Methods for the rapid detection,isolation and sequence determination of “peptaibols” and other aib-containing peptides of fungal origin. I. Gliodeliquescin a fromGliocladium deliquescens

Summary A rapid and simple detection procedure of Aib-containing “peptaibol” polypeptide antibiotics (mycotoxins) in fungal culture broths is described which employs alkylsilica (Sep-Pak?) cartridges for a selective enrichment, and the utilization of the unusualα-aminoisobutyric acid (Aib) and amino alcohols as specific marker constituents, which are easily detectable in total hydrolysates. Preparative isolations from fermentation broths is facilitated by adsorber resin (XAD) column chromatography, and purification of crude isolates is achieved by silica gel- and sephadex LH-20 chromatography. Preparative HPLC using spherical, totally porous ODS-bonded phases enables the isolation of uniform peptides, completely free of sequence analogs, from microheterogeneous peptaibol mixtures, and analytical HPLC of highest resolution allows a “fingerprint” assignment of microheterogeneous peptaibols of different fungal origin. Fast atom bombardment mass spectrometry is demonstrated to be a rapid and precise method for the complete sequence determination of peptaibols when employing sophisticated liquid matrix systems including both, lipophilic tetraethylenglycol and hydrophilic glycerol combined with a selective, trifluoroacetolytic cleavage step carried out directly “on target” (in situ). The versatility of the methods described is demonstrated with the detection and sequence determination of the new membrane-altering eicosapeptide named gliodeliquescin A from the soil fungusGliocladium deliquescens NRRL 3091 establishing the occurence of peptaibols in that fungal genus for the first time. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Purification of ß-glucosidases from <Emphasis Type="Italic">Pichia etchellsii</Emphasis> Using CIM Monolith Columns

β-Glucosidases (EC 3.2.1.21) are industrially important glycosyl hydrolases used for cellulose saccharification as well as for synthesis of glyco-conjugates. Crystal structure of only one β-glucosidase of family 3 of the glycosyl hydrolase families is available due to difficulty in purification of these closely related enzymes from a given source. Multiple steps used during purification result in low yield, making it difficult to study their properties. Conditions for purification of two closely related β-glucosidases (BGL I and BGL II) of family 3 from Pichia etchellsii were investigated in this study. Two weak anion exchange columns convective interaction media-diethyl amino ethyl (CIM-DEAE) and CIM-ethylenediamine (CIM-EDA) were used for this purpose. The results obtained at 0.34 ml disk (CIM-DEAE) level were scaled up to 8 ml CIM-DEAE tube column wherein BGL I and BGL II were separated from the major contaminants in the cell-free extract. The recovered enzymes were completely resolved in the second step using CIM-EDA. A final specific activity of 9,180 IU/mg and 2,345.3 IU/mg was achieved for BGL I and BGL II respectively with an overall yield of 33%. The system should be applicable to resolution of other closely related enzymes from this family.     

Determination of <Superscript>237</Superscript>Np, <Superscript>93</Superscript>Zr and other long-lived radionuclides in medium and low level radioactive waste samples

The majority of long-lived radionuclides produced in the nuclear fuel cycle can be regarded as “difficult-to-measure” nuclides, hence chemical separation is needed before the nuclear measurement of them. A combined radiochemical procedure that enables the simultaneous determination of some “difficult-to-measure” nuclides in medium and low level radioactive wastes has been developed in our laboratory. Recently, this method has been extended for determination of ²³⁷Np and ⁹³Zr. ²³⁷Np and ⁹³Zr are pre-concentrated by co-precipitation on iron(II) hydroxide and zirconium oxide, separated by extraction chromatography using UTEVA, and measured by inductively coupled plasma mass spectrometry (ICP-MS). As even traces of polyatomic ions and isotopes at m/z 237 or 93 cause considerable interferences during ICP-MS detection, a purification step by extraction chromatography was needed. Analyzing real samples (evaporation concentrates of a nuclear power plant) 66–99% and 31–99% chemical yields were achieved for Np and Zr, respectively.     

Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation

Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation have been prepared. They were obtained by a “ post-magnetization” procedure involving a simple treatment of the various affinity gels with magnetic ferrofluid. The magnetic biospecific adsorbents tested include magnetic protein A-Sepharose for isolation of IgG antibodies, magnetic human serum albumin (HSA)-Sepharose for anti-HSA isolation, and magnetic 2′,5′-ADP for isolation of glucose-6-phosphate dehydrogenase from baker’s yeast and hemolyzates of human red blood cells. For the latter enzyme, a 11,000-fold purification was achieved in one step.     

Development and validation of a quantification method for ziyuglycoside I and II in rat plasma: Application to their pharmacokinetic studies

This study provided a novel and generally applicable method to determine ziyuglycoside I and ziyuglycoside II in rat plasma based on liquid chromatography with tandem mass spectrometry. A single step of liquid–liquid extraction with n‐butanol was utilized, and ginsenoside Rg3 was chosen as internal standard. Final extracts were analyzed based on liquid chromatography with tandem mass spectrometry. Chromatographic separation was achieved using a Thermo Golden C₁₈ column, and the applied gradient elution program allowed for the simultaneous determination of two ziyuglycosides in a one‐step chromatographic separation with a total run time of 10 min. The fully validated methodology for both analytes demonstrated high sensitivity (the lower limit of quantitation was 2.0 ng/mL), good accuracy (% RE ≤ ± 15) and precision (% RSD ≤ 15). The average recoveries of both ziyuglycosides and internal standard were all above 75% and no obvious matrix effect was found. This method was then successfully applied to the preclinical pharmacokinetic studies of ziyuglycoside I and ziyuglycoside II. The presently developed methodology would be useful for the preclinical and clinical pharmacokinetic studies for ziyuglycoside I and ziyuglycoside II.     

Solving puzzles of aging: From disposable soma to signal-transduction pathways

Aging is believed to be caused by accumulation of unrepaired damage. The repair is believed to be limited by energetic resources. A testable prediction is: the less resources (calorie restriction), the shorter lifespan. In contrast, calorie restriction extends life span. To explain the conflicting result, paradoxical “allocation of resources” was proposed, thus creating the “odd science of aging.” However, evidence emerges that aging is not driven by damage but instead is driven by nutrient-activated signaling pathways such as TOR (target of rapamycin). This immediately explains how calorie restriction extends life span and solves all “odd” puzzles of aging. Here I suggest that aging is neither random damage nor a program but instead is a quasiprogram (a harmful continuation of valuable developmental programs). It is a genetic process that can be slowed down pharmacologically. And several drugs that directly or indirectly inhibit TOR are available for clinical use.     

Eco1524I, a type II restriction endonuclease

Various strains of Escherichia coli, isolated from different patients, were screened for type II restriction endonuclease activity. In 1 out of 23 patients, a type II restriction endonuclease activity was found. The restriction endonuclease designated Eco1524I was purified to near homogeneity, based on hydroxyapatite and heparin sepharose chromatography. Eco1524I exhibited endonuclease restriction activity in the pH range from 6.0 to 10.0 (maximum level at pH 8.0) and required Mg²⁺ as divalent cation. The enzyme was stable till temperature 55°C and pH range from 6.0 to 10.0. Eco1524I recognized the sequence 6-bp palindromic 5′AGG↑CCT 3′, producing blunt end and is found to be an isoschizomer of Stu I.     

Preparative purification of leuprorelin by HPLC applying a “saw-tooth” gradient

Summary Recently we reported on the development of a new semipreparative HPLC technique for the purification of leuprorelin [1]. This new method, referred to as a “saw-tooth technique” utilizes two successive gradients of two different eluent systems on the same column. Here we show our new results on extending the purification of leuprorelin from the semi-preparative 0.1–2 g) to the preparative (3–25 g) scale. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Multivariate statistical analysis of coastal sediment data

Multivariate statistical analysis of sediment data (information matrix 123 × 16) from the Gulf of Mexico, USA shows that the data structure is defined by four latent factors conditionally called “inorganic natural”, “inorganic anthropogenic”, “bioorganic” and “organic anthropogenic” explaining 39.24%, 23.17%, 10.77% and 10.67% of the total variance of the data system, respectively. The receptor model obtained by the application of the PCR approach makes it possible to apportion the contribution of each chemical component for the latent factor formation. A separation of the contribution of each chemical parameter is achieved within the frames of “natural” and “anthropogenic” origin of the respective heavy metal or organic matter to the sediment formation process. This is a new approach as compared to the traditional “one dimensional” search with a limited number of preliminary selected tracer components. The model suggested divides natural from anthropogenic influences and allows in this way each participant in the sediment formation process to be used as marker of either natural or anthropogenic effects. Received: 20 March 1999 / Revised: 1 June 1999 / Accepted: 3 June 1999     

Adiabatic theory of the vibronic states of the topological isomers of polyatomic molecules. Calculation of optimal conditions for optical stimulation of isomeric rearrangements

The problem of calculating the probability of optical transitions between the topological isomers of polyatomic molecules is considered. It is shown that the adiabatic approximation can be extended to this case. It is suggested that under certain conditions an “unoccupied” isomeric state can be populated or light-stimulated accumulation of one isomer can be achieved. This opens up prospects for designing molecular elements for computer memory. V. I. Vernadskii Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences. Translated fromZhurnal Strukturnoi Khimii, Vol. 37, No. 6, pp. 994–1005, November–December, 1996. Translated by I. Izvekova     

Multivariate statistical analysis of coastal sediment data

Multivariate statistical analysis of sediment data (information matrix 123 × 16) from the Gulf of Mexico, USA shows that the data structure is defined by four latent factors conditionally called “inorganic natural”, “inorganic anthropogenic”, “bioorganic” and “organic anthropogenic” explaining 39.24%, 23.17%, 10.77% and 10.67% of the total variance of the data system, respectively. The receptor model obtained by the application of the PCR approach makes it possible to apportion the contribution of each chemical component for the latent factor formation. A separation of the contribution of each chemical parameter is achieved within the frames of “natural” and “anthropogenic” origin of the respective heavy metal or organic matter to the sediment formation process. This is a new approach as compared to the traditional “one dimensional” search with a limited number of preliminary selected tracer components. The model suggested divides natural from anthropogenic influences and allows in this way each participant in the sediment formation process to be used as marker of either natural or anthropogenic effects. Received: 20 March 1999 / Revised: 1 June 1999 / Accepted: 3 June 1999     

Metal-binding molecules in the organs of <Emphasis Type="Italic">Mus musculus</Emphasis> by size-exclusion chromatography coupled with UV spectroscopy and ICP-MS

Mus musculus mice have been investigated for the total elements content in different organs (lung, liver, spleen, kidney, brain, testicle, heart and muscle) and molecular mass distribution patterns of Mn, Ni, Cu, Zn, As, Pb, Cr, Fe, Co, Se and Cd. Some differences have been found in the organs studied, with especially relevant being the Cu-containing fraction present only in the brain and the As-containing one in the liver. Other differences related to the abundance of the metallospecies have also been found. The present paper is the first step in the study of the “metallome” of this inbred laboratory species from which the genome is completely known. This organism could be used as a model in future studies focused on wild mice and the analytical approach developed could be applied to wild mice to find markers of environmental pollution. Figure The present paper is the first step in the study of the “metallome” of the inbred laboratory specie Mus musculus from which the genome is completely known. Some interesting differences have been found in the extracts from the organs that are discussed along the text. Electronic suplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The present paper is the first step in the study of the “metallome” of the inbred laboratory species Mus musculus from which the genome is completely known. Some interesting differences have been found in the extracts from the organs that are discussed in the text.     

Effect of sol temperature on the structure,morphology, optical and photoluminescence properties of nanocrystalline zirconia thin films

Transparent nanocrystalline zirconia thin films were prepared by sol–gel dip coating technique using Zirconium oxychloride octahydrate as source material on quartz substrates, keeping the sol at room temperature (SET I) and 60 °C (SET II). X-ray diffraction (XRD) pattern shows the formation of mixed phase [tetragonal (T) + monoclinic (M)] in SET I and a pure tetragonal phase in SET II ZrO₂ thin films annealed at 400 °C. Phase transformation from tetragonal to monoclinic was achieved in SET II film annealed at 500 °C. Atomic force microscopy analysis reveals lower rms roughness and skewness in SET II film annealed at 500 °C indicating better optical quality. The transmittance spectra gives a higher average transmittance >85% (UV–VIS region) in SET II films. Optical spectra indicate that the ZrO₂ films contain direct—band transitions. The sub- band in the monoclinic ZrO₂ films introduced interstitial O⁻defect states above the top of the valance band. The energy bandgap increased (5.57–5.74 eV) in SET I films and decreased (5.74–5.62 eV) in SET II films, with annealing temperature. This is associated with the variations in grain sizes. Photoluminescence (PL) spectra give intense band at 384 and 396 nm in SET I and SET II films, respectively. A twofold increase in the PL intensity is observed in SET II film. The “Red” shift of SET I films and “Blue” shift of SET II films with annealing temperature, originates from the change of stress of the film due to lattice distortions.     

Evaluation of high-performance liquid chromatography for determination of phenolic compounds in pear horticultural cultivars

Summary An analytical evaluation of an HPLC method with diode array detection to separate and quantify polyphenolic compounds from pears has been made. The method was applied to the quantitative analysis of phenolics from five pear horticultural cultivars (“Agua”, “Blanquilla”, “Conference”, “Pasagrana” and “Decana”) in both peel and pulp matrices and evaluated for precision and accuracy. Precision was taken as the reproducibility in peak area of the polyphenols of interest as well as in the slope of calibration graphs. Values ranged 2–5%. Accuracy was evaluated by recovery of all polyphenolic compounds from both peel and pulp in all pears investigated. Accuracy values ranged 92–102%, and were independent of the polyphenolic structure, horticultural cultivar and matrix. Identification was by comparing retention times and UV spectra with those of standards when commercially available. When not available commercially, provisional identification was according to spectral characteristics as well as from isolation and hydrolysis data. Application of the method revealed differences between peel and pulp in all cases studied; the higher levels of phenolics were found in the peels. “Decana” and “Pasagrana” cultivars showed the highest phenolic content compounds whereas “Conference” showed the lowest.     

Thin-layer chromatography of tris(1,10-phenanthroline)iron(II) and tris(2,2′-bipyridine)iron(II) on Sephadex G gels

Summary Gel chromatographic behaviour of tris(1, 10-phenanthroline)iron(II), tris(2,2′-bipyridine)iron(II) and tris(glycinato)cobalt(III) on Sephadex G-10 or G-25 was investigated by TLC with 0.001–1.0M NaCl as the eluent. The zone shapes and R_M values of tris(1,10-phenanthroline)iron(II) and tris(2,2′-bipyridine)iron(II) were appreciably dependent on the sample and eluent concentration, while the neutral complex, tris(glycinato) cobalt(III), exhibited the round zones with constant R_M values. The order of R_M values was found to be tris(glycinato)cobalt(III<tris(2,2∔pyridine)iron(II)<tris-(1,10-phenanthroline)iron(II) in all systems studied, although the reverse trend was expected when assuming the chromatographic behaviour of solute compounds to be controlled by the “sieving effect”. The comparison of the behaviour on Sephadex G gels with that on CM-cellulose revealed that the predominant mechanism involved is not the sieving effect, but ion-exchange and/or hydrophobic interaction.     

Normalized Lempel-Ziv complexity and its application in bio-sequence analysis

In this article, we propose a new method to measure DNA similarity based on a normalized Lempel-Ziv complexity scheme. The new method can weaken the effect of sequence length on complexity measurement and save computation time. Firstly, a DNA sequence is transformed into three (0,1)-sequences based on a scheme, which considers “A” and “non-A” , “G” and “non-G”, “C” and “non-C” bases respectively. Then, the normalized Lempel-Ziv complexity of the three (0,1)-sequences constitute a 3D vector. Finally, by the 3D vector, one may characterize DNA sequences and compute similarity matrix for them. The examination of similarities of two sets of DNA sequences illustrates the utility of the method in local and global similarity analysis.     

Actinide targets for neutron cross section measurements

The Advanced Fuel Cycle Initiative (AFCI) and the Generation IV Reactor Initiative have demonstrated a lack of detailed neutron cross sections for certain “minor” actinides. For some closed-fuel-cycle reactor designs more than 50% of reactivity will, at some point, be derived from “minor” actinides that currently have poorly known or in some cases not measured (n,γ) and (n,f) cross sections. Using a combination of resurrected techniques and new developments, we have made a series of targets including highly enriched ²³⁹Pu, ²⁴⁰Pu, and ²⁴²Pu. Thus far, we have electrodeposited these actinide targets. The chemical purification and electodeposition techniques will be described.     

Mimicking catalase and catecholase enzymes by copper(II)-containing complexes

An imidazolate-bridged copper(II)-zinc(II) complex (Cu(II)-diethylenetriamino-μ-imidazolato-Zn(II)-tris(2-aminoethyl)amine perchlorate (denoted as “Cu,Zn complex”) and a simple copper(II) complex (Cu(II)-tris(2-aminoethyl) amine chloride (“Cu-tren”) were prepared and immobilised on silica gel (by hydrogen or covalent bonds) and montmorillonite (by ion exchange). The immobilised substances were characterised by FT-IR spectroscopy and their thermal characteristics were also studied. The obtained materials were tested in two probe reactions: catalytic oxidation of 3,5-di-tert-butyl catechol (DTBC) (catecholase activity) and the decomposition of hydrogen peroxide (catalase activity). It was found that the catecholase activity of the Cu,Zn complex increased considerably upon immobilization on silica gel via hydrogen bonds and intercalation by ion exchange among the layers of montmorillonite. The imidazolate-bridged copper(II)-zinc(II) complex and its immobilised versions were inactive in hydrogen peroxide decomposition. The Cu(II)-tris(2-aminoethyl)amine chloride complex displayed good catalase activity; however, immobilisation could not improve it.     

Preparation of a one-curie <Superscript>171</Superscript>Tm target for the detector for advanced neutron capture experiments (DANCE)

Approximately one curie of ¹⁷¹Tm (T _1/2 = 1.92a) has been produced and purified for the purpose of making a nuclear target for the first measurements of its neutron capture cross section. Target preparation consisted of three key steps: (1) material production; (2) separation and purification; and (3) electrodeposition onto a suitable backing material. Approximately 1.5 mg of the target material (at the time of separation) was produced by irradiating ca. 250 mg of its stable enriched ¹⁷⁰Er lanthanide neighbour with neutrons at the ILL reactor in France. This production method resulted in a “difficult-to-separate” 1:167 mixture of near-neighboring lanthanides, Tm and Er. Separation and purification was accomplished using high-performance liquid chromatography (HPLC), with a proprietary cation-exchange column (Dionex, CS-3) and alpha-hydroxyisobutyric acid (α-HIB) eluent. This technique yielded a final product of ∼95% purity with respect to Tm. A portion (20 μg) of the Tm was electrodeposited onto thin Be foil and delivered to the Los Alamos Neutron Science Center (LANSCE) for preliminary analysis of its neutron capture cross section using the Detector for Advanced Neutron Capture Experiments (DANCE). This paper discusses the major hurdles associated with the separation and purification step, including scale-up issues related to the use of HPLC for material separation and purification of the target material from α-HIB and 4-(2-pyridylazo)resorcinol (PAR) colorant.     

The use of radioiodine in the management of the differentiated tyroid cancer

The use of radioactive iodine (¹³¹I) has substantially improved the prognosis of the differentiated thyroid cancer (DTC); however, its use can be effective only if it is planned with a precise strategy. A “radical” protocol of treatment is described, involving surgical “near total” thyroidectomy, followed by an “ablative” treatment with radioiodine. A total body (TB) scan is performed just after the treatment and, if it is negative, is repeated later with a tracer dose, in order to detect functioning distant metastases, if any, suitable for¹³¹I therapy. Out of a series of 202 patients affected by DTC, several cases (from 9 to 37%, depending on initial stage), who were apparently free of metastases on clinical and radiological basis, resulted positive for functioning metastases not known before, involving lungs, or lymph-nodes, or bones. Patients whose metastases had been discovered only by scan were successfully cured in larger numbers and with lesser amount of radioiodine, compared with others whose metastases were already known on clinical or radiological grounds. Side effects of the treatment were minimal, and not disproportionate to the disease. Patients administered with radioiodine in therapeutic dosage were admitted to the hospital in a special restricted area. Waste handling facilities are described.