Molecular modelling of the photoconduction mechanism by charged solitons intrans-polyacetylene: I

It is possible to rationalize the different steps involved in the photoconductivity observed intrans-polyacetylene (t-PA) in terms of a simple chemical model. The interchain electron transfer (IET) and the intrachain polaron capture by neutral solitons proposed by Orensteinat al. may be represented within a simple molecular orbital (MO) picture. An efficiency index for the IET is proposed in terms of the binding energy of the charged polarons and of the electronic chemical potential. The predictive power of the efficiency index is analyzed in the context of the global softness concept. An MO protocol is described to obtain estimates of the IET hopping probabilities.     

Polaron and bipolaron stability on paraphenylene polymers

Polyparaphenylene is the prototypical conjugated polymer containing phenyl rings and its properties are good references for a family of derived polymers. We investigate the structure, stability, and dynamics of polarons and bipolarons in polyparaphenylene chains under an applied electric field. To do this, we use a bidimensional SSH Hamiltonian model with the Hubbard extension, i.e., with local and nearest-neighbor Coulomb interaction, which has been designed to work with general hexagonal lattices, from which polyparaphenylene can be seen as a prominent case. Using the time-dependent Hartree–Fock approximation, we calculate the structural characteristics, the maximum field strength, supported before polarons and bipolarons gets unstable, and the maximum velocity achieved by these charge carriers. We obtained the polaron and bipolaron terminal velocity to be 0.51 Å/fs and 1.15 Å/fs, respectively. The maximum field strength determined by our calculations is 0.54 mV/Å and 0.80 mV/Å, respectively. Our results are in good agreement with other theoretical methods and experiments.     

Investigation of the Relationship between the Lectin Activity of Azospirilla and Their α-Glucosidase, β-Glucosidase,and β-Galactosidase

The activities of -glucosidase, -glucosidase, and -galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilenseSp7 and Azospirillum lipoferum59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol–acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied for the azospirilla studied. The cofunction of the A. brasilenseSp7 lectin and -galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum59b lectin and glucosidases was revealed. The lectin from A. lipoferum59b may possess saccharolytic activity.     

Restriction fragment analysis of ‘null’ forms at the Gli-1 loci of bread and durum wheats

Summary Wheat accessions lacking some of the - and -gliadin components encoded by the Gli-1 loci on the short arm of chromosome 1D in bread wheat and chromosome 1A in durum wheat were studied by two-dimensional polyacrylamide gel electrophoresis and restriction fragment analysis. Digested genomic DNAs of normal and null forms were probed with a cDNA clone related to -/-gliadins and with a genomic clone encoding an LMW subunit of glutenin. The hybridisation patterns with the -/-gliadin probe were similar to those of cvs Chinese Spring and Langdon used as standards for bread and durum wheats, respectively, but several restriction fragments located on the 1D chromosome of bread wheat and the 1A chromosome of durum wheat were absent in the null forms. In addition, specific LMW glutenin fragments encoded by the same chromosomes were also absent in the null forms, suggesting that simultaneous deletions of blocks of genes for both -/-gliadins and LMW glutenins had occurred. Comparisons of the protein and RFLP patterns enabled some proteins to be mapped to specific restriction fragments.     

Direct and indirect effects of human interferon α on renal cell carcinoma: a new in vitro assay system for evaluating cytokine-mediated antitumor effects

A highly purified natural -interferon (nIFN) was tested in vitro for direct and indirect antiproliferative activity against renal cell carcinoma (RCC), using a modified human tumor clonogenic assay and clinically achievable concentrations. In preclinical experiments, the indirect (cytokine-mediated) antiproliferative activity of nIFN was investigated using ACHN cells (established human RCC cell line). Continuous exposure to nIFN at concentrations of more than 5 IU/ml in the presence of feeder cells (a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from healthy donors) significantly inhibited colony formation of ACHN cells in comparison with growth inhibition in the absence of feeder cells (P<0.05). Various cytokines were measured in the supernatants lying over the medium on the feeder-layer agarose containing the same conditioned feeder cells. With IFN at 500 IU/ml, tumor necrosis factor (TNF) and IFN were detected at markedly high levels for 2–24 h. Neutralizing anti-TNF monoclonal antibody significantly reduced the indirect antiproliferative activity. Using our modified human tumor clonogenic assay technique, sufficient numbers of colonies for drug testing were observed in 19 of 31 surgical specimens (61.3%). In these clinical materials, nIFN at a clinically achievable concentration (50 IU/ml) significantly inhibited colony growth in the presence of feeder cells consisting of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from the patient whose tumor was examined (P<0.05). In colony-forming cases, a significant correlation between the percentage colony survival and TNF concentration in the supernatant was observed (r=–0.95,P<0.01). These results suggest that this assay system may be an appropriate technique for evaluating the antiproliferative activities of nIFN involving cytokine-mediated action, and that TNF may play an important role in this cytokine-mediated activity.This work was supported in part by a grant-in-aid for promoting research (no. 02771010) from the Ministry of Education     

Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of α-3′,4′-anhydrovinblastine: a specific role for a multifunctional enzyme

Summary. We have characterized a basic peroxidase with -3,4-anhydrovinblastine (AVLB) synthase activity, which was purified from Catharanthus roseus leaves. This enzyme was the single peroxidase isoenzyme detected in C. roseus leaves, and the single AVLB synthase activity detected in C. roseus extracts. It was observed that the monomeric substrates of AVLB, vindoline and catharanthine, are both suitable electron donors for the oxidizing intermediates of the basic peroxidase, compounds I and II. Results also showed that the reaction proceeds by a radical-propagated mechanism. Substrate specificity studies of the enzyme revealed that it was also able to oxidize several common peroxidase substrates, indicating a broad range of substrate specificity that is characteristic of class III plant peroxidases. Cytochemical studies showed that the enzyme is localized in C. roseus mesophyll vacuoles, in individual spots at the inner surface of the tonoplast. This particular location suggests a meaningful spatial organization that led to the proposal of a metabolic channeling model for the peroxidase-mediated synthesis of AVLB. The importance of this type of mechanism in the regulation of peroxidase isoenzyme functions in vivo is discussed. In view of the results obtained it is concluded that the basic peroxidase present in C. roseus leaves fulfills all the requirements to be considered as an AVLB synthase, and it is proposed that this specific function of this multifunctional enzyme is determined by metabolic channeling resulting from specific protein–protein interactions.Correspondence and reprints: Institute for Molecular and Cell Biology, University of Porto, rua Campo Alegre 823, 4150-180 Porto, Portugal.Received March 15, 2002; accepted February 4, 2003; published online August 26, 2003     

A glycine-rich sequence in the catalytic site of F-type ATPase

Affinity labeling and genetic studies on the glycine-rich sequence of the subunit ofE. coli F-type ATPase are discussed. A model of the structure of the enzyme near the phosphate moiety is proposed.     

Comparative Functional Analysis of Rat <Emphasis Type="Italic">TGF-β1</Emphasis> and <Emphasis Type="Italic">Xenopus laevis</Emphasis><Emphasis Type="Italic">TGF-β5</Emphasis> Promoters Suggest Differential Regulations

We have carried out a comparative functional analysis of the rat TGF-1 and Xenopus laevis TGF-5 promoters across several mammalian and amphibian cell lines. Progressive deletion constructs of both the promoters have been made using a PCR based approach and the basal promoter activities studied in Xenopus tadpole cell line (XTC), Xenopus adult kidney fibroblast cell line (A6), human hepatoma cell line (HepG2), normal rat kidney cell line (NRK), and Chinese hamster ovary cell line (CHO). Data suggests that the basal promoter activity of TGF-1 is low as compared to TGF-5 promoter in XTC cells but comparable in A6 cells, while TGF-5 promoter shows nearly negligible activity as compared to TGF-5 promoter in all the tested mammalian cell lines. Moreover, TGF-5 promoter is found to be repressed in XTC cells on treatment with TGF-5 protein. Thus, the regulation of TGF-1 and TGF-5 promoters is distinct in amphibian and mammalian species. We therefore suggest that contrary to the suggested functional equivalence of TGF-1 and TGF-5 proteins, TGF-1 and TGF-5 genes have distinct functions in their respective species. Present address (Kartiki V. Desai): Laboratory of Cell Regulation and Carcinogenesis, NCI, NIH Bldg 41, Room C619, Bethesda, MD 20892, USA     

Cholinesterases and cell proliferation in “nonstratified” and “stratified” cell aggregates from chicken retina and tectum

Summary Dissociated single cells from chicken retina or tectum kept in rotation-mediated cell culture aggregate, proliferate and establish a certain degree of histotypical cellto-cell relationships (sorting out), but these systems never form highly laminated aggregates (nonstratified R- and T-aggregates). In contrast, a mixture of retinal plus pigment epithelial cells forms highly stratified aggregates (RPE-aggregates, see Vollmer et al. 1984). The present comparative study of stratified and nonstratified aggregates enables us to investigate the process of cell proliferation uncoupled from that of tissue stratification. Here we try to relate these two basic neurogenetic processes with patterns of expression of cholinesterases (AChE, BChE) during formation of both types of aggregates.During early aggregate formation, in both stratified and nonstratified aggregates an increased butyrylcholinesterase activity is observed close to mitotically active cells. Quantitatively both phenomena show their maxima after 2–3 days in culture. In contrast, AChE-expression in all systems increases with incubation time. In nonproliferative areas, in the center of RPE-aggregates, the formation of plexiform layers is characterized initially by weak BChE and then strong AChE-activity. These areas correspond with the inner (IPL) and outer (OPL) plexiform layers of the retina in vivo. Although by sucrose gradient centrifugation we find that the 6S- and the fiber-associated 11S-molecules of AChE are present in all types of aggregates, during the culture period the ratio of 11S/6S-forms increases only in RPE-aggregates, which again indicates the advanced degree of differentiation within these aggregates.It is thus demonstrated that cholinesterases first correlate with neuronal cell proliferation and later with stratification, which indicates functions of both enzymes during both developmental periods.Abbreviations AChE acetylcholinesterase - BChE butyrylcholinesterase - iso-OMPA specific inhibitor of BChE - BW 284C51 specific inhibitor of AChE - IPL inner plexiform layer - OPL outer plexiform layer     

Synthesis ofα-triazolylα-amino acid derivatives

Summary We report the synthesis of-triazolyl-amino esters by 1,3 dipolar cycloaddition of acetylenic compounds and-azido-amino esters.     

Symmetric interspecies hybrids of mouse and human hemoglobin: Molecular basis of their abnormal oxygen affinity

Interspecies hybrids of HbA and Hb from mouse C57BL/10 [ ₂ ^M ₂ ^H and ₂ ^H ₂ ^M (H=human, M=mouse)], representing 19 and 27 sequence differences per dimers (as compared with human dimer) have been generatedin vitro. The efficiency of the assembly of the interspecies hybrids by the alloplex intermediate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O₂ binding. The intrinsic O₂ affinity of mouse Hb is slightly lower than HbA, while the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingly, the interspecies hybrid ₂ ^M ₂ ^H has high O₂ affinity (compared to either human or mouse Hb), while the interspecies hybrid ₂ ^H ₂ ^M exhibits a very low O₂ affinity. These results suggest that the mouse chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouse and chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouse chain. DPG binds the tetramer in the central cavity formed by the two subunits, hence the DPG effects on the interspecies hybrids should be as in the parent molecule. However, the results of the present study demonstrate that the DPG binding pocket is influenced by the nature of the chain present in the tetramer. The mouse chain reduces considerably the DPG right shift of the O₂ affinity of the human-chain containing hybrid. Sequence analysis suggest that perturbations of the _{1 1} (not the _{1 2}) are communicated to the DPG binding pocket in the presence of the alien subunit, and are the primary determinant of the ligand binding properties. The results have implications for the design of Hb-based blood substitutes and understanding of the inhibitory potential of mouse chains in transgenic mouse expressing human ^S chains.     

Architecture of the Alzheimer’s AβP Ion Channel Pore

We have proposed that the cytotoxic action of Alzheimers amyloid beta protein might be initiated by the interaction with the neuronal cell membrane, and subsequent formation of toxic ion channels. Consequently, AP toxicity can be explained on the basis of harmful ion fluxes across AP channels. The conformation of AP in membranes is not known. However, several models suggests that a transmembrane annular polymeric structure is responsible for the ion channel properties of the membrane-bound AP. To identify that portion of the AP molecule making up the conducting pore we have hypothesized that the region of the AP sequence in the vicinity of the hypothetical pore might interact with complementary regions in the adjacent AP subunits. We have further hypothesized that an interaction by a peptide segment would block AP conductance. To test this hypothesis we synthesized peptides that encompass the histidine dyad (H-H) previously hypothesized to line the pore. We report here that peptides designed to most closely match the proposed pore are, in fact, the most effective at blocking ion currents through the membrane-incorporated AP channel. As previously shown for Zn²⁺ blockade, peptide blockade is also asymmetric. The results also provide additional evidence for the asymmetric insertion of the AP molecules into lipid membranes, and give support to the concept that rings of histidines line the entry to one side of the AP pore.     

Estradiol Protects Against Oxygen and Glucose Deprivation in Rat Hippocampal Organotypic Cultures and Activates Akt and Inactivates GSK-3β

Here we investigated the neuroprotective effect of 17-estradiol in an in vitro model of ischemia. We used organotypic hippocampal slice cultures, acute or chronically treated with 17-estradiol (10 nM), and exposed to oxygen and glucose deprivation (OGD). Cellular death was quantified by measuring uptake of propidium iodide (PI), a marker of dead cells. In OGD exposed cultures, treated only with vehicle, about 70% of the CA1 area of hippocampus was labeled with PI, indicating a great percentage of cellular death. When cultures were treated with 17-estradiol (acute or chronically), this cellular death was reduced to 15%. This effect was prevented by LY294002 but was not by PD98059. Immunoblotting revealed that both, chronic and acute, treatments with 17-estradiol induced the phosphorylation/activation of Akt and the phosphorylation/inactivation of GSK-3. Our results show a clear neuroprotective effect of 17-estradiol and suggest that this effect could involve PI3-K pathway.     

Sensitive Nonradioactive Method for Screening Compounds Interacting with Neuronal α7 Cholinoreceptor

A sensitive nonradioactive method for detection of substances interacting with the neuronal 7-type nicotinic acetylcholine receptor (AChR) was proposed. The method uses biotinylated -cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with -cobratoxin (CTX) as does the whole receptor. LBED produced by heterologous expression of a gene fragment of the 7 subunit of AChR from the rat brain in Escherichia coli cells was sorbed in wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin–peroxidase complex. The ability of other compounds to interact with 7-AChR was checked according to the degree with which they inhibit the Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).     

Regioselective preparation of 2′, 3′-di-O-acylribonucleosides carrying lipophilic acyl groups through a lipase-catalysed alcoholysis

Candida antarctica B lipase-catalysed alcoholysis of 2, 3, 5-tri-O-hexanoyluridine (1a), 2, 3, 5-tri-O-dodecanoyluridine (1b), 2, 3, 5-tri-O-hexanoylinosine (1c) and 2, 3, 5-tri-O-dodecanoylinosine (1d) proceeded regioselectively to produce the corresponding 2, 3-di-O-acylribonucleosides 2a–d, providing a simple and efficient access to these new lipophilic compounds. Contrasting to the alcoholysis, enzymatic hydrolysis of 1a–d using different enzymes and experimental conditions did not proceed regioselectively.     

A reinterpretation of Na channel gating and permeation in terms of a phase transition between a transmembrane S4 α-helix and a channel-helix

A functional model for the S4/IV -helix of the action potential sodium channel is described by means of a thermodynamic approach. The model is based on a phase transition between the -helix and an ion conducting channel-helix which is similar to the well established helix-coil transition in solution. The right hand channel-helix is a peptide chain with an alternating sequence of torsional angles (¹¹)=(87°, 315°) and (²²)=(22°, 107°) which yields a helix of 13.5 Å per turn. The axial dipole moments of the peptide bonds of this chain of l-amino acids nearly cancel each other out in similar way to those in the gramicidin A channel, which is formed by alternating d-and l-amino acids. The helix, which does not contain any H-bonds, is stabilized by a helical file of water molecules which includes the permeating ion(s). This file turns around the channel-helix to form a relatively stable double helix structure which corresponds to the open channel. Since every third side chain in the S4/IV helix carries a positive charge their environments must be polarized. These polarized regions form a left hand screening-helix around the -helix are broken and the internal -carbon atom is considered as fixed, the outer ten residues leave the membrane while the internal ten residues form the channel-helix. In this configuration every positively charged side chain matches nearly exactly every second polarized region of the screening-helix leaving the three regions in-between exposed to the water file containing the ion(s). This further stabilizes the channel and agrees nicely with the idea of cationic selectivity. An analysis of the energetics of the -helix-channel-helix transition showed that the voltage-independent part of the free energy per helix residue could well be close to 0 kcal/mol and thus be in the range where a transition could occur. Two voltage-dependent contributions were included: the break down of the considerable dipole of the -helix and the outward shift of the positive charges of the side chains upon channel-helix formation. Taking into account the fact that the formation of an -helix is a highly cooperative process the degree of voltage dependence of the probability of formation of a channel-helix proved to be in the same range as experimental values for the open probability of modified Na channels whose inactivation had been removed. With regard to gating currents, the model predicts that 2.74 positive charges are moved in an outward direction. Consequences of the model for other experimental findings are discussed.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in<Emphasis Type="Italic"> Xenopus laevis</Emphasis> oocytes

Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken 7, chicken 42 and the Drosophila melanogaster/chicken hybrid receptors SAD/2 and ALS/2. No agonist action of NTX (0.1–100 M) was observed on chicken 7, chicken 42 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (7) or heteromeric (42) nicotinic AChRs. Block by NTX of the chicken 7, chicken 42 and the SAD/2 and ALS/2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.     

Induction of de novo synthesis of phenylalanine ammonia-lyase by L-α-aminooxy-β-phenylpropionic acid in suspension cultures ofDaucus carota L.

Application of L--aminooxy--phenylpropionic acid (L-AOPP), a potent and specific competitive inhibitor of L-phenylalanine ammonia-lyase (PAL), to an anthocyanin-producing cell suspension culture ofDaucus carota results in a dramatic increase in extractable PAL activity and an accumulation of phenylalanine (Noé et al., 1980, Planta149, 283–287). Using an immunoprecipitation technique, evidence was obtained that the increase in PAL activity the result of de-novo synthesis. The activity of the other enzymes of the general phenylpropanoid metabolism, e.g., trans-cinnamate 4-hydroxylase and hydroxycinnamate: CoA ligase, were not affected by L-AOPP. This result strongly supports the view that PAL is regulated independently.Abbreviations CAH trans-cinnamate 4-hydroxylase - L-AOPP L--aminooxy--phenylpropionic acid - PAL L-phenylalanine ammonia-lyase     

A “Bloom” in the Water of Amursky Bay (Sea of Japan) Caused by the Dinoflagellate Oxyrrhis marina Dujardin, 1841

A bloom in the water of Amursky Bay (Sea of Japan) caused by the dinoflagellate Oxyrrhis marina Dujardin, 1841 was recorded for the first time. The highest density of this species in the bloom area was 443.3 million cells/liter. The abundant development of microalga was observed from July to September 2002 at a water temperature of 17–24.5°C and a salinity of 7–18. Changes in the density of O. marina and other species of phytoplankton during the bloom period are analyzed. Possible reasons for the blooms of O. marina in Amursky Bay are discussed.