Involvement of tryptophan residues in colchicine binding and the assembly of tubulin

Chemical modification of tubulin with 2-hydroxy-5-nitrobenzyl bromide, a reagent selective for tryptophan, inhibits tubulin's colchicine binding and in vitro assembly activities. Loss of colchicine binding shows a linear relationship with the modification of tryptophan residues, and is complete when not more than five residues are modified. GTP affords partial protection against this loss of colchicine binding. The in vitro assembly of tubulin is somewhat less sensitive, since microtubules are formed from tubulin dimers possessing 3–4 but not five modified residues. Furthermore, two of the eight tryptophans per dimer are reactive when tubulin is assembled into microtubules.     

4,4'-Bis dimethylaminodiphenylcarbinol: a new reagent for selective chemical modification. Interaction with porcine malate dehydrogenase

4,4-bis Dimethylaminodiphenylcarbinol (BDC-OH) has recently been reported to be a highly sensitive reagent for the quantitative determination of sulfhydryl residues in biological materials (1). In this communication the effectiveness of BDC-OH as a reagent for selective chemical modification of “active center” cysteine residues was investigated. The supernatant and mitochondrial forms of malate dehydrogenase were chosen for investigation by this reagent. Supernatant malate dehydrogenase which has never been found to contain an “active center” cysteine is unaffected by this reagent. Mitochondrial malate dehydrogenase (L malate: NAD⁺ oxidoreductase, EC 1.1.1.37) from porcine heart can be irreversibly inactivated by a 20 fold M excess of the reagent. Chemical modification of two essential sulfhydryl residues is prevented by the presence of the coenzyme, NAD⁺, suggesting that the site of interaction is located at or near the coenzyme binding site and hence at or near the enzymatic center of this enzyme.     

The number and nature of , -unsaturated amino acids in subtilin

In subtilin, a peptide produced by Bacillus subtilis, there are present three α,β-unsaturated amino acids, namely, two residues of dehydroalanine and one residue of β-methyldehydroalanine (dehydrobutyrine). Subtilin and nisin, a polypeptide produced by Streptococcus lactis, thus have in common not only the COOH-terminal sequence dehydroalanyllysine but also the number and nature of α,β-unsaturated amino acids.     

Ecological fish production in the inland delta of the Middle Danube, a floodplain river

Synopsis Ecological fish production in one parapotamal arm of the Middle Danube inland delta in Slovakia, based on 10 samplings during 5 consecutive years, ranged from 349 to 3272 (bar|x = 1066) kg ha^–1 in total production (P_T) and from 39 to 662 (bar|x = 204) kg ha^–1 in available production (P_A). Young-of-the-year fish made up an estimated 49–62% (bar|x = 54) of the P_T. According to the food production/food consumption budget calculated by three different methods, less than half of the P_T is of autochthonous origin, while most is from other sources. Floodplain fish production includes two components: fish originating within the floodplain and fish that are temporary immigrants. The latter component varies considerably in response to the hydrological regime. To assess the P_T in riverine ecosystems, different seasons and years are needed to give realistic values.This paper is dedicated to my friend and sometime co-worker, Eugene K. Balon, at the occasion of his 65th birthday and transition to University Professor Emeritus. He was one of the first ichthyologists to undertake quantitative studies in large rivers in the late 1950's, when most fish biologists thought that fishes inhabit only the sea, lakes, reservoirs and small streams.     

Phosphorus-31 NMR of rat brain in vivo with bloodless perfluorocarbon perfused rat

Rat brain in vivo has been examined by ³¹p NMR under conditions of normal blood perfusion (hematocrit 38%) and under conditions in which a perfluorocarbon blood substitute, devoid of any phosphorus containing compounds, largely replaced the animal's normal blood supply (hematocrit 7%). These studies demonstate that 2,3-diphosphoglycerate does not — as has been suggested — contribute to, and thus does not interfere with, the ³¹p NMR analysis of rat brain in vivo. However, low intensity ³¹P resonances assigned to choline phosphate, glycerol 3-phosphorylethanolamine, and glycerol 3-phosphorylcholine are observed. “High energy phosphorus” metabolite levels show no marked change over two hours with perfluorocarbon blood substitution from those of the normal blood perfused animal. This supports use of perfluorocarbon media for tissue perfusion in vitro and for ¹⁹F NMR vascular imaging in vivo.     

Preparation of New Glycoheptofurano[2,1-d]Imidazolidine-2-Thiones., Isomerizations to Acyclic-Sugar C-Nucleoside Analogues of Imidazole

Abstract

1-Methyl- and 1-aryl-(1,2-dideoxy-D-glyofurano)[2,1-d]-imidazolidine-2-thiones having the configurations β-D-glycero-L-gluco (4), β-D-glycero-D-ido (5—8), α-D glycerol-D-galacto (9—10) and β-D-glycero-D-talo (11, 12) are prepared by reaction of 2-amino-2-deoxy-aldoses with methyl and aryl isothiocyanates. 1-Aryl-(1,2-dideoxy–β-D-glycero-L-gluco-heptofurano)[2,1-d]imidazolidine-2-thiones (1—3) have been converted into 1-aryl-4-(D-galacto-pentitol-1-yl)-4-imidazo-line-2-thiones (24—26) by acid catalysed isomerization.     

Editor's Note

This issue of Soviet Psychology — Vol. V, No. 1 — marks a new point in the development of English translations of Soviet psychology and psychiatry. Our original journal, published in Volumes I-IV as Soviet Psychology and Psychiatry, has given birth to two new journals: Soviet Psychology and Soviet Psychiatry. This will give International Arts and Sciences Press the opportunity to publish twice as much material from the fund of Soviet theory and research in the study of human behavior. The increased space in this new journal will allow for a broader coverage of Soviet work in psychology, as outlined in our last issue, the special Handbook of Soviet Psychology.     

The Neuronal Kv4 Channel Complex

Kv4 channel complexes mediate the neuronal somatodendritic A-type K⁺ current (I_SA), which plays pivotal roles in dendritic signal integration. These complexes are composed of pore-forming voltage-gated α-subunits (Shal/Kv4) and at least two classes of auxiliary β-subunits: KChIPs (K ⁺-Channel-Interacting-Proteins) and DPLPs (Dipeptidyl-Peptidase-Like-Proteins). Here, we review our investigations of Kv4 gating mechanisms and functional remodeling by specific auxiliary β-subunits. Namely, we have concluded that: (1) the Kv4 channel complex employs novel alternative mechanisms of closed-state inactivation; (2) the intracellular Zn²⁺ site in the T1 domain undergoes a conformational change tightly coupled to voltage-dependent gating and is targeted by nitrosative modulation; and (3) discrete and specific interactions mediate the effects of KChIPs and DPLPs on activation, inactivation and permeation of Kv4 channels. These studies are shedding new light on the molecular bases of I_SA function and regulation. Special issue article in honor of Dr. Ricardo Tapia.     

The Zα domain of PKZ from Carassius auratus can bind to d(GC)n in negative supercoils

PKZ was the most recently discovered member of eIF2α kinase family in fish. CaPKZ, the first identified fish PKZ, possessed a conserved eIF2α kinase catalytic domain in C-terminal and two Z-DNA binding domains (Zα) in N-terminal. The Zα of CaPKZ closely resembled that of other Z-DNA binding proteins: ADAR1, DLM-1, and E3L. In order to understand more about the function of CaPKZ, we expressed and purified three constructed peptides of CaPKZ (P_Zα): P_Zα1Zα2, P_Zα1Zα1 and P_Zα2Zα2. Moreover, most of the plasmids containing d(GC)_n inserts were maintained in the Z-conformation, as confirmed by using inhibition of methylation experiments and anti-Z-DNA antibody. Gel mobility shift assays were then used to examine the affinity of these P_Zα to the recombinant plasmids. Meanwhile, a competition experiment using P_Zα1Zα2 and anti-Z-DNA antibody was performed. The results revealed that P_Zα1Zα2 and P_Zα1Zα1 were able to bind to the recombinant plasmids with high affinity, whereas P_Zα2Zα2 could not bind to it. In addition, dimerization of P_Zα1Zα2 indicated the function unit of Zα of CaPKZ would be a dimer.     

Genetic transformation of a pasture legume,Lotus corniculatus,L. (birdsfoot trefoil)

Summary A rapid procedure for introducing foreign genes inLotus corniculatus based on the induction of hairy roots byAgrobacterium rhizogenes was developed. Expression of chloramphenicol acetyltransferase and neomycin phosphotransferase II was revealed in transgenic plants. Southern blot hybridization was used to confirm the genetic transformation. The transgenic plants looked normal and did not show any morphological modification compared to the seed grown plants.     

Prediction of a common structural scaffold for proteasome lid,COP9-signalosome and eIF3 complexes

Background  

The 'lid' subcomplex of the 26S proteasome and the COP9 signalosome (CSN complex) share a common architecture consisting of six subunits harbouring a so-called PCI domain (proteasome,CSN, eIF3) at their C-terminus, plus two subunits containing MPN domains (Mpr1/Pad1N-terminal). The translation initiation complex eIF3 also contains PCI- and MPN-domain proteins, but seems to deviate from the 6+2 stoichiometry. Initially, the PCI domain was defined as the region of detectable sequence similarity between the components mentioned above.     

Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins

Background  

In mammals, there is evidence suggesting that methyl-CpG binding proteins may play a significant role in histone modification through their association with modification complexes that can deacetylate and/or methylate nucleosomes in the proximity of methylated DNA. We examined this idea for the X chromosome by studying histone modifications on the X chromosome in normal cells and in cells from patients with ICF syndrome (Immune deficiency, Centromeric region instability, and Facial anomalies syndrome). In normal cells the inactive X has characteristic silencing type histone modification patterns and the CpG islands of genes subject to X inactivation are hypermethylated. In ICF cells, however, genes subject to X inactivation are hypomethylated on the inactive X due to mutations in the DNA methyltransferase (DNMT3B) genes. Therefore, if DNA methylation is upstream of histone modification, the histones on the inactive X in ICF cells should not be modified to a silent form. In addition, we determined whether a specific methyl-CpG binding protein, MeCP2, is necessary for the inactive X histone modification pattern by studying Rett syndrome cells which are deficient in MeCP2 function.     

Molecular Basis of Substrate Polyspecificity of the Candida albicans Mdr1p Multidrug/H+ Antiporter

The molecular basis of polyspecificity of Mdr1p, a major drug/H⁺ antiporter of Candida albicans, is not elucidated. We have probed the nature of the drug-binding pocket by performing systematic mutagenesis of the 12 transmembrane segments. Replacement of the 252 amino acid residues with alanine or glycine yielded 2/3 neutral mutations while 1/3 led to the complete or selective loss of resistance to drugs or substrates transported by the pump. Using the GlpT-based 3D–model of Mdr1p, we roughly categorized these critical residues depending on their type and localization, 1°/ main structural impact (“S” group), 2°/ exposure to the lipid interface (“L” group), 3°/ buried but not facing the main central pocket, inferred as critical for the overall H⁺/drug antiport mechanism (“M” group) and finally 4°/ buried and facing the main central pocket (“B” group). Among “B” category, 13 residues were essential for the large majority of drugs/substrates, while 5 residues were much substrate-specific, suggesting a role in governing polyspecificity (P group). 3D superposition of the substrate-specific MFS Glut1 and XylE with the MDR substrate-polyspecific MdfA and Mdr1p revealed that the B group forms a common substrate interaction core while the P group is only found in the 2 MDR MFS transporters, distributed into 3 areas around the B core. This specific pattern has let us to propose that the structural basis for polyspecificity of MDR MFS transporters is the extended capacity brought by residues located at the periphery of a binding core to accomodate compounds differing in size and type.     

Structural and functional aspects of oligo(uridylic acid)-containing and poly(adenylic acid)-containing ribonucleic acids from rat liver

Uridylate tracts were released from rat liver mRNA by nuclease digestion and terminally-labeled invitro with ³²P using polynucleotide kinase. The pattern of fragments released from A⁺ and U⁺ mRNAs were the same as judged by electrophoresis on urea-polyacrylamide gels. The bulk of the fragments were in the size range 20–40 residues, but larger components (up to 70 residues in length) could also be seen. The ability of U⁺ and A⁺ mRNAs to direct the synthesis of proteins in a rabbit reticulocyte cell-free system was evaluated. The translation products were resolved by two-dimensional polyacrylamide gel electrophoresis. A comparison of the proteins made by the two classes of RNA showed that the U⁺ mRNA fraction represents a subset of A⁺ mRNA species, although the proportions of the protein products were quite different and several proteins were found to be unique to the U⁺ mRNA class.     

Oligomers Containing 2′-Deoxyinosine Isosteres as Ambiguous Nucleoside or 1,3-Propanediol as Nucleoside Substitute

Abstract

Three new appropriately protected phosphoramidites have been synthesized. Two of them (1 and 2) are isosteric to that of inosine (3) [1], one is a derivative of 1′3-propanediol (4). Whereas the inosine isosteres contain an ambiguous base recognizing adenine, guanine as well as cytosine residues in double stranded DNA-fragments the 1,3-propanediol unit can be seen as a simple nucleoside substitute in a DNA chain. It contains only those structural elements necessary to form the sugar/phosphate backbone, without supplying the DNA with either a base [21 or a 2′-deoxyribofuranosyl moiety.     

The use of molecular dynamics simulations to evaluate the DNA sequence-selectivity of G–A cross-linking PBD–duocarmycin dimers

The pyrrolobenzodiazepine (PBD) and duocarmycin families are DNA-interactive agents that covalently bond to guanine (G) and adenine (A) bases, respectively, and that have been joined together to create synthetic dimers capable of cross-linking G–G, A–A, and G–A bases. Three G–A alkylating dimers have been reported in publications to date, with defined DNA-binding sites proposed for two of them. In this study we have used molecular dynamics simulations to elucidate preferred DNA-binding sites for the three published molecular types. For the PBD–CPI dimer UTA-6026 (1), our simulations correctly predicted its favoured binding site (i.e., 5′-C(G)AATTA-3′) as identified by DNA cleavage studies. However, for the PBD–CI molecule (‘Compound 11’, 3), we were unable to reconcile the results of our simulations with the reported preferred cross-linking sequence (5′-ATTTTCC(G)-3′). We found that the molecule is too short to span the five base pairs between the A and G bases as claimed, but should target instead a sequence such as 5′-ATTTC(G)-3′ with two less base pairs between the reacting G and A residues. Our simulation results for this hybrid dimer are also in accord with the very low interstrand cross-linking and in vitro cytotoxicity activities reported for it. Although a preferred cross-linking sequence was not reported for the third hybrid dimer (‘27eS’, 2), our simulations predict that it should span two base pairs between covalently reacting G and A bases (e.g., 5′-GTAT(A)-3′).     

Cell adhesion and spreading factor. Chemical modification studies

The purified fetal calf serum factor that promotes cell adhesion and spreading of baby hamster kidney cells on tissue culture substrata has been subjected to a variety of chemical modifications and then tested for activity. These studies have shown that modification of the carbohydrate portions of the factor by glycosidic enzymes or by periodate oxidation did not alter its ability to promote cell spreading. On the other hand, modification of some protein portions of the factor by proteolytic enzymes or by specific modification of —COOH groups, tyrosine residues, or tryptophan residues resulted in a marked inhibition of factor activity. Modification of protein —SH groups, —NH₂ groups, or methionine residues did not affect factor activity. Control experiments indicate that the various modifications were directed at the activity of the factor and not its adsorption onto the substrata.     

Genome sequence analyses of two isolates from the recent <Emphasis Type="Italic">Escherichia coli</Emphasis> outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EAHEC)

The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).     

Design,synthesis, and DNA binding characteristics of a group of orthogonally positioned diamino,N-formamido,pyrrole- and imidazole-containing polyamides

Orthogonally positioned diamino/dicationic polyamides (PAs) have good water solubility and enhanced binding affinity, whilst retaining DNA minor groove and sequence specificity compared to their monoamino/monocationic counterparts. The synthesis and DNA binding properties of the following diamino PAs: f-IPI (3a), f-IPP (4), f-PIP (5), and f-PPP (6) are described. P denotes the site where a 1-propylamino group is attached to the N1-position of the heterocycle. Binding of the diamino PAs to DNA was assessed by DNase I footprinting, thermal denaturation, circular dichroism titration, biosensor surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) studies. According to SPR studies, f-IPI (3a) bound more strongly (K_eq = 2.4 × 10⁸ M^?1) and with comparable sequence selectivity to its cognate sequence 5′-ACGCGT-3′ when compared to its monoamino analog f-IPI (1). The binding of f-IPI (3a) to 5′-ACGCGT-3′ via the stacked dimer motif was balanced between enthalpy and entropy, and that was quite different from the enthalpy-driven binding of its monoamino parent f-IPI (1). f-IPP (4) also bound more strongly to its cognate sequence 5′-ATGCAT-3′ (K_eq = 7.4 × 10⁶ M^?1) via the side-by-side stacked motif than its monoamino analog f-IPP (2a). Although f-PPP (6) bound via a 1:1 motif, it bound strongly to its cognate sequence 5′-AAATTT-3′ (K_eq = 4.8 × 10⁷ M^?1), 15-times higher than the binding of its monoamino analog f-PPP (2c), albeit f-PPP bound via the stacked motif. Finally, f-PIP (5) bound to its target sequence 5′-ATCGAT-3′ as a stacked dimer and it has the lowest affinity among the diamino PAs tested (K_eq <1 × 10⁵ M^?1). This was about two times lower in affinity than the binding of its monoamino analog f-PIP (2b). The results further demonstrated that the ‘core rules’ of DNA recognition by monoamino PAs also apply to their diamino analogs. Specifically, PAs that contain a stacked IP core structure bind most strongly (highest binding constants) to their cognate GC doublet, followed by the binding of PAs with a stacked PP structure to two degenerate AT base pairs, and finally the binding of PAs with a PI core to their cognate CG doublet.     

Comparative studies of the phytochrome system in cell cultures from different plants

Phytochrome contents have been assayed in vivo in cell suspension cultures of Petroselinum hortense, Daucus carota and Glycine max. After transferring the cells to fresh medium phytochrome increased in parallel with the increase in cell number, whereas the amount of phytochrome per cell remained constant. The rate of phytochrome reaccumulation after pretreatment with 15 h red light was very similar in all three systems (2.8–3.6 (e) 10^–5/h). Dark reversion and a fast and slow P_fr destruction were observed in all systems. The rate constants of these reactions varied strongly between the systems. The phytochrome systems of the cell cultures were compared with those of etiolated and light-grown seedlings and it was concluded that the cell suspension cultures of Petroselinum hortense and Daucus carota behaved similarly to light-grown seedlings. In contrast, those of Glycine max behaved similarly to a dark grown seedling.Abbreviations P_r'fr red, far-red absorbing forms of phytochrome - P_tot P_r+P_fr total amount of phytochrome - fwt fresh weight