycerol was present in addition to sucrose. This result was interpreted due to more flexible movement of the protein backbones upon S₂ formation with a higher cryoprotectant content. Light-induced difference spectra measured at 150 K using either blue light without near-infrared light or red plus near-infrared light also did not show any detectable difference. In addition, a different spectrum upon near-infrared illumination at 150 K of the PS II sample in which the S₂ state had been photogenerated at 200 K exhibited no meaningful signals. These results indicate that the two S₂ forms that give rise to the multiline and g = 4.1 signals have only minor differences, if any, in the structures of amino-acid ligands and polypeptide backbones. This conclusion suggests that conversion between the two spin states is caused by a spin-state transition in the Mn(III) ion rather than valence swapping within the Mn-cluster that would considerably affect the vibrations of ligands.This revised version was published online in October 2005 with corrections to the Cover Date.     

Modulation of the Chaperone DnaK Allosterism by the Nucleotide Exchange Factor GrpE

Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70_NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70_SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaK_NBD, with ATP inducing the open, substrate-receptive DnaK_SBD conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaK_NBD. DnaK_SBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaK_NBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaK_SBD near DnaK_NBD. This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK-GrpE complex bends and its tip contacts DnaK_SBD, whereas the DnaK_NBD-DnaK_SBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.     

Chemical structure of the ether lipids of thermophilic acidophilic bacteria of the Caldariella group

The lipids of the Caldariella group of extremely thermophilic acidophilic bacteria are based on a 72-membered macrocyclic tetraether made up from two C₄₀ diol units and either two glycerol units or one glycerol and one nonitol. The C₄₀ components have the 16,16′-biphytanyl skeleton and the detailed structure of three of them is established.     

Identification of immunogenic determinants of the spike protein of SARS-like coronavirus

Bat SARS-like coronavirus (SL-CoV) has a genome organization almost identical to that of SARS-CoV, but the N-terminus of the Spike (S) proteins, which interacts with host receptor and is a major target of neutralizing antibodies against CoVs, of the two viruses has only 63–64% sequence identity. Although there have been reports studying the overall immunogenicity of S_SL, knowledge on the precise location of immunodominant determinants for S_SL is still lacking. In this study, using a series of truncated expressed S_SL fragments and S_SL specific mouse sera, we identified two immunogenic determinants for S_SL. Importantly, one of the two regions seems to be located in a region not shared by known immunogenic determinants of the S_SARS. This finding will be of potential use in future monitoring of SL-CoV infection in bats and spillover animals and in development of more effective vaccine to cover broad protection against this new group of coronaviruses.     

A2 (N2) Neuraminidase of the X-7 Influenza Virus Recombinant: Determination of Molecular Size and Subunit Composition of the Active Unit

Neuraminidase activity of influenza virus was directly seen on sodium dodecyl sulfate polyacrylamide gels with the aid of the synthetic substrate, methoxyphenol neuraminic acid. Neuraminidase (NA) appeared as a high-molecular-weight fraction with a size in the range of 220,000 to 250,000 daltons. Isolation of this fraction from the X-7 strain of influenza virus, dissociation with sodium dodecyl sulfate, and reduction showed the presence of two polypeptides of 66,000 (NA₁) and 58,000 (NA₂) molecular weights in equimolar concentration. We postulate that the minimum active unit for the viral A₂ neuraminidase is a tetramer composed of two NA₁ and two NA₂ subunits.     

Production of monoclonal antibodies for the detection of potato virus Y

Monoclonal antibodies (McAb) were obtained to potato virus Y (PVY) after immunisation of BALB/c mice with purified PVY, tobacco necrotic strain (PVY_n). Spleen cells from a mouse showing a high serum titre were used for fusion with X63NS1 myeloma cells. Hybridomas were selected in medium containing HAT. Culture supernatants were screened for antibody production against PVY, ordinary strain (PVY₀) and PVY_n using indirect ELISA. Clones of interest were further cross-reacted with 12 isolates each of PVY₀ and PVY_n and two isolates of potato virus A (PVA) and healthy sap. For further trials, two clones which reacted specifically with PVY_n isolates and one which detected all PVY isolates except two of potato virus C (PVC) were selected.     

Characterisation of neuropeptides of the AKH/RPCH-family from corpora cardiaca of Coleoptera

Summary Extracts of corpora cardiaca from two members of the family Tenebrionidae,Zophobas rugipes andTenebrio molitor, from one member of the Chrysomelidae,Leptinotarsa decemlineata, and from three members of the Scarabaeidae,Pachnoda marginata, P. sinuata andMelolontha hippocastani, were assayed for adipokinetic and hypertrehalosaemic activity in acceptor locusts (Locusta migratoria) and cockroaches (Periplaneta americana), respectively. All corpus cardiacum material tested, except that from the cockchafer,M. hippocastani, gave positive bioassay results. Biological activities of corpus cardiacum extracts from all species investigated can be resolved on reversed-phase high performance liquid chromatography (RP-HPLC). Gland extracts from the two tenebrionid species each show a single peak of biological activity associated with a single peak of UV absorbance having an identical retention time in both species. The two biologically active fractions from the corpora cardiaca of the potato beetle,L. decemlineata, coelute with exogenous (synthetic) hypertrehalosaemic hormones I and II of the American cockroach. The two species of the genusPachnoda contain two active compounds in their glands; compound I of each species is more abundant and elutes just ahead of the (synthetic) hypertrehalosaemic hormone of the cockroachBlaberus discoidalis. The gland material ofM. hippocastani exhibits and absorbance peak with the same retention time as the major peak from thePachnoda-species; however, this peak material does not elicit biological activity in the assays used here. After fractionation by RP-HPLC the main biologically active compounds were subjected to amino acid analyses. All factors are peptidic and contain 8 amino acid residues. The peptides from the tenebrionid species have the amino acid residues Asx₍₂₎, Glx₍₁₎, Ser₍₁₎, Pro₍₁₎, Leu₍₁₎, Phe₍₁₎ and Trp_(i), whereas the main peptide from corpora cardiaca ofP. marginata contains the residues Asx₍₂₎, Glx₍₁₎, Ser₍₁₎, Pro₍₁₎, Tyr₍₁₎, Leu₍₁₎ and Trp₍₁₎. Amino acid composition analyses of the two active fractions fromL. decemlineata reveal the residues Asx₍₂₎, Glx₍₁₎, Ser₍₁₎, Pro₍₁₎, Val₍₁₎, Phe₍₁₎ and Trp₍₁₎ for compound I and Asx₍₁₎, Glx₍₁₎, Thr₍₂₎, Pro₍₁₎, Leu₍₁₎, Phe₍₁₎ and Trp₍₁₎ for compound II.     

Evolution of the multidomain protein wheat germ agglutinin

Summary We compared the homologous amino acid sequences of hevein and each of the four domains (A, B, C, and D) of wheat germ agglutinin and used them to construct a pseudophylogenetic tree relating these sequences to a hypothetical common ancestor sequence. In the crystal structure of the wheat germ agglutinin dimer, six pseudo-twofold rotational symmetry axes have previously been located in addition to the true twofold axis. Four of these relate two nonidentical domains to each other in each of the four possible pairs constituting the sugar-binding sites (A₁D₂, A₂D₁, B₁C₂, and B₂C₁). The remaining two relate contiguous unique pairs of sugar-binding sites to each other (A₁D₂ to B₁C₂, and A₂D₁ to B₂C₁). These latter two sets of pairs are related to each other by the true twofold axis. Side chains that mediate sugar binding in the interfaces of each of the four pairs were found to be largely conserved. The sequence homology, taken together with these pseudo-symmetry elements in the dimer structure, suggests a pathway for the evolution of the four-domain molecule from a single-domain dimer that can be correlated with simultaneous development of the saccharide-binding sites.     

Characterization of cholinesterase molecular forms in the mucosal cells along the intestine of the chicken

The presence of a butyrylcholinesterase (BuChE, EC 3.1.1.8) in the musocal cells of the chicken intestine was demonstrated by histochemical and biochemical methods. The study of its distribution, along the intestine from duodenum to rectum, showed that the jejuno-ileum possesses the highest activity. Sucrose gradient centrifugation revealed, in all intestinal areas, two globular forms with sedimentation coefficients of 4.3 S (G₁ form) and 10.8 S (G₄ form). The presence of Triton X-100 in the preparations did not modify the sedimentation profiles of these two forms which can be considered as soluble BuChE. The ratio of G₁/G₄-forms progressively decreases along the intestine from duodenum to rectum indicating a predominance of the G₄ form in the areas where the activity is low. Our results are discussed in relation to other studies of globular forms of chicken BuChE.Abbreviations AchE Acetylcholinesterase - BuChe Butyrylcholinesterase - LSS Low-Salt-Soluble - DS Detergent-Soluble - HSS High-Salt-Soluble     

Localization of the violet and yellow receptor cells in the crayfish retinula

Cellular identification of color receptors in crayfish compound eyes has been made by selective adaptation at 450 nm and 570 nm, wavelengths near the λ_max''s of the two retinular cell classes previously demonstrated. By utilizing earlier evidence, the concentration of lysosome-related bodies (LRB) was used to measure relative light adaptation and thus wavelength sensitivity in 665 retinular cells from six eyes. The observed particle distributions demonstrate the following. Both violet and yellow receptors occur ordinarily in each retinula. Of the seven regular retinular cells two (R₃ and R₄ using Eguchi''s numbering [1965]) have mean sensitivities significantly greater to violet and less to yellow than the other five. The latter apparently comprise "pure" yellow receptors (R₁ and R₇) and mixed yellow and violet receptors (R₂, R₅, and R₆). Explanations of such ambiguity requiring two visual pigments in single retinular cells or intercellular coupling of adjacent neuroreceptors are apparently precluded by previous evidence. Present data imply alternatively some positional variability in the violet pair''s location in individual retinulas. Thus R₃ and R₄ are predominantly the violet receptors but in some retinulas R₂ and R₃ or R₄ and R₅ (or rarely some other cell pairs) may be. The retinal distribution of such variations has yet to be determined. In agreement with intracellular recordings the blue and yellow cells here identified belong to both the vertical and horizontal e-vector sensitive channels.     

The Chemical Structure of the Intermediate Metabolites of Catechin I–IV

Examinations were made on two intermediate metabolites excreted after administration of (+)-catechin to the rabbit. From the infrared and ultraviolet spectra of these two substances (C₁₁H₁₂O₃ and C₁₁H₁₂O₄) and chemical properties of their various derivatives, they were identified as 3-hydroxyphenyl lactone and 3, 4-dihydroxyphenyl lactone. However, it still remains unknown whether they are δ- or γ-lactone.     

Evidence for the presence in tobacco leaves of multiple enzymes for the oxidation of glycolate and glyoxylate

The enzymic oxidation of glycolate to glyoxylate and glyoxylate to oxalate by preparations purified from tobacco (Nicotiana tabacum var Havana Seed) leaves was studied. The K_m values for glycolate and glyoxylate were 0.26 and 1.0 millimolar, respectively. The ratio of glycolate to glyoxylate oxidation was 3 to 4 in crude extracts but decreased to 1.2 to 1.5 on purification by (NH₄)₂SO₄ fractionation and chromatography on agarose A-15 and hydroxylapatite. This level of glyoxylate oxidation activity was higher than that previously found for glycolate oxidase (EC 1.1.3.1). The ratio of the two activities was changed by reaction with the substrate analog 2-hydroxy-3-butynoate (HBA) which at all concentrations inhibited glyoxylate oxidation to a greater extent than glycolate oxidation. The ratio of the two activities could also be altered by changing the O₂ concentration. Glycolate oxidation increased 3.6-fold when the O₂ atmosphere was increased from 21 to 100%, whereas glyoxylate oxidation increased only 1.6-fold under the same conditions. These changes in ratio during purification, on inhibition by HBA, and under varying O₂ concentrations imply that tobacco leaves contain at least two enzymes capable of oxidizing glycolate and glyoxylate.     

Mapping of the two mitochondrial antimycin A resistance loci in Saccharomyces cerevisiae.

Summary Retention or loss of the two new mitochondrial antimycin A resistance loci A_I and A_II has been analyzed in a large number of stable cytoplasmic petite mutants. Using these deletion mutants it was possible to localize the two antimycin A resistance loci in the O_I-O_II region of mitochondrial DNA. The genetic loci are mapped in the following order: O_II-A_I-A_II-cobl-O_I. The mapping relationship of mutants resistant to antimycin A or funiculosin to various cob mutants is described.     

Pliocene climates: the nature of the problem

The Pliocene may have been the last time period when global temperatures were greater than present. The Pliocene data base is sufficiently complete to provide a valuable test for climate model predictions for warm time periods. This paper reviews some key issues with respect to understanding and verifying theories for the origin of Pliocene warmth. There are two main factors cited to explain Pliocene warmth—higher CO₂ levels or higher levels of ocean heat transport. The two explanations may not be exclusive; for example CO₂ increases may drive ocean heat transport changes. However, initial proxy-CO₂ reconstructions suggest that the CO₂ perturbation is very small (~100 ppm) to effect such large changes in climate. Considerably more data are needed to evaluate the magnitude of Pliocene CO₂ changes. Although more modeling work is also required, it is necessary to continue evaluation of input data used to force model results (particularly sea surface temperatures). Continued simulations and evaluation of pCO₂ and SST results should enable us to better understand how the earth responds during a global warming and determine whether our models are properly simulating such changes.     

The effect of the diameter of cyclic peptide nanotube on its chirality discrimination

In this work, the transport behaviors of the enantiomers of lactic acid (LA) in two cyclic peptide nanotubes (CPNTs) with different diameters were studied using steered molecular dynamic (SMD) simulation to investigate the effect of the diameter of CPNT on the discrimination of the enantiomers of LA. For this purpose, two cyclic peptides with two different sizes ([Ala-_D-Ala-_L]₅ and [Ala-_D-Ala-_L]₄) were used for constructing two CPNTs so that each CPNT was composed of eight cyclic peptide units. The docking calculations were performed to obtain the appropriate position of each enantiomer at the lumen of each CPNT. The variation of the pulling force versus time, exerted on the enantiomers moving in the CPNTs was calculated using the SMD simulations with two different strategies (positional and directional).The obtained results showed that the diameter of CPNT has considerable effect on the discrimination of the LA enantiomers so that the increase of the diameter of CPNT, increased the velocity difference between two enantiomers and improved the performance of CPNT for the chirality discrimination. The SMD simulations indicated that the velocity of S-enantiomer became more than R-enantiomer and its motion became more comfortable than R-enantiomer when the diameter of CNPT increased. The RDFs of the H and O atoms of the LA enantiomers relative to the O atoms of CPNT were calculated and it was found that the increase of the diameter of CPNT creates the significant changes in the RDFs of H1, H2 and H3 atoms of the enantiomers.     

Some genetic aspects of the resistance of Spodoptera frugiperda to a nuclear polyhedrosis virus

A probit analysis of the dosage-response of two isolates of Spodoptera frugiperda to a single nuclear polyhedrosis virus revealed a greater than 5-fold difference in the LD₅₀. The response of F₁ progeny of reciprocal crosses between the two parental isolates demonstrated that resistance to the virus is not sex-linked. The LD₅₀ values of F₁ and F₂ backcrosses suggest that the major variation in resistance is due to a single gene or genes that lack dominance.