Spectrophotometric studies on complexes formed between tri-, di- and monobutyltin(IV) chlorides and 3-hydroxyflavone in different solvents

The complex formation of tri-, di- and monobutyltin(IV) chloride with 3-hydroxyflavone was studied spectrophotometrically in methanol, chloroform and toluene (the last two after extraction with buffer pH 9.0) by the molar ratio method. The composition of the chelates formed was found to be solvent and molar ratio (C_M/C_L) dependent. The complex formation constants were calculated.     

Structural interpretations of F(0) rotary function in the Escherichia coli F(1)F(0) ATP synthase

F(1)F(0) ATP synthases are known to synthesize ATP by rotary catalysis in the F(1) sector of the enzyme. Proton translocation through the F(0) membrane sector is now proposed to drive rotation of an oligomer of c subunits, which in turn drives rotation of subunit gamma in F(1). The primary emphasis of this review will be on recent work from our laboratory on the structural organization of F(0), which proves to be consistent with the concept of a c(12) oligomeric rotor. From the NMR structure of subunit c and cross-linking studies, we can now suggest a detailed model for the organization of the c(12) oligomer in F(0) and some of the transmembrane interactions with subunits a and b. The structural model indicates that the H(+)-carrying carboxyl of subunit c is located between subunits of the c(12) oligomer and that two c subunits pack in a front-to-back manner to form the proton (cation) binding site. The proton carrying Asp61 side chain is occluded between subunits and access to it, for protonation and deprotonation via alternate entrance and exit half-channels, requires a swiveled opening of the packed c subunits and stepwise association with different transmembrane helices of subunit a. We suggest how some of the structural information can be incorporated into models of rotary movement of the c(12) oligomer during coupled synthesis of ATP in the F(1) portion of the molecule.     

An exact coordination mode of 6-thiopurine with an organotin(IV): preparation and crystal structure of triphenyl(6-thiopurinyl)tin(IV)

Triphenyl(6-thiopurinyl)tin has been prepared and its structure reinvestigated by X-ray diffraction. In the structure, a new coordination mode was observed that was different from those reported previously from investigations by infrared and Mössbauer spectroscopy. In the molecule, 6-thiopurine coordinated to tin by the S and N(1) atoms. The discrete molecules were connected to form a zigzag 1D network through intermolecular H?N hydrogen bonds. The tin environment is pentacoordinated with cis-trigonal bipyramidal geometry.     

Rotation of the c subunit oligomer in EF(0)EF(1) mutant cD61N

ATP synthases (F(0)F(1)-ATPases) mechanically couple ion flow through the membrane-intrinsic portion, F(0), to ATP synthesis within the peripheral portion, F(1). The coupling most probably occurs through the rotation of a central rotor (subunits c(10)epsilon gamma) relative to the stator (subunits ab(2)delta(alpha beta)(3)). The translocation of protons is conceived to involve the rotation of the ring of c subunits (the c oligomer) containing the essential acidic residue cD61 against subunits ab(2). In line with this notion, the mutants cD61N and cD61G have been previously reported to lack proton translocation. However, it has been surprising that the membrane-bound mutated holoenzyme hydrolyzed ATP but without translocating protons. Using detergent-solubilized and immobilized EF(0)F(1) and by application of the microvideographic assay for rotation, we found that the c oligomer, which carried a fluorescent actin filament, rotates in the presence of ATP in the mutant cD61N just as in the wild type enzyme. This observation excluded slippage among subunit gamma, the central rotary shaft, and the c oligomer and suggested free rotation without proton pumping between the oligomer and subunit a in the membrane-bound enzyme.     

Heat Resistant Characteristics of Major Royal Jelly Protein 1 (MRJP1) Oligomer

Soluble royal jelly protein is a candidate factor responsible for mammiferous cell proliferation. Major royal jelly protein 1 (MRJP1), which consists of oligomeric and monomeric forms, is an abundant proliferative protein in royal jelly. We previously reported that MRJP1 oligomer has biochemical heat resistance. Therefore, in the present study, we investigated the effects of several heat treatments (56, 65 and 96°C) on the proliferative activity of MRJP1 oligomer. Heat resistance studies showed that the oligomer molecular forms were slightly maintained until 56℃, but the molecular forms were converted to macromolecular heat-aggregated MRJP1 oligomer at 65℃ and 96℃. But, the growth activity of MRJP1 oligomer treated with 96°C was slightly attenuated when compared to unheated MRJP1 oligomer. On the other hand, the cell proliferation activity was preserved until 96℃ by the cell culture analysis of Jurkat cells. In contrast, those of IEC-6 cells were not preserved even at 56°C. The present observations suggest that the bioactive heat-resistance properties were different by the origin of the cells. The cell proliferation analysis showed that MRJP1 oligomer, but not MRJP2 and MRJP3, significantly increased cell numbers, suggesting that MRJP1 oligomer is the predominant proliferation factor for mammiferous cells.     

X-ray crystal structure determinations of two thiourea tin(II) complexes: Diacetatobis(thiourea)tin(II) and ditin(II)tetrabromopenta(thiourea)dihydrate

The crystal structures of diacetatobis(thiourea)tin(II) (I) and ditin(II)tetrabromopenta(thiourea)dihydrate (II) have been determined by X-ray diffraction analysis. The compound I crystallizes in the monoclinic space group Pc with a = 11.932(6), b = 10.937(5), c = 21.919(8) Å, β = 96.5(1), Z = 8. The compound II crystallizes in the orthorhombic space group Pnma with a = 27.83(3), b = 16.13(4), c = 6.11(6) Å, Z = 4. In compound I the tin atom has a square pyramidal environment. It is bonded to two thiourea sulphur atoms and to two carboxylate oxygens. In the compound II there are two tin sites both with trigonal pyramidal coordination. The ¹¹⁹Sn Mössbauer data for thiourea tin(II) compounds are discussed, in terms of their crystal structures.     

Stable octameric structure of recombinant hemoglobin alpha(2)beta(2)83 Gly-->Cys

We have engineered a recombinant hemoglobin (rHb betaG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb betaG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric betaG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric betaG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer betaG83C was not modified after incubation with fresh plasma.     

Conformation of the oligonucleotide d(AAAAAATTTTTT)2 by two-dimensional nuclear Overhauser effect spectroscopy and its relevance to poly(dA).poly(dT)

Conformational analysis from the pattern and intensities of cross-peaks in the two-dimensional nuclear Overhauser effect proton nmr spectra of the homopolymer, poly(dA) · poly(dT), and the analogous oligomer, d(AAAAAATTTTTT)₂, indicate that they both exist in the B-conformation. The conformation of the ApT/TpA junction in the oligomer is significantly different from the rest of the base pairs.     

The synthesis and crystal structures of triphenyl(1,2,3-trimethylallyl)tin and triphenyl( 1,1,2-trimethylallyl)tin

In an attempt to isolate new allylating reagents, two new tin compounds, triphenyl(1,2,3-trimethylallyl)tin (3) and triphenyl(1,1,2-trimethylallyl)tin (4) have been prepared and their crystal structures determined. Both compounds are examples of σ-bound allyl complexes. However, the infrared spectra of both compounds do not show the absorptions characteristic of other σ-bound allyl complexes. The chemistry of these new compounds also differs significantly from that of similar triphenyltin(allyl) reagents.     

The molecular structure of Okazaki fragment r(CGCA)d(AAAAAGCG):d(CGCTTTTTTGCG) in solution

The hetero duplex molecule, r(CGCA)d(AAAAAGCG):d(CGCTTTTTTGCG) which corresponds to Okazaki fragment was synthesized and its molecular structure has been analyzed by NMR study. The RNA strand of RNA-DNA hybrid region adopts A-form and DNA strand of the same region deviates from the standard B-form. The conformation of DNA-DNA duplex segment belongs to B-form. The hybrid-DNA duplex junction shows a structural discontinuities, A-B junction. The same conformational characteristic of oligo(dA): oligo(dT) tract as that of DNA oligomer which has same base sequence has been observed.     

Synthesis of d(GC) and d(CG) octamers containing alternating phosphorothioate linkages: effect of the phosphorothioate group on the B-Z transition

The synthesis of four oligonucleotides containing alternating phosphorothioate groups, (Rp)-and (Sp)-d[G(p(S)CpG)3p(S)C] and (Rp)- and (Sp)-d[C(p(S)GpC)p(S)G], by the phosphite approach is described. Silica gel to which 2'(3')-O-acetyluridine and 5'-succinyl groups were bound served as support for oligomer synthesis. The syntheses were carried out by dimer addition with presynthesized diastereomerically pure dinucleoside phosphorothioates as building blocks. The products were characterized by 31P NMR, nuclease P1 digestion, and oxidation to the corresponding all-phosphate-containing oligomers. The ability of each oligomer to adopt the Z conformation under high-salt conditions was screened for by circular dichroism spectroscopy. Both (Rp)-d[G(p(S)CpG)3p(S)C] and (Sp)-d[C(p(S)GpC)3p(S)G] are capable of forming Z-type structures at high NaCl concentrations. In the case of (Rp)-d[G(p(S)CpG)3p(S)C] where a phosphorothioate of the Rp configuration occurs 5' to a deoxycytidine residue, the B----Z transition is potentiated in comparison to the unmodified oligomer. (Sp)-d[G(p(S)CpG)3p(S)C] and (Rp)-d[C(p(S)GpC)3p(S)G] retain the B conformation even at high NaCl concentration.     

Synthesis of RNA oligomer using 9-fluorenylmethoxycarbonyl (Fmoc) group for 5'-hydroxyl protection

An approach using a new combination of protecting groups in RNA oligomer synthesis is proposed, in which 5'-hydroxyl group of ribose moiety is temporarily protected with the alkaline labile 9-fluorenylmethoxycarbonyl (Fmoc) group and the 2'-hydroxyl group is protected with the acid labile 1-ethoxyethyl (EE) group. The adoption of this method presented great selectivity in removing the 5'-hydroxyl protecting group and facilitated the RNA oligomer synthesis. A RNA pentamer was synthesized by the phosphotriester method in solution.     

Organotin compounds and aquatic bacteria: A review

Organotins are toxic to microorganisms. Trisubstituted organotins (R₃SnX) are considered more toxic than disubstituted (R₂SnX₂) or monosubstituted (RSnX₃) compounds, and tetrasubstituted compounds (R₄Sn) are not considered toxic. In the R₃Sn series propyl-, butyl-, pentyl-, phenyl- and cyclohexyltins are the most toxic to microorganisms. Toxicity towards aerobes in the R₃Sn series is related to total molecular surface area and to the octanol: water partition coefficient,Kow, which is a measure of hydrophobicity. Care must be taken when testing the toxicity of tin compounds in the laboratory, for a number of biological, chemical and physical factors can influence the apparent toxicity. Although TBT is generally the most toxic of the butyltins, there are instances where monobutyltin (MBT) is as toxic, or more toxic, than TBT to microorganisms. Thus, debutylation in the sequence TBT→DBT→MBT→Sn does not detoxity TBT for all microorganisms. Some microorganisms can methylate inorganic or organic tins under aerobic or anaerobic conditions. Methylation can also occur by chemical means and the relative contributions of biotic and abiotic mechanisms are not clear. It is difficult to isolate a pure culture which can methylate tin compounds aerobically, and it is difficult to isolate a pure culture which degrades TBT, suggesting that microbial consortiums may be involved in transformations of organotins in the aquatic environment. Methylation and debutylation alter the adsorbtivity and solubility of tin compounds; thus microorganisms can influence the environmental mobility of tin. TBT-resistant microorganisms can be isolated, and in some of them resistance to TBT can be plasmid-mediated. The literature review for this paper was completed in July, 1992.     

Inhibition of proliferation and induction of apoptosis by abrogation of heat-shock protein (HSP) 70 expression in tumor cells

Tumor cells often express elevated levels of heat-shock protein (HSP) 70. The present study was designed to invesitgate the role of HSP70 in the proliferation and survival of tumor cells in the human system. When Molt-4 and other tumor cells were treated in vitro with HSP70 antisense oligomer, they displayed propidiumiodide-stained condensed nuclei (intact or fragmented). A ladder-like pattern of DNA fragments was observed with HSP70 antisense-oligomer-treated tumor cells in agrose gel electrophoresis, which was consistent with internucleosomal DNA fragmentation. Flow cytometry analysis revealed the hypodiploid DNA peak of propidium-iodide-stained nuclei in the antisense-oligomer-treated cells. The apoptosis induced by HSP antisense oligomer was dose- and time-dependent. The antisense oligomer induced apoptosis mainly in tumor cells at G1 and S phase, resulting in an inhibition of cell proliferation. HSP70 antisense oligomer caused DNA-sequence-specific inhibition of HSP70 expression, which preceded apparent apoptosis. These results indicate that HSP70 antisense treatment inhibits the expression of HSP70, which in turn inhibits cell proliferation and induces apoptosis in tumor cells and suggest that HSP70 is required for tumor cells to proliferate and survive under normal condition.     

Isothiocyanatotriphenyl(pyridinium-2-carboxylato)tin(IV) monohydrate

The crystal structure of isothiocyanatotriphenyl- (pyridinium-2-carboxylato)tin(IV) monohydrate is reported. The crystals are monoclinic, space group P2₁/n, a = 10.349(2), b =12.003(2), c = 19.325(4) Å, β = 97.68(2)°, Z = 4, refined to R_F = 0.024 on 4249 observed reflections.The tin(IV) atom is five-coordinate, being bound to three phenyl groups, the isothiocyanato nitrogen atom and an oxygen from the picolinic acid. The geometry around the tin atom is trigonal bipyramidal, with the three phenyl groups occupying the equatorial positions, while the picolinic acid oxygen and the isothiocyanato nitrogen are coordinated axially. The acidic proton of picolinic acid has shifted position in the complex, and is bound to the heterocyclic nitrogen atom. The acid is thus coordinated in the form of a zwitterion. These trigonal bipyramidal units are linked together as dimers by pairs of water molecules, each of which hydrogen- bonds to the non-coordinated carboxylate oxygen atoms of both picolinic acid molecules, plus the heterocyclic nitrogen atom of one picolinic acid molecule. For complex formation with the protonated acid, theheterocyclic nitrogen should be alpha to the carboxylic acid group.     

Chlorotriphenyl(quinolinium-2-carboxylato)tin(IV) monohydrate

The crystal structure of chlorotriphenyl(quinolinium-2-carboxylato)tin(IV) monohydrate is reported. The crystals are monoclinic, space group C2/c with cell parameters a = 20.048(3) Å, b = 11.724(1) Å, c = 23.291(3) Å, ]gb = 113.42(1), Z = 8, refined to R_F = 0.034 on 3331 observed reflections. The tin(IV) atom is five-coordinate, being found to three phenyl groups, the chlorine atom and an oxygen from the quinaldic acid. The geometry around the tin atom is trigonal bipyramidal, with the three phenyl groups occupying the equatorial positions, and the chlorine and quinaldic acid oxygen, the apical ones. The acidic proton of quinaldic acid has shifted position in the complex, and is bound to the heterocyclic nitrogen atom.The acid is thus coordinated in the form of a zwitterion. These trigonal bipyramidal units are linked together as dimers by pairs of water molecules, each of which hydrogen-bonds to the non-coordinated carboxylate oxygen atoms of both quinaldic acid molecules, plus the heterocyclic nitrogen atom of one quinaldic acid molecule. For complex formation with the protonated acid, the heterocyclic nitrogen should be alpha to the carboxylic acid group.     

Preparation and characterization of oligonucleotides containing S-[2-(N7-guanyl)ethyl]glutathione.

S-[2-(N7-Guanyl)ethyl]glutathione is the major adduct derived from modification of DNA with 1,2-dibromoethane in biological systems and is postulated to be a mutagenic lesion [Humphreys, W. G., Kim, D.-H., Cmarik, J. L., Shimada, T., & Guengerich, F. P. (1990) Biochemistry 29, 10342-10350]. Oligonucleotides containing this modified base were prepared by treatment of oligonucleotides with S-(2-chloroethyl)glutathione and purified by chromatography. The self-complementary oligonucleotide d(ATGCAT), when thus modified at the single guanine, appeared to associate with itself as judged by UV measurements, but CD and NMR measurements indicated a lack of hybridization, with a decrease in the melting temperature of greater than 10 degrees C. The same lack of self-association was noted when d(ATGCAT) was modified to contain an N-acetyl-S-[2-(N7-guanyl)ethyl]cysteine methyl ester moiety. The oligomer d-(C1A2T3G4C5C6T7) was modified to contain a single S-[2-(N7-guanyl)ethyl]glutathione moiety at the central position, and UV, CD, and 1H NMR studies indicated that this oligomer hybridized to its normal complement d(A8G9G10C11A12T13G14), although the binding was considerably weakened by adduction (imino proton NMR spectroscopy in the presence of H2O indicated that the hydrogen bond signals seen in the oligomer were all broadened upon modification). All proton resonances were identified using two-dimensional 1H NMR spectroscopy. Adduct formation affected the chemical shifts of the base and 1', 2', and 2" protons of T3 and C5, the 2" proton of C6, and the 8 and 1' protons of C11, while little effect was observed on other protons. No cross-peaks were detected between the glutathione and oligomer moieties in two-dimensional nuclear Overhauser enhanced NMR studies. These results suggest that a rather local structural perturbation occurs in the DNA oligomer upon modification and that the glutathione moiety appears to be relatively unperturbed by its placement in the duplex. When the cytosine in the normal d(AGGCATG) complement to d-(CATGCCT) was changed to each of the other three potential bases at the central position, no hybridization with the oligomer d(CATGCCT) containing S-[2-(N7-guanyl)ethyl]glutathione was detected. We conclude that these N7-guanyl derivatives destabilize hybridization and that bases other than cytosine do not appear to show preferential thermodynamic bonding to these adducts, at least in the sequences examined to date.(ABSTRACT TRUNCATED AT 400 WORDS)     

4-(2-aminooxyethoxy)-2-(ethylureido)quinoline-oligonucleotide conjugates: synthesis,binding interactions,and derivatization with peptides

Oligo-2'-O-methylribonucleotides conjugated with 4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ) and 4-ethoxy-2-(ethylureido)quinoline (EOQ) were prepared by reaction of the AOQ or EOQ phosphoramidite with the protected oligonucleotide on a controlled pore glass support. Deprotection with ethylenediamine enabled successful isolation and purification of the highly reactive AOQ-conjugated oligomer. Polyacrylamide gel electrophoresis mobility shift experiments showed that the dissociation constants of complexes formed between an AOQ- or EOQ-conjugated 8-mer and complementary RNA or 2'-O-methyl-RNA targets (9- and 10-mers) were in the low nM concentration range at 37 degrees C, whereas no binding was observed for the corresponding nonconjugated oligomer, even at a concentration of 500 nM. Fluorescence studies suggested that this enhanced affinity is most likely due to the ability of the quinoline ring of the AOQ or EOQ group to stack on the last base pair formed between the oligomer and target, thus stabilizing the duplex. The binding affinity of a 2'-O-methyl RNA 15-mer, which contained an alternating methylphosphonate/phosphodiester backbone, for a 59-nucleotide stem-loop HIV TAR RNA target, increased 2.3 times as a consequence of conjugation with EOQ. The aminooxy group of AOQ-conjugated oligomers is a highly reactive nucleophile, which reacts readily with aldehydes and ketones to form stable oxime derivatives. This feature was used to couple an AOQ-oligomer with leupeptin, a tripeptide that contains a C-terminus aldehyde group. A simple method was developed to introduce a ketone functionality into peptides that contain a cysteine residue by reacting the peptide with bromoacetone. The resulting keto-peptide was then coupled to the AOQ-oligomer. This procedure was used to prepare oligonucleotide conjugates of a tetrapeptide, RGDC, and a derivative of HIV tat peptide having a C-terminus cysteine. The combination of the unique reactivity of the aminooxy group and enhanced binding affinity conferred by its quinoline ring suggests that AOQ may serve as a useful platform for the preparation of novel oligonucleotide conjugates.     

The effect of stannous and stannic (tin) chloride on DNA in Chinese hamster ovary cells

Tin(II) at concentrations up to 500 μM stannous chloride (SnCl₂), produced extensive DNA damage, as detected by alkaline sucrose gradient (ASG) analysis in Chinese hamster ovary (CHO) cells treated for 1 h at 37°C in serum-free minimal essential medium (MEM). However treatment of cells with tin(IV), as stannic chloride (SnCl₄), produced no such DNA damage. There was no loss in colony formation 6 days after either treatment suggesting that the DNA damage induced by the tin(II) was rapidly repaired and/or that DNA synthesis proceeded on the damaged templates permitting cell division to occur. Alternatively, the type of DNA damage caused by tin(II) may not lead to a reduction in colony-forming ability. Tin(II) produced about 200 times more ASG detectable DNA damage on an equi-molar basis than did Cr(VI), a known human carcinogen. This study indicates that tin(II) may be potentially genotoxic.     

The synthesis and functional evaluation of RNA and DNA polymers having the sequence of Escherichia coli tRNA(fMet)

Stepwise, solid-phase chemical synthesis has provided long RNA and DNA polymers related to the sequence of Escherichia coli tRNA(fMet). The 34-ribonucleotide oligomer corresponding to the sequence of the 5'-half tRNA molecule has been synthesized and then characterized by gel purification, terminal nucleotide determinations and sequence analysis. This 34-nucleotide oligomer serves as an acceptor in the RNA-ligase-catalyzed reaction with a phosphorylated 43-ribonucleotide oligomer corresponding to the sequence of the 3'-half molecule of tRNA(fMet). The DNA molecule having the sequence of tRNA(fMet) is a 76-deoxyribonucleotide oligomer with a 3'-terminal riboadenosine residue and all U residues replaced by T. These polymers have been compared with an oligodeoxyribonucleotide lacking all 2'-hydroxyl groups except for the 3'-terminal 2'-OH, an oligoribonucleotide lacking modified nucleosides and E. coli tRNA(fMet). The all-RNA 77-nucleotide oligomer can be aminoacylated by E. coli methionyl-tRNA synthetase preparation from E. coli with methionine and threonylated in the A37 position using a yeast extract. In agreement with work by Khan and Roe using tDNA(Phe) and tDNA(Lys), the rA77-DNA(fMet) can be aminoacylated, and preliminary evidence suggests that it can be threonylated to a small extent. Kinetic data support the notion that aminoacylation of tRNA(fMet) does not depend on the presence of 2'-hydroxyl groups with the exception of that in the 3'-terminal nucleotide.