Purification of telomerase from Euplotes aediculatus: requirement of a primer 3' overhang.

Telomerase is a ribonucleoprotein enzyme that uses its internal RNA moiety as a template for synthesis of telomeric repeats at chromosome ends. Here we report the purification of telomerase from Euplotes aediculatus by affinity chromatography with antisense 2'-O-methyl oligonucleotides, a method that was developed for small nuclear ribonucleoprotein particles (snRNPs). Elution of bound ribonucleoprotein from the antisense oligonucleotide under nondenaturing conditions was achieved by a novel approach, using a displacement oligonucleotide. Polypeptides of 120 kDa and 43 kDa (a doublet) copurify with the active telomerase and appear stoichiometric with telomerase RNA. A simple model for DNA end replication predicts that after semiconservative DNA replication, telomerase will extend the newly synthesized, blunt-ended leading strand. We show that purified Euplotes telomerase has no activity with blunt-ended primers. Instead, efficient extension requires 4 to 6 single-stranded nucleotides at the 3' end. Therefore, this model predicts the existence of other activities such as helicases or nucleases that generate a single-stranded 3' end from a blunt end, thus activating the end for telomerase extension.

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium.

We describe a protease, named "thiocalsin," that is activated by calcium but only after reductive activation by thioredoxin, a small protein with a redox-active disulfide group that functions widely in regulation. Thiocalsin appeared to be a 14-kDa serine protease that functions independently of calmodulin. The enzyme, purified from germinating wheat grain, specifically cleaved the major indigenous storage proteins, gliadins and glutenins, after they too had been reduced, preferentially by thioredoxin. The disulfide groups of the enzyme, as well as its protein substrates, were reduced by thioredoxin via NADPH and the associated enzyme, NADP-thioredoxin reductase. The results broaden the roles of thioredoxin and calcium and suggest a joint function in activating thiocalsin, thereby providing amino acids for germination and seedling development.

Definition of the HLA-A29 peptide ligand motif allows prediction of potential T-cell epitopes from the retinal soluble antigen, a candidate autoantigen in birdshot retinopathy.

The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy.

The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.

Investigation of the prebiotic synthesis of amino acids and RNA bases from CO2 using FeS/H2S as a reducing agent.

An autotrophic theory of the origin of metabolism and life has been proposed in which carbon dioxide is reduced by ferrous sulfide and hydrogen sulfide by means of a reversed citric acid cycle, leading to the production of amino acids. Similar processes have been proposed for purine synthesis. Ferrous sulfide is a strong reducing agent in the presence of hydrogen sulfide and can produce hydrogen as well as reduce alkenes, alkynes, and thiols to saturated hydrocarbons and reduce ketones to thiols. However, the reduction of carbon dioxide has not been demonstrated. We show here that no amino acids, purines, or pyrimidines are produced from carbon dioxide with the ferrous sulfide and hydrogen sulfide system. Furthermore, this system does not produce amino acids from carboxylic acids by reductive amination and carboxylation. Thus, the proposed autotrophic theory, using carbon dioxide, ferrous sulfide, and hydrogen sulfide, lacks the robustness needed to be a geological process and is, therefore, unlikely to have played a role in the origin of metabolism or the origin of life.

The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction.

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.

Sticky ions in biological systems.

Aqueous gel sieving chromatography on Sephadex G-10 of the Group IA cations (Li+, Na+, K+, Rb+, Cs+) plus NH4+ as the Cl- salts, in combination with previous results for the halide anions (F-, Cl-, Br-, I-) as the Na+ salts [Washabaugh, M.W. & Collins, K.D. (1986) J. Biol. Chem. 261, 12477-12485], leads to the following conclusions. (i) The small monovalent ions (Li+, Na+, F-) flow through the gel with water molecules attached, whereas the large monovalent ions (K+, Rb+, Cs+, Cl-, Br-, I-) adsorb to the nonpolar surface of the gel, a process requiring partial dehydration of the ion and implying that these ions bind the immediately adjacent water molecules weakly. (ii) The transition from strong to weak hydration occurs at a radius of about 1.78 A for the monovalent anions, compared with a radius of about 1.06 A for the monovalent cations (using ionic radii), indicating that the anions are more strongly hydrated than the cations for a given charge density. (iii) The anions show larger deviations from ideal behavior (an elution position corresponding to the anhydrous molecular weight) than do the cations and dominate the chromatographic behavior of the neutral salts. These results are interpreted to mean that weakly hydrated ions (chaotropes) are "pushed" onto weakly hydrated surfaces by strong water-water interactions and that the transition from strong ionic hydration to weak ionic hydration occurs where the strength of ion-water interactions approximately equals the strength of water-water interactions in bulk solution.

Inactivation of ethanol-inducible cytochrome P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal, a major product of membrane lipid peroxidation.

Of the microsomal P450 cytochromes, the ethanol-inducible isoform, P450 2E1, is believed to be predominant in leading to oxidative damage, including the generation of radical species that contribute to lipid peroxidation, and in the reductive beta-scission of lipid hydroperoxides to give hydrocarbons and aldehydes. In the present study, the sensitivity of a series of P450s to trans-4-hydroxy-2-nonenal (HNE), a known toxic product of membrane lipid peroxidation, was determined. After incubation of a purified cytochrome with HNE, the other components of the reconstituted system (NADPH-cytochrome P450 reductase, phosphatidylcholine, and NADPH) were added, and the rate of oxygenation of 1-phenylethanol to yield acetophenone was assayed. Inactivation occurs in a time-dependent and HNE concentration-dependent manner, with P450s 2E1 and 1A1 being the most sensitive, followed by isoforms 1A2, 3A6, and 2B4. At an HNE concentration of 0.24 microM, which was close to the micromolar concentration of the enzyme, four of the isoforms were significantly inhibited, but not P450 2B4. In other experiments, the reductase was shown to be only relatively weakly inactivated by HNE. P450s 2E1 and 2B4 in microsomal membranes from animals induced with acetone or phenobarbital, respectively, are as readily inhibited as the purified forms. Evidence was obtained that the P450 heme is apparently not altered and the sulfur ligand is not displaced, that substrate protects against HNE, and that the inactivation is reversed upon dialysis. Higher levels of reductase or substrate do not restore the activity of inhibited P450 in the catalytic assay. Our results suggest that the observed inhibition of the various P450s is of sufficient magnitude to cause significant changes in the metabolism of foreign compounds such as drugs and chemical carcinogens by the P450 oxygenase system at HNE concentrations that occur in biological membranes. In view of the known activities of P450 2E1 in generating lipid hydroperoxides and in their beta-scission, its inhibition by this product of membrane peroxidation may provide a negative regulatory function.

Desenvolvimento e avaliação de métodos de extração e separação cromatográfica em colunas monolíticas e superficialmente porosas para determinação de herbicidas triazínicos em solos e águas

O uso de pesticidas levou ao aumento da produtividade e qualidade dos produtos agrícolas, porém o seu uso acarreta na intoxicação dos seres vivos pela ingestão gradativa de seus resíduos que contaminam o solo, a água e os alimentos. Dessa forma, há a necessidade do monitoramento constante de suas concentrações nos compartimentos ambientais. Para isto, busca-se o desenvolvimento de métodos de extração e enriquecimento de forma rápida, com baixo custo, gerando um baixo volume de resíduos, contribuindo com a química verde. Dentre estes métodos destacam-se a extração por banho de ultrassom e a extração por ponto nuvem. Após o procedimento de extração, o extrato obtido pode ser analisado por técnicas de Cromatografia a Líquido de Alta Eficiência (HPLC) e a Cromatografia por Injeção Sequencial (SIC), empregando fases estacionárias modernas, tais como as monolíticas e as partículas superficialmente porosas. O emprego de SIC com coluna monolítica (C18, 50 x 4,6 mm) e empacotada com partículas superficialmente porosas (C18, 30 x 4,6 mm, tamanho de partícula 2,7 µm) foi estudado para separação de simazina (SIM) e atrazina (ATR), e seus metabólitos, desetilatrazina (DEA), desisopropilatrazina (DIA) e hidroxiatrazina (HAT). A separação foi obtida por eluição passo-a-passo, com fases móveis compostas de acetonitrila (ACN) e tampão Acetato de Amônio/Ácido acético (NH4Ac/HAc) 2,5 mM pH 4,2. A separação na coluna monolítica foi realizada com duas fases móveis: MP1= 15:85 (v v-1) ACN:NH4Ac/HAc e MP2= 35:65 (v v-1) ACN:NH4Ac/HAc a uma vazão de 35 µL s-1. A separação na coluna com partículas superficialmente porosas foi efetivada com as fases móveis MP1= 13:87 (v v-1) ACN: NH4Ac/HAc e MP2= 35:65 (v v-1) ACN:NH4Ac/HAc à vazão de 8 µL s-1. A extração por banho de ultrassom em solo fortificado com os herbicidas (100 e 1000 µg kg-1) resultou em recuperações entre 42 e 160%. A separação de DEA, DIA, HAT, SIM e ATR empregando HPLC foi obtida por um gradiente linear de 13 a 35% para a coluna monolítica e de 10 a 35% ACN na coluna com partículas superficialmente porosas, sendo a fase aquosa constituída por tampão NH4Ac/HAc 2,5 mM pH 4,2. Em ambas as colunas a vazão foi de 1,5 mL min-1 e o tempo de análise 15 min. A extração por banho de ultrassom das amostras de solo com presença de ATR, fortificadas com concentrações de 250 a 1000 µg kg-1, proporcionou recuperações entre 40 e 86%. A presença de ATR foi confirmada por espectrometria de massas. Foram realizados estudos de fortificação com ATR e SIM em amostras de água empregando a extração por ponto nuvem com o surfactante Triton-X114. A separação empregando HPLC foi obtida por um gradiente linear de 13 a 90% de ACN para a coluna monolítica e de 10 a 90% de ACN para a coluna empacotada, sempre em tampão NH4Ac/HAc 2,5 mM pH 4,2. Em ambas as colunas a vazão foi de 1,5 mL min-1 e o tempo de análise 16 min. Fortificações entre 1 e 50 µg L-1 resultaram em recuperações entre 65 e 132%.

Análise de flavanóides por cromatografia líquida de alta eficiência e eletroforese capilar - otimização de separação e aplicações tecnológicas

No presente trabalho foram estudadas as separações de 18 flavonóides (9 agliconas e 9 glicosídeos) pelas técnicas de Cromatografia Líquida de Alta Eficiência em fase reversa (RP-HPLC) e Cromatografia Micelar Eletrocinética em fluxo reverso (RF-Meck). Em ambas as técnicas foram avaliados solventes puros (metanol, acetonitrila e tetrahidrofurano) e suas misturas como formas de promover a variação de seletividade, através da modificação da fase móvel em HPLC, e da natureza do aditivo orgânico em RF-Meck. Nos estudos efetuados em HPLC utilizando-se gradiente, pode-se comprovar a possibilidade da modelagem do fator de retenção em funçã da proporção de solvente utilizados (MeOH, ACN, THF e suas misturas). Pode-se ainda, com base nos dados de retenção e na análise hierárquica de c1usters, diferenciar quatro diferentes grupos de sistemas cromatográficos com diferentes seletividades para flavonóides agliconas, e outros quatro com diferentes seletividades para glicosídeos. Os sistemas cromatográficos mais ortogonais (cada um pertencente a um grupo de seletividade) foram aplicados na separação de uma planta modelo (Azadirachta indica), de onde pode-se escolher a fase móvel mais seletiva para se otimizar a separação dos flavonóides glicosilados presentes nas folhas desta planta. No método final otimizado pode-se identificar e quantificar cinco dos flavonóides majoritários presentes, sendo três glicosídeos de quercetina (rutina, isoquercitrina e quercitrina) e dois glicosídeos de kaempferol (astragalin e nicotiflorin), em amostras de duas diferentes procedências (Piracicaba-SP e Silvânia-GO). Nos estudos envolvendo a separação dos dezoito flavonóides por RFMEKC pode-se comprovar diferenças significativas de seletividade quando se varia a natureza do solvente orgânico utilizado como aditivo, além de se observar tendências na migração em função das propriedades do solvente adicionado e da estrutura molecular do flavonóide. O solvente de menor eficiência para separação dos flavonóides foi o MeOH. Através da análise dos eletroferogramas obtidos através de um planejamento experimental de misturas, e das trocas de pares críticos observadas nos vários eletrólitos utilizados, obteve-se um método de separação com apenas um par crítico em menos de 12 minutos de corrida. O coeficiente de variação obtido para o fator de retenção foi de 1,5% e para área de 3%, considerando-se cinco injeções. O método desenvolvido foi aplicado com sucesso na identificação dos flavonóides majoritários presentes na planta modelo (Neem), obtendo-se o mesmo resultado do estudo anterior. Como forma de avaliar a concentração de flavonóides totais presentes em espécies vegetais é comum a análise de extratos após hidrólise ácida (conversão de todos glicosídeos em agliconas). Desta forma otimizou-se uma metodologia de separação em RP-HPLC de 8 flavonóides agliconas comumente presentes em alimentos e extratos vegetais de uso cosmético. A otimização foi efetuada mediante um planejamento experimental de misturas, para escolha da fase móvel mais seletiva, e de um planejamento fatorial composto central, para otimização das condições de gradiente. O método obtido foi o mais rápido já visto dentro da literatura consultada. A separação em linha de base foi efetuada em menos de 15 minutos, com coeficientes de variação de área entre 0,1 e 1,8%, coeficiente de correlação de 0,9993 a 0,9994 na faixa de 5 a 100 µg/mL, e limites de quantificação estimados na faixa de 0,1 a 0,21µg/mL. O método desenvolvido foi aplicado na otimização das condições de hidrólise de um extrato de Neem. A otimização foi efetuada através de metodologia de superfície de resposta, levando-se em consideração a concentração de ácido adicionada, o tempo de reação, a temperatura, e a concentração de um antioxidante (ácido ascórbico) adicionado. O resultado da otimização foi uma metodologia de hidrólise com tempo de reação igual a 5 minutos, utilizando-se 1,4 mol/L de HCI, 119°C e 500 µg/mL de ácido ascórbico. Através das metodologias de análise e de hidrólise desenvolvidas pode-se constatar a presença e quantificar no extrato de Neem os flavonóides agliconas quercetina, kaempferol e miricetina. Com o objetivo de se avaliar quais os componentes presentes em extratos vegetais são os responsáveis pelo poder antioxidante atribuído a determinadas plantas, foi montado um sistema de avaliação de poder antioxidante \"on-line\" com reação pós-coluna em HPLC (baseado na literatura) utilizando-se como \"radical livre modelo\" o ABTS. A análise da planta modelo (Neem) neste sistema mostrou que os flavonóides glicosilados identificados nas partes anteriores deste trabalho são os responsáveis pelo poder antioxidante atribuído a esta planta. De posse desta informação, e visando a obtenção de extratos para aplicações cosméticas com poder antioxidante, modelou-se a extração dos flavonóide do Neem em função da composição do solvente extrator (água, etanol , propilenoglicol e suas misturas), de acordo com um planejamento simplex centróide ampliado. Além da previsão da concentração dos princípios ativos pode-se ainda prever outras propriedades dos extratos obtidos, tais como, índice de refração e densidade, muitas vezes constituintes de especificações técnicas de acordo com as aplicações a que se destinam (cremes, xampús, etc).

A Hydrodynamic Method For Measuring Aqueous Nanoparticle Surface Interactions

The objectives of this research dissertation were to develop and present novel analytical methods for the quantification of surface binding interactions between aqueous nanoparticles and water-soluble organic solutes. Quantification of nanoparticle surface interactions are presented in this work as association constants where the solutes have interacted with the surface of the nanoparticles. By understanding these nanoparticle-solute interactions, in part through association constants, the scientific community will better understand how organic drugs and nanomaterials interact in the environment, as well as to understand their eventual environmental fate. The biological community, pharmaceutical, and consumer product industries also have vested interests in nanoparticle-drug interactions for nanoparticle toxicity research and in using nanomaterials as drug delivery vesicles. The presented novel analytical methods, applied to nanoparticle surface association chemistry, may prove to be useful in assisting the scientific community to understand the risks, benefits, and opportunities of nanoparticles. The development of the analytical methods presented uses a model nanoparticle, Laponite-RD (LRD). LRD was the proposed nanoparticle used to model the system and technique because of its size, 25 nm in diameter. The solutes selected to model for these studies were chosen because they are also environmentally important. Caffeine, oxytetracycline (OTC), and quinine were selected to use as models because of their environmental importance and chemical properties that can be exploited in the system. All of these chemicals are found in the environment; thus, how they interact with nanoparticles and are transported through the environment is important. The analytical methods developed utilize and a wide-bore hydrodynamic chromatography to induce a partial hydrodynamic separation between nanoparticles and dissolved solutes. Then, using deconvolution techniques, two separate elution profiles for the nanoparticle and organic solute can be obtained. Followed by a mass balance approach, association constants between LRD, our model nanoparticle, and organic solutes are calculated. These findings are the first of their kind for LRD and nanoclays in dilute dispersions.

Quaternary ammonium-functionalized silica sorbents for the solid-phase extraction of aromatic amines under normal phase conditions

Quaternary ammonium-functionalized silica materials were synthesized and applied for solid-phase extraction (SPE) of aromatic amines, which are classified as priority pollutants by US Environmental Protection Agency. Hexamethylenetetramine used for silica surface modification for the first time was employed as SPE sorbent under normal phase conditions. Hexaminium-functionalized silica demonstrated excellent extraction efficiencies for o-toluidine, 4-ethylaniline and quinoline (recoveries 101–107%), while for N,N-dimethylaniline and N-isopropylaniline recoveries were from low to moderate (14–46%). In addition, the suitability of 1-alkyl-3-(propyl-3-sulfonate) imidazolium-functionalized silica as SPE sorbent was tested under normal phase conditions. The recoveries achieved for the five aromatic amines ranged from 89 to 99%. The stability of the sorbent was evaluated during and after 150 extractions. Coefficients of variation between 4.5 and 10.2% proved a high stability of the synthesized sorbent. Elution was carried out using acetonitrile in the case of hexaminium-functionalized silica and water for 1-alkyl-3-(propyl-3-sulfonate) imidazolium-functionalized silica sorbent. After the extraction the analytes were separated and detected by liquid chromatography ultraviolet detection (LC-UV). The retention mechanism of the materials was primarily based on polar hydrogen bonding and π–π interactions. Comparison made with activated silica proved the quaternary ammonium-functionalized materials to offer different selectivity and better extraction efficiencies for aromatic amines. Finally, 1-alkyl-3-(propyl-3-sulfonate) imidazolium-functionalized silica sorbent was successfully tested for the extraction of wastewater and soil samples.

Detritylation of Protected Tetrazoles by Naphthalene-Catalyzed Lithiation

Treatment of N-tritylated tetrazoles bearing aliphatic, aromatic, or heteroaromatic substituents (including functionalized ones) with lithium powder and a catalytic amount of naphthalene led to reductive removal of the trityl group to give excellent yields of the corresponding free tetrazoles without decomposition of the tetrazole ring. The detritylation process was successfully extended to several tetrazoles that are components of sartans, an interesting class of drugs. The chemoselectivity between trityl–tetrazole and trityl­–amine bond-cleavage reactions was also studied. This method represents an efficient technique for deprotection of tritylated tetrazoles under non-acidic conditions.

Sm-Nd isotope and Mn/Ca ratios for cleaned Neogloboquadrina pachyderma from ODP Hole 105-647A (Table 1)

The neodymium (Nd) isotope composition of ancient seawater is a potentially useful tracer of changes in continental inputs and ocean circulation on timescales of a few ka. Here we present the first Nd isotope record for seawater using sedimentary foraminifera cleaned using standard oxidative-reductive techniques. The data, along with Mn/Ca ratios, suggest that cleaned foraminifera provide a reliable record of Nd in seawater and hold out the prospect of using Nd in foraminifera to examine changes in seawater that accompany glacial-interglacial climatic cycles. The principal potential problem to be overcome with the use of forams as records of trace elements in ancient seawater is their diagenetic Fe-Mn coatings. These contain large amounts of Nd and other trace elements but can be cleaned off using highly reducing reagents. Mn(Ca ratios for the majority of the cleaned sedimentary foraminifera analysed here lie within the range (10-100 µmol/mol) that has yielded success in studies of transition elements in forams. Mass-balance modelling suggests that for residual Mn/Ca ratios <100 µmol/mol, Nd added to the foram in the coating will never shift the measured Nd isotope composition significantly away from the seawater value acquired by the foram test in the water column. Additionally, Nd concentrations measured in cleaned sedimentary foraminifera are comparable with those for a modern sample that has never encountered diagenetic fluids. Finally, core-top planktonic foraminifera for two sites have Nd isotope compositions that are identical to local surface seawater. The data we present here for Labrador Sea forams over the past 2.5 m.y. are interpreted in terms of changes in the seawater isotopic composition. The data show a pronounced shift from epsilon-Nd values of ~-12 to ~-19 in the period 2.5-1.5 Ma. This change is interpreted to result from the initiation of Northern Hemisphere glaciation and the increased derivation of Labrador Sea Nd via ice-rafting from Archaean terranes in central Canada. In combination with stable isotope and foraminiferal relative species abundance data, the new Nd data are consistent with the surface hydrography of the Labrador Sea being dominated by a fluctuating balance between cold, polar waters containing unradiogenic Nd and warm, subtropical waters containing more radiogenic Nd. The major change in Labrador Sea Nd that is observed in the past 2.5 Ma can, on its own, account for the change in the Nd isotope composition of North Atlantic Deep Water over the same time period.