In vitro investigation of the geometric contraction behavior of chemo-mechanical P-protein aggregates (forisomes)

We investigated the contracting behavior of forisomes from Vicia faba by carrying out precise measurements of their changing geometric parameters in vitro in the absence and in the presence of dissolved oxygen. Furthermore, we investigated the fine structure of forisomes by scanning electron microscopy. For the first time, single forisomes were titrated with Ca(2+), protons, and hydroxide ions recording the complete progression of their contractions. An apparent Ca(2+)-binding constant of (22+/-3) muM was calculated from two complete titration curves. The forisomes also contracted in the presence of Ba(2+) and Sr(2+) ions, but the amplitudes of contraction were smaller under the same measuring conditions. The time taken to change from the longitudinally expanded into the longitudinally contracted state was up to 2 s shorter in 10 mM Ca(2+) in comparison to 0.2mM Ca(2+). However, the contraction time was prolonged by decreasing the Ca(2+) concentration. In the absence of dissolved oxygen, the transition between the two final states of the forisomes was almost reversible and the amplitude of contraction remained almost constant during the first 25 contraction cycles. In the presence of dissolved oxygen the forisomes denaturated after a few cycles and lost their ability to contract, just after only a few cycles with 10 min in the contracted state. Denaturation of the forisomes occurred appreciably in the contracted state. We propose a cycle process to explain the thermodynamic basis of the Ca(2+)-induced contraction and its reversal by EDTA. Reducing the pH-value from 7.3 to 4.0 caused the forisomes to shorten by approximately 15%, while increasing the pH to 11.0 caused them to shorten by 28 to 30%. In both cases, the increases of the forisomes volume were greater than during the Ca(2+) induced contraction. The pH values of 4.7+/-0.3, and 10.2+/-0.2 marked the inflection points of the acid base titration of different forisomes.     

Formation of aggregates by plant roots in homogenised soils

The influence of root growth and water regime on the formation of aggregates was studied in modified minirhizotrons under controlled conditions. Two soils, a black earth (67% clay) and a red-brown earth (19% clay) were ground and forced through a 0.5 mm sieve. Ryegrass, pea and wheat were grown for fifteen wetting and drying (wd) cycles for 5 months. Another set of minirhizotrons was not planted and served as a control. Measurements of aggregate size distribution (ASD), aggregate tensile strength (ATS), aggregate stability (AS), aggregate bulk density (ABD) and organic carbon (OC) were made on single aggregates of the 2–4 mm fraction. The results showed that aggregates of the black earth which has a high clay content and shrink/swell properties had more smaller aggregates with higher ATS, AS and ABD than those from the red-brown earth. It was also found that for both soils: (1) w/d cycles and higher root length density (RLD) increased the proportions of smaller aggregates and aggregate strength; (2) differences in the ability of the plant species to influence aggregation was evident and seemed to be related to the RLD. The RLD was in the order ryegrass > wheat > pea. Mechanisms likely to be involved in processes of aggregate formation and stabilization are discussed. They include cracking of soil due to tensile stresses generated during drying of a shrinking soil; changes in pore water pressure within the soil mass caused by water uptake by plant roots generating effective stresses; and biological processes associated with plant roots and root exudates.     

Ultrastructural observation of paracrystalline aggregates of microtubules in anterior pituitary cells of the chinchilla (Chinchilla laniger)

Summary Unusual paracrystalline aggregates of microtubules which have not been described in any other mammalian species were observed in cultured anterior pituitary cells of normal chinchillas as well as in situ in the pituitary glands of these animals. These aggregates appeared as regularly arranged tubular structures in the longitudinal plane, and as a checkerboard pattern of closely and regularly packed microtubules when examined in transverse section. Supplementation with vinblastine, colcemide or colchicine in the culture medium did not change these structures morphologically. Each unit of tubules consisted of an outer wall or parellelogram profile and an inner wall composed of a single hexagonal doublet or in a figure 8 form. The outer wall of the parallelogram was 35×28 nm in length for both sides, while the diagonal of the inner wall was 18×28 nm. These paracrystalline aggregates of microtubules in the chinchilla pituitary cells are morphologically distinct from the paracrystalline assembly of cytoplasmic microtubules induced by vinblastine or other alkaloids.The function and significance of these paracrystalline aggregates in anterior pituitary cells of the chinchilla are uncertain.Supported by USPHS Grant HD 11826     

Ultrastructure and surface topography of aggregates of human limb mesenchymal cells (HLM15) in vitro

Summary Tissue-like aggregates of human embryo fibroblasts can be created in vitro by limited aspiration of cells released from substrate during subcultivation. Aggregates increase in size, exhibit intercellular junctions, display a surface topography characteristic of cellular movement, elaborate an extracellular matrix and possess features of cellular death and phagocytosis. These cells, when introduced to a new culture environment, do not migrate away from one another as is common when a primary culture is started from tissue fragments. Instead, cells exhibit continued contact with each other, and develop complex junctional structures during that association. Cellular aggregates generated in this manner may provide a useful system for providing further information on cellular adhesion, intercellular communication, morphogenetic cell movements and the mechanisms of cell death. Dr. Kelley is the recipient of a Research Career Development Award (HD70407).     

Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: A review

A review concerning the definition, extraction, characterization, production and functions of extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment reactors is given in this paper. EPS are a complex high-molecular-weight mixture of polymers excreted by microorganisms, produced from cell lysis and adsorbed organic matter from wastewater. They are a major component in microbial aggregates for keeping them together in a three-dimensional matrix. Their characteristics (e.g., adsorption abilities, biodegradability and hydrophilicity/hydrophobicity) and the contents of the main components (e.g., carbohydrates, proteins, humic substances and nucleic acids) in EPS are found to crucially affect the properties of microbial aggregates, such as mass transfer, surface characteristics, adsorption ability, stability, the formation of microbial aggregates etc. However, as EPS are very complex, the knowledge regarding EPS is far from complete and much work is still required to fully understand their precise roles in the biological treatment process.     

花鼠外部形态及部分内脏器官的测量和分析

对捕捉的花鼠进行了外部形态和内脏器官的测量和分析,结果表明:外部形态和内脏器官的变异系数较大,各器官之间存在着显著的相关关系;不同年龄组间外部形态和内脏器官的大小存在着显著差异,同年龄组不同性别间差异不显著。     

Conformation-dependent recognition of mutant HTT (huntingtin) proteins by selective autophagy

Protein misfolding is the common theme for neurodegenerative disorders including Huntington disease (HD), which is mainly caused by cytotoxicity of the mutant HTT (huntingtin) protein (mHTT). The soluble mHTT has an expanded polyglutamine (polyQ) stretch that may adopt multiple conformations, among which the one recognized by the polyQ antibody 3B5H10 is the most toxic due to unknown mechanisms. In a recent study, we showed that the 3B5H10-recognized mHTT species has a slower degradation rate due to its resistance to selective macroautophagy/autophagy. In HD mouse brain tissues as well as HD patient fibroblasts and post-mortem brain tissues, the 3B5H10-recognized mHTT species lacks Lys63-polyubiquitination and SQSTM1/p62 interaction, which are essential for cargo recognition by selective autophagy. Collectively, we discovered that the mHTT protein is subject to conformation-dependent recognition by selective autophagy, which is more selective than what we perceived: the process can be selective among different conformations of the same protein, leading to conformation-dependent differences in protein degradation and toxicity.     

Real-Time Measurement of Intracellular Reactive Oxygen Species Using Mito Tracker Orange (CMH2TMRos)

We have investigated a novel method to monitor real changes of intracellular ROS by the use of CMH₂TMRos (a reduced form of MitoTracker orange) in Swiss 3T3 fibroblasts. Arachidonic acid induced a rapid increase of CMTMRos fluorescence with a maximal elevation at 120–150 sec, which was determined by scanning every 10 sec with a confocal microscope. The fluorescence increase by arachidonic acid was completely inhibited by 2-MPG but not by catalase, indicating a major contribution of superoxide to the oxidation of CMH₂TMRos. Incubation with glucose oxidase, exogenous H₂O₂, KO₂ and lysophosphatidic acid also increased the CMTMRos fluorescence, which was blocked by 2-MPG. These results suggested that CMH₂TMRos is a useful fluorophore for real-time monitoring of intracellular ROS and also indicated that CMH₂TMRos detects primarily superoxide in cells even though the fluorophore can be oxidized by both superoxide and H₂O₂.     

Measurement of biological aerosol with a fluorescent aerodynamic particle sizer (FLAPS): correlation of optical data with biological data

Biological aerosol measurement in real time is anurgent military requirement that also has manypotential non-military applications. Such detectioncapabilities will be useful in environmentalmonitoring, for example, in gathering information inperceived hazardous areas like housing developmentsdownwind of sewage treatment plants.Experience gained from measuring fluorescence signalsof single bacterial spores under flow cytometry usingUV excitation at 340--360 nm, was applied to concepttesting of a prototype instrument, built to do thesame for aerosols. This machine was capable ofresolving particle size as well as fluorescenceintensity of each particle under laboratory and fieldconditions; it was called the fluorescent aerodynamicparticle sizer (FLAPS). A second generation FLAPS(FLAPS2) was designed to be smaller, power efficientand field portable. FLAPS2 was challenged underrefereed conditions in blind trials to determine if itcould detect biological aerosols in natural fieldenvironment. This paper describes practical aspects ofmeasuring biological aerosols when the results must becompared to reference samplers that provide culturableor ``live' data. Treatment of particle size andfluorescence information is discussed with respect toFLAPS and reference data fidelity. Finally, anobjective method is introduced to evaluate FLAPS datacorrelation to reference data. The measurementssuggest that there is positive correlation betweenFLAPS measurements and live biological aerosolparticles.     

Hydrogen peroxide can be generated by tau in the presence of Cu(II)

Alzheimer's disease has been closely related with oxidative stress, which might be responsible for the dysfunction or death of neuronal cells that contributes to disease pathogenesis. Impaired copper homeostasis makes contribution to the oxidative stress and consequently to several neurodegenerative conditions. Inappropriate binding of Cu(II) to cellular proteins are currently being explored as sources of pathological oxidative stress in several neurodegenerative disorders. Here we report that a fragment of tau protein possesses copper reduction activity and initiates the copper-mediated generation of hydrogen peroxide. The tau peptide was found to be oxidized to form disulfide bond-linked dimer. The hydrogen peroxide generated was quantified by TCEP/DTNB (tris(2-carboxyethyl) phosphine hydrochloride/5,5'-dithio-bis(2-nitrobenzoic acid). Since the copper reduction capacity and the generation of hydrogen peroxide were believe to be a major toxicological pathway of Abeta peptide, the functional similarity shared by tau and Abeta implies a new perspective of tau pathology.     

SERS Study of Tetrodotoxin (TTX) by Using Silver Nanoparticle Arrays

The optical properties of tetrodotoxin (TTX), a scarce toxin with anesthetic properties, were studied using nanoparticle arrays-assisted surface-enhanced Raman scattering (SERS). The nanoparticles arrays were fabricated using nanosphere lithography and a metallic lift-off process to control the particle size, shape, and spacing in the arrays. Using density functional methods, the Raman spectrum of TTX was also calculated with Gaussian03 software. The main peaks of the spectrum are originated from the vibration of the NH₂ molecule group. In the SERS experiment, we were able to measure the Raman spectrum with a TTX concentration as less as 0.9 ng/mL. This sensitivity is comparable to that from high performance liquid chromatography.     

Specific biodegradation of polychlorinated biphenyls (PCBs) facilitated by plant terpenoids

The aim of this study was to examine how plant terpenoids, as natural growth substrates or inducers, would affect the biodegradation of PCB congeners. Various PCB degraders that could grow on biphenyl and several terpenoids were tested for their PCB degradation capabilities. Degradation activities of the PCB congeners, 4,4′-dichlorobiphenyl (4,4′-DCBp) and 2,2′-dichlorobiphenyl (2,2′-DCBp), were initially monitored through a resting cell assay technique that could detect their degradation products. The PCB degraders,Pseudomonas sp. P166 andRhodococcus sp. T104, were found to grow on both biphenyl and terpenoids ((S)-(−) limonene,p-cymene and α-terpinene) whereasArthrobacter sp. B1B could not grow on the terpenoids as a sole carbon source. The B1B strain grown on biphenyl exhibited good degradation activity for 4,4′-DCBp and 2,2′-DCBp, while the activity of strains P166 and T104 was about 25% that of the B1B strain, respectively. Concomitant GC analysis, however, demonstrated that strain T104, grown on (S)-(−) limonene,p-cymene and α-terpinene, could degrade 4,4′-DCBp up to 30%, equivalent to 50% of the biphenyl induction level. Moreover, strain T104 grown on (S)-(−) limonene, could also degrade 2,2′-DCBp up to 30%. This indicates that terpenoids, widely distributed in nature, could be utilized as both growth and/or inducer substrate(s) for PCB biodegradation in the environment.     

Microbial synthesis of cis,cis-muconic acid by Sphingobacterium sp. GCG generated from effluent of a styrene monomer (SM) production plant

Sphingobacterium sp. GCG was isolated from the sewage of a styrene monomer manufacturing factory in Taiwan. This bacterium could utilize benzoic acid to synthesize cis,cis-muconic acid. Production of cis,cis-muconic acid by Sphingobacterium sp. strain is growth-associated. It was found that the strain through the BS-type (benzoic acid/succinic acid) culture medium with succinic acid as a growth carbon source exhibits better growth rate than the other mediums. It was also found that glutamate used as a nitrogen source could optimize the production of cis,cis-muconic acid. In addition, an increment of ca. 75–100% of cis,cis-muconic acid could be obtained by adding EDTA–FeCl₃ complex solution, since the enzyme catA, a necessary oxygenase to convert catechol to cis,cis-muconic acid, consists of 2 Fe(III) ions as the cofactor. On the other hand, a five-fold increase in the yield of cis,cis-muconic acid was observed when proceeded under fed-batch culture.     

Ancestry of plant MADS-box genes revealed by bryophyte (Physcomitrella patens) homologues

Three MADS-box cDNA clones and two corresponding genomic sequences (gDNAs) have been isolated from the bryophyte Physcomitrella patens and sequenced. Our findings indicate that the genes may be expressed in a tissue- or age-specific manner, and that expression of one of them is regulated by an alternative splicing mechanism. Conceptual translation of the clones reveals that the encoded MADS-domain proteins have the typical plant-domain pattern (MIKC). Additionally, there is a high degree of conservation of intron number and positions between angiosperm MADS-box genes and the moss loci. These observations confirm the homology of moss and higher plant MADS-box genes. We conclude that the MIKC pattern evolved in MADS-box genes after the separation of the plant lineage from that of fungi and animals, and that it must have been present in the common ancestor of mosses, ferns and seed plants. Therefore it evolved at least 400 million yr ago. Phylogenetic analysis of a large subset of the sequenced plant MADS-box genes, incorporating those from P. patens , indicates that the bryophyte genes are not orthologues of spermatophyte genes belonging to any of the well recognized higher plant gene subfamilies. This conclusion accords well with reports that the known fern MADS-box genes also comprise subfamilies distinct from those of higher plants. Therefore we tentatively propose that the gene duplication and diversification events that created the MADS-box gene subfamilies, discernible in extant angiosperm and other spermatophyte groups, occurred after separation of the moss and fern lineages from the lineage which produced the higher plants.     

Measurement of human granulocyte-macrophage colony-stimulating factor (GM-CSF) by enzyme-linked immunosorbent assay

Summary An IgG monoclonal antibody against recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), designated HGMI, was produced by fusion of immune mouse splenocytes with HAT-sensitive murine myeloma cells. A sandwich enzyme-linked immunosorbent assay (ELISA) for measurement of human GM-CSF was developed using this HGMI and a polyclonal antibody against GM-CSF raised in a rabbit. GM-CSF in culture supernatants of phytohemagglutinin (PHA)- or concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) were measured by this ELISA system and the conventional CFU-GM colony formation method. The data indicated that the ELISA was highly efficient and sensitive for the detection of as little as 50 pg/ml recombinant GM-CSF. The CFU-GM colony assay may be influenced by other cytokines which can enhance or suppress colony formation, and ELISA for GM-CSF is more useful for kinetic studies of precise levels of production from PBMC.     

Characterization of Trypanosoma (Duttonella) vivax by isoenzyme analysis

Stocks derived from 10 different primary isolates of T. vivax were subjected to isoenzyme analysis for 34 enzymes by both isoelectric focusing in agarose and electrophoresis in starch gel. Trypanosomes were measured and their morphology examined for comparison with the biochemical data. Thirteen enzymes (14 zymograms) were selected to construct isoenzyme profiles. Nine different zymodemes were identified and only two stocks were identical. Both rodent infectivity and the production of the haemorrhagic syndrome could be correlated with the isoenzyme profiles.     

Chemical detoxification vs mechanical removal of host plant toxins in Eucalyptus feeding sawfly larvae (Hymenoptera: Pergidae)

The essential oils that characterize the eucalypts and related Myrtaceae pose a challenge for herbivores. Phytophagous insects that feed on oil-rich Myrtaceae have developed specific mechanisms to deal with these oils, some of which are notoriously toxic (e.g. 1,8-cineole). Some of the eight Australian subfamilies in the sawfly family Pergidae are associated exclusively with Eucalyptus and Melaleuca species that often have high concentrations of essential oils. Unexpectedly, the Perginae and Pterygophorinae use different mechanisms to deal with the same toxic components in their respective host plants. Larvae of the Perginae have the inner surface of their mandibles equipped with soft brush-like structures that are unique among phytophagous insects in general. The proposed role of these ancillary mandibular structures in separating leaf oils from nutritive plant matter could be confirmed in experiments with larvae of two pergine species. The oil sequestration is, however, incomplete and chemical gut content analyses by gas-chromatography (GC) revealed that 1,8-cineole does enter the midgut and is metabolised to hydroxycineole. Although the related Pterygophorinae also feed mainly on oil-rich Myrtaceae, they do not sequester the oil and lack morphological structures on their mandibles. Chemical analysis of the gut content of two pterygophorine species showed that they rely solely on chemical detoxification of the relevant plant compounds, with GC demonstrating that the 1,8-cineole is removed far more rapidly and completely than in the pergine species.     

Inhibition of plant cell membrane transport phenomena induced by zearalenone (F-2)

Zearalenone (F-2), an estrogenic factor produced by a number of Fusarium spp., stimulates a leakage of electrolytes, -cyanin and aminoacids from three plant tissues. F-2 inhibits rubidium uptake in roots of Zea mays L. and Beta vulgaris L. var. rubra. However the effect in the latter tissue is evident after long-term treatments with the toxin. Rubidium uptake is not affected in Solanum tuberosum L. var. Bintje. The toxin also causes inhibition of H⁺ extrusion, of root elongation, of ATPase activity of plasmalemma-enriched fractions and depolarization of transmembrane potentials of corn roots. The evidence presented supports the hypothesis that F-2 affects plasma membranes of several plant species.Abbreviations F-2 Zearalenone - PD Potential difference - FC Fusicoccin - DCCD Dicyclohexylcarbodiimide     

Measurement of k(L)a by dynamic pressure method in pilot-plant fermentor

The dynamic pressure method (DPM) is used for measurement of k(L)a in a 1-m(3) pilot scale fermentor in coalescing (distilled water) and noncoalescing (0.3 M Na(2)SO(4) aqueous solution) batches. The method consists in recording oxygen concentration in a batch after a small pressure change (20 kPa) in the fermentor. The upward pressure change is brought about by temporary closing and subsequent throttling of outlet gas stream and the downward change by full reopening of the gas outlet. Absorption of pure oxygen yields the same k(L)a values as absorption of air. In noncoalescing batch, the downward k(L)a values are always higher than the upward values owing to spontaneous nucleation of bubbles. The experiments performed in a stirred cell confirm this behavior. Thus, only upward pressure change should be used for measurement. The correlation of k(L)a data measured in small (18-L) and large (1000-L) vessels based on power dissipated and superficial gas velocity are in a good agreement. Unlike the DPM, the classical dynamic methods yield, under the same conditions, excessively low values of k(L)a (the dynamic startup method) or fail to produce data at all (the dynamic method with interchange of air for N(2)). (c) 1994 John Wiley & Sons, Inc.     

Gold nanoparticles decorated with oligo(ethylene glycol) thiols: kinetics of colloid aggregation driven by depletion forces

We have studied the kinetics of the phase-separation process of mixtures of colloid and protein in solutions by real-time UV-vis spectroscopy. Complementary small-angle X-ray scattering (SAXS) was employed to determine the structures involved. The colloids used are gold nanoparticles functionalized with protein resistant oligo(ethylene glycol) (OEG) thiol, HS(CH(2))(11)(OCH(2)CH(2))(6)OMe (EG6OMe). After mixing with protein solution above a critical concentration, c*, SAXS measurements show that a scattering maximum appears after a short induction time at q = 0.0322 A(-1), which increases its intensity with time but the peak position does not change with time, protein concentration and salt addition. The peak corresponds to the distance of the nearest neighbor in the aggregates. The upturn of scattering intensities in the low q-range developed with time indicating the formation of aggregates. No Bragg peaks corresponding to the formation of colloidal crystallites could be observed before the clusters dropped out from the solution. The growth kinetics of aggregates is followed in detail by real-time UV-vis spectroscopy, using the flocculation parameter defined as the integral of the absorption in the range of 600-800 nm wavelengths. At low salt addition (<0.5 M), a kinetic crossover from reaction-limited cluster aggregation (RLCA) to diffusion-limited cluster aggregation (DLCA) growth model is observed, and interpreted as being due to the effective repulsive interaction barrier between colloids within the depletion potential. Above 0.5 M NaCl, the surface charge of proteins is screened significantly, and the repulsive potential barrier disappeared, thus the growth kinetics can be described by a DLCA model only.