The use of β-galactosidase as a tracer in immunocytochemistry

Summary A new immunocytochemical method using -galactosidase as a tracer is described. The positive staining appears blue on an unstained background. The present method has the high sensitivity and specificity of the immunoperoxidase method and appears to be a practical alternative. The substrate has no carcinogenic activity. Staining is permanent and the sections can be dehydrated and mounted in synthetic media. Enzyme and substrate solutions are stable for several months.     

Elucidation of the Hydrolytical Properties of α-Hydroxybenzylphosphonates as a New Potential Pro-Oligonucleotide Concept

Abstract

The synthesis of Fpmp-protected α-hydroxybenzylphosphonate modified oligonucleotides as potential new pro-oligonucleotides is described. The proposed hydrolytic pathways of the oligonucleotides were studied using two dimers 2 and 4 and the tetramer 6 containing one α-hydroxybenzyl modification as model compounds.     

Cation-π interaction of alkali metal ions with C24 fullerene: a DFT study

Using first principle calculations, we investigated cation-π interactions between alkali cations (Li(+), Na(+), and K(+)) and pristine C(24) or doped fullerenes of BC(23), and NC(23). The most suitable adsorption site is found to be atop the center of a six-membered ring of the exterior surface of C(24) molecule. Interaction energies of these cations decreased in the order: Li(+)?>?Na(+)?>?K(+), with values of -31.82, -22.36, and -15.68 kcal mol(-1), respectively. It was shown that the interaction energies are increased and decreased by impurity doping of B and N atoms in adjacent wall of adsorption site, depending on electron donating or receptivity of the doping atoms.     

Evolution of insect-plant relationships — a devil''s advocate approach

Most hypotheses concerning the evolution of insect-plant relationships are based on the assumptions that, (1) phytophagous insects reduce plant fitness, and that (2) insect-plant relationships are the result of unconstrained selection. It can be shown, however, that there is little evidence to support these assumptions. As an alternative, it is proposed that the evolution of insect-plant relationships results primarily from autonomous evolutionary events; namely from heritable functional changes within the insects' nervous system that determine plant recognition and ultimately host plant specificity. These changes cannot be evoked by selective ecological agents. They originate from intrinsic changes (mutationssensu lato) within the insect genome. Ecological factors play a secondary role: by either supporting or preventing the establishment of the new genotype with the novel food preference. This paper has been dedicated in warm friendship to Professor Louis M. Schoonhoven, the leading scientist in sensory physiology of phytophagous insects, on the occasion of his 60th birthday.     

Soil moisture redistribution as a mechanism of facilitation in savanna tree–shrub clusters

Plant–soil water relations were examined in the context of a selective removal study conducted in tree–shrub communities occupying different but contiguous soil types (small discrete clusters on shallow, duplex soils versus larger, extensive groves on deep, sandy soils) in a subtropical savanna parkland. We (1) tested for the occurrence of soil moisture redistribution by hydraulic lift (HL), (2) determined the influence of edaphic factors on HL, and (3) evaluated the significance of HL for overstory tree–understory shrub interactions. Diel cycling and nocturnal increases in soil water potential (Ψ_soil), characteristic signatures of HL, occurred intermittently throughout an annual growth cycle in both communities over a range of moisture levels (Ψ_soil=−0.5 to −6.0 MPa) but only when soils were distinctly stratified with depth (dry surface/wet deep soil layers). The magnitude of mean (±SE) diel fluctuations in Ψ_soil (0.19±0.01 MPa) did not differ on the two community types, though HL occurred more frequently in groves (deep soils) than clusters (shallow soils). Selective removal of either Prosopis glandulosa overstory or mixed-species shrub understory reduced the frequency of HL, indicating that Prosopis and at least one other woody species was conducting HL. For Zanthoxylum fagara, a shallow-rooted understory shrub, Prosopis removal from clusters decreased leaf water potential (Ψ_leaf) and net CO₂ exchange (A) during periods of HL. In contrast, overstory removal had neutral to positive effects on more deeply-rooted shrub species (Berberis trifoliolata and Condalia hookeri). Removal of the shrub understory in groves increased A in the overstory Prosopis. Results indicate the following: (a) HL is common but temporally dynamic in these savanna tree–shrub communities; (b) edaphic factors influencing the degree of overstory/understory development, rooting patterns and soil moisture distribution influence HL; (c) net interactions between overstory and understory elements in these woody patches can be positive, negative and neutral over an annual cycle, and (d) Prosopis-mediated HL is an important mechanism of faciliation for some, but not all, understory shrubs.     

Can we use diffusion MRI as a bio-marker of neurodegenerative processes?

Magnetic resonance imaging (MRI) is an imaging technique with a rapidly expanding application range. This methodology, which relies on quantum physics and substance magnetic properties, is now being routinely used in the clinics and medical research. With the advent of measuring functional brain activity with MRI (functional MRI), this methodology has reached a larger section of the neuroscience community (e.g. psychologists, neurobiologists). In the past, the use of MRI as a biomarker or as an assay to probe tissue pathophysiological condition was limited. However, with the new applications of MRI: molecular imaging, contrast-enhanced imaging and diffusion imaging, MRI is turning into a powerful tool for in vivo characterization of tissue pathophysiology. This review focuses on the diffusion MRI. Although it only measures the averaged Brownian translational motion of water molecules, using different analysis schemes, one can extract a wide range of quantitative indices that represent tissue morphology and compartmentalization. Statistical and visualization routines help to relate these indices to biologically relevant measures such as cell density, water content and size distribution. The aim of this review is to shed light on the potential of this methodology to be used in biological research. To that end, this review is intended for the non-MRI specialists who wish to pursue biological research with this methodology. We will overview the current applications of diffusion MRI and its relation to cellular biology of brain tissue.     

Amyloid-β Peptide-specific DARPins as a Novel Class of Potential Therapeutics for Alzheimer Disease

Passive immunization with anti-amyloid-β peptide (Aβ) antibodies is effective in animal models of Alzheimer disease. With the advent of efficient in vitro selection technologies, the novel class of designed ankyrin repeat proteins (DARPins) presents an attractive alternative to the immunoglobulin scaffold. DARPins are small and highly stable proteins with a compact modular architecture ideal for high affinity protein-protein interactions. In this report, we describe the selection, binding profile, and epitope analysis of Aβ-specific DARPins. We further showed their ability to delay Aβ aggregation and prevent Aβ-mediated neurotoxicity in vitro. To demonstrate their therapeutic potential in vivo, mono- and trivalent Aβ-specific DARPins (D23 and 3×D23) were infused intracerebroventricularly into the brains of 11-month-old Tg2576 mice over 4 weeks. Both D23 and 3×D23 treatments were shown to result in improved cognitive performance and reduced soluble Aβ levels. These findings demonstrate the therapeutic potential of Aβ-specific DARPins for the treatment of Alzheimer disease.     

Manipulation of β-glucuronidase for use as a reporter in vacuolar targeting studies

It has been documented that when furnished with an endomembrane signal sequence for the endoplasmic reticulum, -glucuronidase (GUS) is N-glycosylated, resulting in the nearly complete loss of enzymatic activity. To enable use of -glucuronidase as a reporter protein in secretory and vacuolar targeting studies, one of the two putative N-linked glycosylation sites within the GUS gene was altered by site-directed mutagenesis. The second N-linked glycosylation site was not altered because sequence analysis of nucleotide sequences around the second putative glycosylation site revealed that the published sequence was incorrect, and that no such site existed.     

High Potential of a Transposon mPing as a Marker System in japonica �� japonica Cross in Rice

Although quantitative traits loci (QTL) analysis has been widely performed to isolate agronomically important genes, it has been difficult to obtain molecular markers between individuals with similar phenotypes (assortative mating). Recently, the miniature inverted-repeat transposable element mPing was shown to be active in the japonica strain Gimbozu EG4 where it had accumulated more than 1000 copies. In contrast, most other japonicas, including Nipponbare, have 50 or fewer mPing insertions in their genome. In this study we have exploited the polymorphism of mPing insertion sites to generate 150 PCR markers in a cross between the closely related japonicas, Nipponbare × Gimbozu (EG4). These new markers were distributed in genic regions of the whole genome and showed significantly higher polymorphism (150 of 183) than all other molecular markers tested including short sequence repeat markers (46 of 661). In addition, we performed QTL analysis with these markers using recombinant inbred lines derived from Nipponbare × Gimbozu EG4, and successfully mapped a locus involved in heading date on the short arm of chromosome 6. Moreover, we could easily map two novel loci involved in the culm length on the short arms of chromosomes 3 and 10.Key words: Linkage mapping, Transposon, japonica, Oryza sativa L., QTL analysis     

Identification of N δ-acetylornithine as a novel osmolyte in some Gram-positive halophilic eubacteria

In the course of a screening programme for novel natural substances acting as compatible solutes, an unusual amino acid derivative was detected in several halophilic Gram-positive eubacteria. The compound was isolated and subsequently identified as N -acetylornithine using spectroscopic (NMR, MS) and chromatographic methods. So far, N -acetylornithine has only been described as an excretion product of some bacteria under certain growth conditions. This report describes, for the first time, its intracellular accumulation and use as a compatible solute in halophilic/halotolerant eubacteria. Correspondence to: E. A. Galinski     

Strategies of a Bornean tropical rainforest water use as a function of rainfall regime: isohydric or anisohydric?

Although Bornean tropical rainforests are among the moistest biomes in the world, they sporadically experience periods of water stress. The observations indicate that these ecosystems tend to have little regulation of water use, despite episodes of relatively severe drought. This water-use behaviour is often referred to as anisohydric behaviour, as opposed to isohydric plants that regulate stomatal movement to prevent hydraulic failure. Although it is generally thought that anisohydric behaviour is an adaptation to more drought-prone habitats, we show that anisohydric plants may also be more favoured than isohydric plants under very moist environments where there is little risk of hydraulic failure. To explore this subject, we examined the advantages of isohydric and anisohydric species as a function of the hydroclimatic environment using a stochastic model of soil moisture and carbon assimilation dynamics parameterized by field observations. The results showed that under very moist conditions, anisohydric species tend to have higher productivity than isohydric plants, despite the fact that the two plant types show almost the same drought-induced mortality. As precipitation decreases, the mortality of anisohydric plants drastically increases whereas that of isohydric plants remains relatively constant and low; in these conditions, isohydric plants surpass anisohydric plants in their productivity.     

A preliminary study of the use of benthic algae as biological indicators of heavy metal pollution in Sørfjorden,Norway

Concentrations of zinc, copper, lead and cadmium in seawater and in some benthic algae from Sørfjorden are reported. The concentration factors for Fucus vesiculosus and Ascophyllum nodosum are in good agreement with values given by other authors, even though the zinc concentrations are about ten times larger than those found in most previous work. The concentration factors obtained for zinc and cadmium are essentially the same in the whole area; however, for copper and lead the values decrease towards the mouth of the fjord.     

Discovery of 4-aminomethylphenylacetic acids as γ-secretase modulators via a scaffold design approach

Starting from literature examples of nonsteroidal anti-inflammatory drugs (NSAIDs)-type carboxylic acid γ-secretase modulators (GSMs) and using a scaffold design approach, we identified 4-aminomethylphenylacetic acid 4 with a desirable γ-secretase modulation profile. Scaffold optimization led to the discovery of a novel chemical series, represented by 6b, having improved brain penetration. Further SAR studies provided analog 6q that exhibited a good pharmacological profile. Oral administration of 6q significantly reduced brain Aβ42 levels in mice and rats.     

Bioremediation of multi-metal contaminated soil using biosurfactant — a novel approach

An unconventional nutrient medium, distillery spent wash (1:3) diluted) was used to produce di-rhamnolipid biosurfactant by Pseudomonas aeruginosa strain BS2. This research further assessed the potential of the biosurfactant as a washing agent for metal removal from multimetal contaminated soil (Cr-940 ppm; Pb-900 ppm; Cd-430 ppm; Ni-880 ppm; Cu-480 ppm). Out of the treatments of contaminated soil with tap water and rhamnolipid biosurfactant, the latter was found to be potent in mobilization of metal and decontamination of contaminated soil. Within 36 hours of leaching study, di-rhamnolipid as compared to tap water facilitated 13 folds higher removal of Cr from the heavy metal spiked soil whereas removal of Pb and Cu was 9–10 and 14 folds higher respectively. Leaching of Cd and Ni was 25 folds higher from the spiked soil. This shows that leaching behavior of biosurfactant was different for different metals. The use of wastewater for production of biosurfactant and its efficient use in metal removal make it a strong applicant for bioremediation.     

Identification of chilling and heat requirements of cherry trees—a statistical approach

Most trees from temperate climates require the accumulation of winter chill and subsequent heat during their dormant phase to resume growth and initiate flowering in the following spring. Global warming could reduce chill and hence hamper the cultivation of high-chill species such as cherries. Yet determining chilling and heat requirements requires large-scale controlled-forcing experiments, and estimates are thus often unavailable. Where long-term phenology datasets exist, partial least squares (PLS) regression can be used as an alternative, to determine climatic requirements statistically. Bloom dates of cherry cv. ‘Schneiders späte Knorpelkirsche’ trees in Klein-Altendorf, Germany, from 24 growing seasons were correlated with 11-day running means of daily mean temperature. Based on the output of the PLS regression, five candidate chilling periods ranging in length from 17 to 102 days, and one forcing phase of 66 days were delineated. Among three common chill models used to quantify chill, the Dynamic Model showed the lowest variation in chill, indicating that it may be more accurate than the Utah and Chilling Hours Models. Based on the longest candidate chilling phase with the earliest starting date, cv. ‘Schneiders späte Knorpelkirsche’ cherries at Bonn exhibited a chilling requirement of 68.6?±?5.7 chill portions (or 1,375?±?178 chilling hours or 1,410?±?238 Utah chill units) and a heat requirement of 3,473?±?1,236 growing degree hours. Closer investigation of the distinct chilling phases detected by PLS regression could contribute to our understanding of dormancy processes and thus help fruit and nut growers identify suitable tree cultivars for a future in which static climatic conditions can no longer be assumed. All procedures used in this study were bundled in an R package (‘chillR’) and are provided as Supplementary materials. The procedure was also applied to leaf emergence dates of walnut (cv. ‘Payne’) at Davis, California.     

Characterization of polychlorinated alkane mixtures—a Monte Carlo modeling approach

A Monte Carlo model was developed to characterize the molecular composition of polychlorinated alkane mixtures. The model is based upon a simulation of the free-radical chlorination process by which polychlorinated alkane mixtures are produced industrially from n-alkanes. In the model, the free-radical chlorination reaction was simulated by randomly selecting a position on a partially converted alkane molecule for target by chlorine free-radical attack. The relative reactivities of the hydrogen atoms on the alkane chain towards chlorine free-radical substitution were either determined experimentally or extrapolated from experimental results and incorporated into the model. The result of the simulation is the prediction of the detailed molecular composition of any PCA mixture. Good agreement was found when comparing the distribution of molecules predicted by the model to analytically determined distributions of real PCA mixtures. Results from the model were then coupled with rules describing the action of biological enzymes to estimate the upper limit possible for the aerobic biodegradation of PCA mixtures.     

Mechanisms underlying the cytotoxic effects of Tachpyr—a novel metal chelator

Tachpyr (N,N′N″-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane), a novel metal chelator, was previously shown to deplete intracellular iron and exert a cytotoxic effect on cultured bladder cancer cells. Tachpyr binds Fe(II) and readily reduces Fe(III). The iron(II)–Tachpyr chelate undergoes intramolecular oxidative dehydrogenation resulting in mono- and diimino Fe(II) complexes. The present study investigates the redox-activity of the Tachpyr–iron complex to better define the mechanism of Tachpyr's cytotoxicity. Tachpyr's mechanism of cytotoxicity was studied using cell-free solutions, isolated DNA, and cultured mammalian cells by employing UV–VIS spectrophotometry, oximetry, spin-trapping technique, and electron paramagnetic resonance (EPR) spectrometry. The results show that: (1) Tachpyr by itself after 24 h of incubation had a cytotoxic effect on cultured cells; (2) fully oxidized Tachpyr had no cytotoxic effects on cultured cells even after 24 h of incubation; (3) Tachpyr protected isolated DNA against H₂O₂-induced damage, but not against HX/XO-induced damage; and (4) Tachpyr–Fe(II) chelate slows down but does not block oxidation of Fe(II), allows O₂⁻-induced or Tachpyr-induced reduction of Fe(III), and consequently promotes production of OH through the Haber–Weiss reaction cycle. The results indicate that Tachpyr can protect cells against short-term, metal-mediated damage. However, upon prolonged incubation, Tachpyr exerts cytotoxic effects. Therefore, in addition to iron depletion, low-level oxidative stress, which in part occurs because of redox cycling of the coordinated iron ion, may contribute to the cytotoxic effects of Tachpyr.