Structure and biological properties of first row d-transition metal complexes with N-substituted sulfonamides

Cobalt (II), copper (II), nickel (II) and zinc (II) complexes with 2-hydroxy-1-naphthaldehyde derived N-substituted sulfonamides have been synthesized and the nature of bonding and structure of compounds have been deduced from physical, analytical and spectral (IR, ¹H NMR, ¹³C NMR, Mass and electronic) data. An octahedral geometry has been suggested for the complexes. Complexes along with the ligands were assessed for their antibacterial and antifungal activities on different species of pathogenic bacteria and fungi. The results revealed the ligands to possess moderate to significant antibacterial activity which was, in many cases, enhanced on chelation. Similar results were observed for antifungal activity. Brine shrimp bioassay was also carried out for in vitro cytotoxic properties against Artemia salina.     

Nuclear magnetic resonance study of ring conformations of cyclic tetradepsipeptide AM-toxins and related peptides

Cyclic tetradepsipeptides, AM-toxin I and II, are the host-specific phytotoxins of Alternaria mali. In order to elucidate conformation-toxicity relationships, we analyzed the 270-MHz proton nmr spectra of AM-toxins and hydrogenated analogs, (D -Ala²)AM-toxin I (toxic) and (L -Ala²)AM-toxin I (not toxic), in (C²H₃)₂SO. These cyclic tetradepsipeptides do not contain N-substituted amino acid residues, and all the peptide and ester groups have been found to be transoid. Two conformers with very unequal populations have been found for AM-toxin I and II; the C^β?C^α? C?O conformations of the Dha² residues are nonplanar S-trans in the major conformer and nonplanar S-cis in the minor conformer. Only one ring conformation has been found for each of (L -Ala²) and (D -Ala²)AM-toxin I. (L -Ala²)AM-toxin I takes a C₄-type ring conformation; all the C?O groups and C^α-H bonds are oriented to the same side of the ring. (D -Ala²)AM-toxin I takes a new ring conformation; the side chain and C?O group of the L -Amp¹ residue are oriented to the same side of the ring. This new conformation is also found for the major conformers of AM-toxin I and II and thus appears to be required for the toxicity. The ring conformations of Tyr(OCH₃)¹-bearing analog tetradepsipeptides have been found to be much the same as those of Amp¹-bearing depsipeptides. Furthermore, on the basis of the two distinct conformations of (D -Ala²) and (L -Ala²)AM-toxin I, an empirical rule is proposed for the stable ring conformations of cyclic tetra-D ,L -peptides, not containing N-substituted amino acid residues.     

Synthesis and characterization of fluorinated homocysteine derivatives as potential molecular probes for ¹⁹F magnetic resonance spectroscopy and imaging

A N-trifluoroacetyl-protected amino acid containing a thioester function, 2,2,2-trifluoro-N-(2-oxo-tetrahydrothiophen-3-yl)acetamide (TFA-tHcy), has been synthesized and characterized. It was then used to prepare a fluorine-labeled N-homocysteinylated protein, ¹⁹F-Hcy-εN-Lys-albumin, that was characterized by SDS-PAGE, MALDI-TOF-MS, UV-vis and ¹⁹F NMR spectroscopy. On average, four N-trifluoroacetylhomocysteine residues were covalently conjugated to human serum albumin through the N-substituted homocysteine thiolactone. The in situ homocysteinylation of human plasma proteins with TFA-tHcy has also been performed and has led to the formation of N-homocysteinylated proteins, with albumin modification accounting for ca. 75% of all fluorine-labeled human plasma proteins. The synthesized fluorinated molecular probes can be potentially used as informative molecular probes for in vivo ¹⁹F magnetic resonance spectroscopy and imaging.     

6β-Amidinopenicillanic Acids—a New Group of Antibiotics

SINCE 6-aminopenicillanic acid became available¹, many semisynthetic penicillins carrying an acyl moiety on the 6-amino-group have been prepared. Thereby penicillins with improved oral absorption, resistance to penicillinase and to a lesser degree increased activity towards Gram-negative bacilli have been made available². Many other N-substitutions are possible, however, but these have not so far resulted in useful compounds^{2, 3}. We report here some of our findings on a new type of N-substituted 6-aminopenicillanic acids.     

Effects of ethidium bromide and berenil on protein synthesis

The effects of ethidium bromide, an intercalating dye and berenil, a nonintercalating dye on the biological activities ofEscherichia coli ribosomes have been studied. Ethidium bromide treatment drastically reduced both enzymatic and nonenzymatic initiation complex formation, enzymatic as well as nonenzymatic binding of phenylalanyl tRNA, peptidyl transferase, GTPase as well as the overall protein synthesising activity as measured by the poly U-dependent polymerization of phenylalanine. On berenil treatment, however, only enzymatic formation of the initiation complex is marginally reduced. Other reactions are not markedly affected except the enzymatic phenylalanyl tRNA binding which is slightly decreased only at high Mg²⁺ concentration; the treated ribosome has lowered polymerizing activity at sub-optimal Mg²⁺ concentration (10 mM). Although it has already been shown in this laboratory that treatment with either dye leads to the unfolding of the structure of the ribosome, the present studies indicate that berenil treatment does not alter the structure of the ribosome drastically in contrast to ethidium bromide treatment.     

New Plant Growth Stimulants Based on Water-Soluble Nanoparticles of N-Substituted Monoamino-Acid Derivatives of Fullerene C60 and the Study of their Mechanisms of Action

Growth-stimulating effects of water-soluble nanoparticles of N-substituted monoamino-acid derivatives of fullerene C₆₀ (L- and D-alanine, L- and D-valine, L- and D-aspartic acid, β-alanine, and γ-aminobutyric and ε-aminocaproic acids in potassium salt form) were investigated. It was found that the nanoparticle size and relative antiradical activity of fullerene derivatives were factors that influence such physiological parameters of field peas as seed germination rate, germination energy, and root growth capacity. It was shown that the relative antiradical activity of nanoparticles in the selected group of compounds was determined by the total surface area of the nanoparticles regardless of the structure of the amino-acid substituent. The possibility of using amino-acid derivatives of fullerene as effective growth stimulating substances has been demonstrated. A dose-dependent effect of N-(monohydrofullerenyl)-D-alanine potassium salt in a concentration range of 10^–9–10^–11 M on the seed-germination rate and germination energy of field peas has been demonstrated.

     

Modification and Inheritance of Pleiotropic Cross Resistance and Collateral Sensitivity in SACCHAROMYCES CEREVISIAE

A meiotic segregant (oli^PR1) was isolated with a phenotype of multiple cross resistance and collateral sensitivity. Strain oli^PR1 has increased sensitivity to ethidium bromide, dequalinium chloride, acriflavin, paromomycin and neomycin, and increased resistance to oligomycin, rutamycin, venturicidin, triethyltin bromide, antimycin, carbonylcynamide-m-chlorophenylhydrazone, tetra-N-butylammonium bromide, dibenzyldimethylammonium chloride, triphenylmethylphosphonium bromide, chloramphenicol, carbomycin, tetracycline, triton-X-165 and cycloheximide. Single gene inheritance of the cross resistance and collateral sensitivity was shown by 2:2 parental ditype segregation and reversion of the complete phenotype by a spontaneous revertant. The locus conferring the oli^PR1 phenotype was mapped 11.7 units from an unspecified centromere. Antibiotic resistance showed incomplete dominance, with the level of hybrid resistance dependent upon the inhibitor tested. Resistant diploids that produced four resistant ascospores were the result of mitotic recombination prior to meiosis. A partial revertant phenotype (sensitive to all inhibitors except oligomycin, antimycin and carbonylcyanide-m-chlorophenylhydrazone) was shown to be due to a single nuclear gene causing partial suppression of oli^PR1. Anaerobic pretreatment, 37° and 0.5 M KCl were observed to reduce the growth of oli^PR1 when challenged with seven diverse inhibitors (antimycin, carbonylcyanide-m-chlorophenylhydrazone,-chloramphenicol, cycloheximide, oligomycin, triethyltin bromide, and triphenylmethylphosphonium bromide). Resistance to cycloheximide was not altered by the [rho—] state. A revertant of oli^PR1 (sensitive to the above inhibitors but resistant to ethidium bromide, paromycin and neomycin) showed anaerobic and temperature sensitization to ethidium bromide, paromomycin and neomycin. Continuous monitoring of oxygen uptake by the revertant after anaerobic pretreatment revealed that anaerobiosis sensitized respiratory adaptation of the revertant to neomycin. It is proposed that oli^PR1 is a mutation resulting in the alteration of plasma membrane premeability to many diverse inhibitors.     

Determination of membrane-bound chloroplast RNA by the ethidium bromide fluorometric method

The method of El-Hamalawi et al. [(1975) Anal. Biochem.67, 384–391] for the fluorometric determination of nucleic acids with ethidium bromide has been adapted for the assay of membrane-associated chloroplast RNA. Membranes are stripped of RNA by incubation in a high-salt buffer lacking Mg²⁺, and the RNA is collected by magnesium phosphate-ethanol coprecipitation. RNA levels are determined by measuring the degree of enhancement of ethidium bromide fluorescence.     

Interactions of [NSI +] prion-like determinant with SUP35 and VTS1 genes in Saccharomyces cerevisiae

Previously we characterized [NSI ⁺], determinant, that possesses the features of a yeast prion. This determinant causes the nonsense suppression in strains that bear different N-substituted variants of Sup35p, which is a translation release factor eRF3. As a result of the genomic screen, we identified VTS1, the overexpression of which is a phenotypic copy of [NSI ⁺]. Here, we analyzed the influence of SUP35 and VTS1 on [NSI ⁺]. We demonstrated nonsense suppression in the [NSI ⁺] strains, which appears when SUP35 expression was decreased or against a background of general defects in the fidelity of translation termination. [NSI ⁺] has also been shown to increase VTS1 mRNA amounts. These findings facilitate the insight into the mechanisms of nonsense suppression in the [NSI ⁺] strains and narrow the range of candidates for [NSI ⁺] determinant.     

Mode of action of antitumour antibiotics-III: Modulation of permeability of nuclear membrane in the presence of the antibiotics

The binding characteristics of the antibiotics to nuclei and their effect on the permeability of nuclear membrane with respect to histones and ribonucleic acids have been investigated. The binding constant for chromomycin A₃ was found to be 1.4 × 10⁴M^?1 and number of binding sites was equal to 3.48 ± 1.08 × 10¹² molecules/nuclei. The antibiotic chromomycin A₃ enhanced the uptake of lysine-rich histone, actinomycin D decreased the uptake and ethidium bromide had no effect. Chromomycin A₃ also enhanced the release of acid insoluble fraction containing RNA from the nuclei, actinomycin D and ethidium bromide inhibited the release of acid insoluble fraction containing RNA. The relevance of this finding to the role of nuclear envelope in understanding the mechanism of action of the antibiotic has been discussed.     

Involvement of a Primary Electrogenic Pump in the Mechanism for HCO(3) Uptake by the Cyanobacterium Anabaena variabilis

The response of the membrane potential to HCO₃⁻ supply has been studied in the cyanobacterium Anabaena variabilis strain M-3 under various conditions. Changes in potential were followed with the aid of the lipophilic cation tetraphenyl phosphonium bromide.     

Decavanadate protonation degrees in aqueous solution

Aqueous solutions of vanadium(V) have been prepared at different OH⁻ vs. vanadium concentrations from both decavanadic acid and metavanadate solutions. These solutions have been extracted with an excess of tetraheptylammonium bromide in benzene. The bromide displaced has been determined in order to gain information on the average charge of the extracting species already known to be variously protonated decavanadate anions. The addition of a constant excess of supporting electrolytes has been avoided, as it is known it could affect the position of equilibria.Besides the already well known H₂V₁₀O₂₈⁴⁻, HV₁₀ O₂₈⁵⁻ and V₁₀O₂₈⁶⁻ species, strong evidence has also been obtained for the existence of H₃V₁₀O₂₈³⁻.Moreover, a slow disproportionation of V₁₀O₂₈⁶⁻ into HV₁₀O₂₈⁵⁻ and V₂O₇⁴⁻ (or HVO₄²⁻) during a first stage, and then into metavanadate, resulted.Therefore, H₆V₁₀O₂₈ proves a strong acid as triprotic instead of tetraprotic, as reported in the literature. Inconsistent results are almost certainly due to the high ionic media usually employed.     

Polypeptides. LVI. Effect of lithium bromide and of temperature on the conformation of a copolymer of glutamic acid and lysine

By using optical rotatory dispersion measurements, the helix content of poly Glu⁵⁰Lys⁵⁰ has been investigated and compared with that of poly Glu²⁰Lys²⁰Ala⁶⁰ in aqueous solutions. Measurements were made at pH 3 and at pH 8 in various concentrations of lithium bromide. Various factors affecting helix stabilization are considered and their perturbation by lithium bromide is related to the shape of the observed transition curves. A residual helix content of 12% in 8M LiBr, based upon a b₀ of +100 for a fully random conformation, was observed for poly Glu⁵⁰Lys⁵⁰ at pH 3 and 8. The loss of helix content of poly Glu⁵⁰Lys⁵⁰ as a function of temperature is also reported. ΔH is approximately ?6.9 kcal./mole for the overall transition, compared to ?6.5 kcal./mole for poly Glu²⁰Lys²⁰Ala⁶⁰. The midpoint of the broad transition is near 40°C. at pH 3, but much lower, at ?10 to 0°C., at pH 8. These results are discussed in terms of the stabilizing factors for the partial helix content of the polypeptides.     

Synthesis,characterization and antitumor activity of a series of N-substituted iminodiacetato(diammine) platinum(II) complexes

A series of water-soluble N-substituted iminodiacetato (diammine)platinum(II) complexes [Pt(NRIDA)(NH₃)₂] have been synthesized and characterized by measurement of physical properties (conductivity and pH) and by various spectroscopic techniques (infrared, ¹H and ¹³C{¹H} nuclear magnetic resonance). The iminodiacetate ligand is coordinated to platinum through an O,N linkage. The results obtained suggest that these complexes are relatively stable for more than 24 h in aqueous solution. Preliminary in vitro and in vivo screening test for antitumor activity of these complexes against L1210 murine leukemia were performed. Many of complexes had acceptable in vitro cytotoxicity, but none displayed a significant level of in vivo antitumor efficacy.     

Covalent binding of ethidium azide analogs to Salmonella DNA in vivo: competition by ethidium bromide.

The photoreactive analogs of ethidium bromide (ethidium mono- and diazide) have been developed as drug probes to determine the actual molecular details of ethidium bromide interactions with DNA. In an effort to demonstrate that the analogs in fact mimic the parent ethidium, competition experiments were designed using ³H thymidine-labeled DNA in intact $\text{Salmonella}$ TA1538, which is reverted by the azide analogs. ¹⁴C-labeled ethidium azide analogs were used in combination with the non-labeled ethidium bromide. The results presented here demonstrate that the parent ethidium competes with the azide analogs as a DNA intercalating drug using CsCl density gradient ultracentrifugation.     

High level production of a peptide hormone analogue [Leu13]motilin inE. coli

Summary A peptide hormone analogue [Leu¹³]motilin has been produced in high yield by recombinant DNA techniques. The peptide was expressed from a multicopied [Leu¹³]motilin gene fused to a salmon growth hormone gene fragment. The monomeric [Leu¹³]motilin was obtained by treatment of the fusion protein with cyanogen bromide, carboxypeptidase A and B. [Leu¹³]motilin showed the equivalent biological activity to that of the natural form.     

Development of a protocol for the automated analysis of amino acids in brain tissue samples and microdialysates

An automated precolumn derivatisation method has been developed for the measurement of fourteen amino acids in brain tissue and microdialysate samples. The method involves labelling amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide (CN⁻). The resulting highly stable N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives were separated using a binary gradient elution profile and detected fluorometrically. The order of elution of the derivatised amino acids was confirmed by using liquid chromatography with fluorescence and mass spectrometric detection in tandem. Linear calibration plots were obtained for all amino acids in the range studied (0.2–12.5 μM). The limit of detection for CBI derivatives of amino acids was in the range 5–20 fmol (S/N=2) using a 5 μl injection volume. The method has been used for the measurement of amino acids in microdialysates from rat brain and tissue homogenates from different regions of mouse brain.     

Plasmid chromosome isolation: An improved batch procedure for large plasmids

A one-step, batch fractionation procedure for the isolation of Escherichia coli plasmids with molecular weights up to at least 1.3 × 10⁸ has been devised. A 15-ml lysate of 5 × 10¹⁰ bacteria is gently prepared and underlain with 15 ml of CsCl-ethidium bromide solution in a 30-ml centrifuge bottle. During an 18- to 24-h 100,000g centrifugation in an anglehead rotor, an aggregate of dense cellular debris including almost all of the host DNA pellets. Plasmid chromosomes are banded in the supernatant isopycnic gradient and are detected by fluorescence of their intercalated ethidium bromide. Plasmid recoveries of 40–70% are achieved. The preparations are pure enough to be used for plasmid circumference measurements by electron microscopy.     

Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli

The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4′-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the ? 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.     

Synthèse,conformation et affinité tréhalasique des α-d-glucopyranosyl-α-d-xylopyranoside, α-d-glucopyranosyl-α-d-mannopyranoside et α-d-allopyranosyl-α-d-glucopyranoside

α-d-Glucopyranosyl α-d-xylopyranoside has been synthesized in 49% yield by treatment of 2,3,4-tri-O-benzyl-α-d-xylopyranosyl bromide with 2,3,4,6-tetra-O-acetyl-d-glucopyranose in nitromethane-benzene with mercuric cyanide and bromide, followed by catalytic hydrogenolysis and O-deacetylation. Condensation with 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl bromide in acetonitrile-dichloromethane with mercuric cyanide, followed by catalytic hydrogenolysis and O-deacetylation, gave α-d-glucopyranosyl α-d-mannopyranoside and β-d-glucopyranosyl β-d-mannopyranoside in 44 and 25% yield, respectively. The mixture was resolved by column chromatography of the fully acetylated derivatives. Selective acetylation of the di-O-benzylidene derivative of trehalose with N-acetylimidazole, followed by oxidation with dimethyl sulfoxide-acetic anhydride at C-3 and stereoselective reduction gave, after removal of the protecting groups, α-d-allopyranosyl α-d-glucopyranoside in 20% overall yield. The structure of the compounds was confirmed by ¹H- and ¹³C-n.m.r., and mass spectrometry. α-d-Glucopyranosyl α-d-xylopyranoside and α-d-allopyranosyl α-d-glucopyranoside are less efficient substrates than trehalose for cockchafer trehalase, but α-d-glucopyranosyl α-d-mannopyranoside is a competitive inhibitor of the enzyme.