Electron paramagnetic resonance study of the migratory ant Pachycondyla marginata abdomens

Electron paramagnetic resonance was used to investigate the magnetic material present in abdomens of Pachycondyla marginata ants. A g congruent with 4.3 resonance of high-spin ferric ions and a very narrow g congruent with 2 line are observed. Two principal resonance broad lines, one with g > 4.5 (LF) and the other in the region of g congruent with 2 (HF), were associated with the biomineralization process. The resonance field shift between these two lines, HF and LF, associated with magnetic nanoparticles indicates the presence of cluster structures containing on average three single units of magnetite-based nanoparticles. Analysis of the temperature dependence of the HF resonance linewidths supports the model picture of isolated magnetite nanostructures of approximately 13 nm in diameter with a magnetic energy of 544 K. These particles are shown to present a superparamagnetic behavior at room temperature. The use of these superparamagnetic particle properties for the magnetoreception process of the ants is suggested.     

Infectious necrotizing enteritis and mortality caused by Vibrio carchariae in summer flounder Paralichthys dentatus during intensive culture

An epizootic causing mortality among cultured summer flounder Paralichthys dentatus occurred in summer of 1998 at a land-based facility on Narragansett Bay, Rhode Island, USA. The disease, flounder infectious necrotizing enteritis (FINE), was characterized by reddening around the anal area, distended abdomens filled with opaque serosanguineous fluid, enteritis and necrosis of the posterior intestine. In extreme cases of the disease, the posterior intestine was detached from the anus and was observed coming out the vent. The intestine of individuals that recovered from the disease ended in a blind-sac; the abdomens of these fish were distended, due to food and water inside the intestinal blind-sac. A bacterium was isolated from ascites fluid and kidney of moribund flounder and identified as the causative agent in challenge experiments. The pathogen was identified as Vibrio carchariae by morphological and biochemical characteristics and sequence of the 16S rRNA. The LD50 estimate was 5 x 10(5) colony-forming units injected intraperitoneally into 100 to 200 g summer flounder.     

The roles of juvenile hormone and 20-hydroxy-ecdysone during vitellogenesis in isolated abdomens of Drosophila melanogaster

Both juvenile hormone and 20-hydroxy-ecdysone seem to be involved in the regulation of vitellogenesis in Drosophila melanogaster. It is the purpose of this paper to begin to define the functions of these two hormones. Although vitellogenin synthesis does not occur at a high rate in 1-day-old female abdomens isolated from the head and thorax before 0.75 hr after eclosion, both ZR515 (a juvenile hormone analogue) and 20-hydroxy-ecdysone can cause in these preparations vitellogenin synthesis and secretion into the haemolymph. The synthesis and secretion into the haemolymph of all three vitellogenins which are detectable by electrophoresis in sodium dodecyl sulphate-containing gels of polyacrylamide is promoted by both hormones. That result excludes the hypothesis that these two hormones regulate the synthesis of different vitellogenins. A dose-response curve showed that an injection of 0.2 μl of a 10^?6 M 20-hydroxy-ecdysone solution was sufficient to promote vitellogenin synthesis and secretion in isolated abdomens. Ovaries from isolated female abdomens treated with juvenile hormone analogue showed nearly normal amounts of all three vitellogenins and morphologically normal advanced vitellogenic follicles, whereas ovaries from isolated abdomens treated with 20-hydroxy-ecdysone contained little vitellogenin and no vitellogenic follicles. We conclude that under the conditions used, juvenile hormone permits vitellogenin uptake into the oöcyte much more readily than does 20-hydroxy-ecdysone.     

Quantitative analysis of the metabolism in the isolated pupal abdomens of the silkworm, Bombyx mori, infected with nuclear polyhedrosis virus

The rate of oxygen uptake and the amount of several main cell constituents were determined in NPV-infected and uninfected isolated pupal abdomens of the silkworm, Bombyx mori. The oxygen uptake in uninfected isolated abdomens increases for the first 2 days due to the effect of water injection, but thereafter it remains almost unchanged at a relatively low level. When isolated abdomens are infected with an NPV, the oxygen uptake increases markedly from 3 days postinfection onward, being about three times that of uninfected isolated abdomens at late stages of infection. The amounts of cell constituents analyzed in the present study change little in uninfected isolated abdomens throughout the experiment. Infection of NPV leads to a marked accumulation of both DNA and RNA, and a loss of glycogen. The amount of protein, lipid, and sugar are scarcely affected by NPV infection. These results indicate that the activity of metabolism in uninfected isolated abdomens is not only low but also stable, and that infection of NPV results in an activation of host cell metabolism. It is considered that such isolated pupal abdomens provide an excellent opportunity to analyze both the process of NPV replication and the alteration of host cell metabolism resulting from NPV infection.     

Ecdysteroid synthesis and molting by the tobacco hornworm, Manduca sexta, in the absence of prothoracic glands

When a pair of prothoracic glands (PGs) were removed from Manduca sexta pupae on the day of pupation, the hemolymph ecdysteroid titer remained at a low level. When a portion of the gland pair was extirpated from pupae after the critical period for prothoracicotropic hormone release, the maximum hemolymph ecdysteroid titer was reduced in proportion to the mass of the PGs removed. These findings clearly showed that the PGs in intact pupae are responsible for the elevated ecdysteroid titer required to elicit adult development on schedule. When brains were removed on the day of pupation, the initiation of adult development was delayed for weeks or months. In contrast, pupae whose PGs were removed on the day of pupation initiated development only 7 days late, indicating the existence of an additional source of pupal ecdysteroids. Further, abdomens of male M. sexta that were isolated on the day of pupation initiated adult development spontaneously within 70 days. The implantation of day 0 pupal brains into these isolated abdomens accelerated the initiation of adult development and elicited synchronous adult development. The hemolymph ecdysteroid titer of those isolated abdomens receiving implants of brains increased within 5 days and reached a maximum level of 1.5 micrograms/ml. The analysis of hemolymph ecdysteroids by reverse-phase HPLC revealed that ecdysone was the major moiety and that the ecdysteroid composition was similar to that of normal, intact pupae that had just initiated adult development. These results demonstrate that the PGs are not requisite for adult development. An increased hemolymph ecdysteroid titer was also observed in isolated abdomens from which the testes were removed and in abdomens devoid of their digestive tract. Indeed, in the latter case, the ecdysteroid titer attained much higher levels than those observed for abdomens with intact guts. Despite numerous attempts to identify the tissue(s) in the isolated abdomens responsible for the increase in ecdysteroid titer, its identity remains unknown.     

Double abdomen induction by UV in Bradysia tritici (syn. Sciara ocellaris,Sciaridae): sensitive stages and conditions for photoreversal

Summary The pattern anomaly double abdomen was induced in embryos of Bradysia tritici (syn. Sciara ocellaris) by irradiation of the anterior egg pole with far UV (254 or 285 nm) using low UV fluences. The maximum yield of 18% of double abdomens was obtained when 2.5 h embryos were irradiated (late intravitelline cleavage stage); earlier irradiation failed to yield double abdomens, as did irradiations after the early syncytial blastoderm stage. Exposing irradiated embryos to photoreverting light (366 nm) reduced the yield of malformations. Most double abdomens were symmetrical and the number of segments ranged from 3 to 8 in each set, with the mean value at 6.4 segments.     

The Regulation of yolk polypeptide synthesis in Drosophila ovaries and fat body by 20-hydroxyecdysone and a juvenile hormone analog

In adult female Drosophila melanogaster an increase in the synthesis and secretion of three yolk polypeptides (YPs) occurs during the first 24 hr after eclosion. During organ culture, these same polypeptides are synthesized and secreted into the medium by both fat body and ovaries. Two hormones, 20-hydroxyecdysone (20-HE) and a juvenile hormone analog (ZR-515) stimulate synthesis and secretion of YPs into the hemolymph of isolated female abdomens. The present experiments were undertaken to compare synthesis of YPs in normal females with YP synthesis in preparations deprived of anterior endocrine glands, and to find which hormone stimulates synthesis in the different organs. Separation of hemolymph proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that at eclosion incorporation of [³⁵S]methionine into YP1 and YP2 was low and was barely detectable in YP3. Over the next 24 hr the rate of label incorporation increased for all the YPs. Isolation of female abdomens at eclosion prevented this increase in label incorporation but did not entirely abolish YP synthesis. Application of either ZR-515 or 20-HE to isolated abdomens stimulated up to ninefold label incorporation into three polypeptides which comigrated with YPs from normal vitellogenic females. The response of isolated abdomens to ZR-515 or 20-HE was first detectable between 90 and 135 min after hormone application. The stimulated bands were confirmed to be YPs by a comparison of peptide digests of each of the three labeled polypeptides with those of the yolk polypeptides from intact vitellogenic females. The hypothesis that the two hormones might act on different organs was tested by treating isolated female abdomens with various concentrations of either ZR-515 or 20-HE and then culturing the stimulated organ in vitro with [³⁵S]methionine. The fat body responded to both hormones by synthesizing and secreting into the culture medium polypeptides which comigrated with the YPs found in hemolymph, whereas the ovary produced similar polypeptides only after ZR-515. These secreted polypeptides were confirmed to be YPs by repeating the experiment using organs from heterozygotes for both YP2 and YP3 electrophoretic variants. Such organs synthesized five polypeptides which comigrated with the corresponding yolk polypeptides. These findings are discussed in relation to a hypothesis for the action of the two hormones.     

Cytoplasmic motions, rheology, and structure probed by a novel magnetic particle method

The motions of magnetic particles contained within organelles of living cells were followed by measuring magnetic fields generated by the particles. The alignment of particles was sensed magnetometrically and was manipulated by external fields, allowing non-invasive detection of particle motion as well as examination of cytoplasmic viscoelasticity. Motility and rheology data are presented for pulmonary macrophages isolated from lungs of hamsters 1 d after the animals had breathed airborne gamma-Fe2O3 particles. The magnetic directions of particles within phagosomes and secondary lysosomes were aligned, and the weak magnetic field produced by the particles was recorded. For dead cells, this remanent field was constant, but for viable macrophages, the remanent field decreased rapidly so that only 42% of its initial magnitude remained 5 min after alignment. A twisting field was applied perpendicular to the direction of alignment and the rate at which particles reoriented to this new direction was followed. The same twisting was repeated for particles suspended in a series of viscosity standards. Based on this approach, the low-shear apparent intracellular viscosity was estimated to be 1.2-2.7 X 10(3) Pa.s (1.2-2.7 X 10(4) poise). Time-lapse video microscopy confirmed the alignment of ingested particles upon magnetization and showed persistent cellular motility during randomization of alignment. Cytochalasin D and low temperature both reduced cytoplasmic activity and remanent-field decay, but affected rheology differently. Magnetic particles were observed in association with the microtubule organizing center by immunofluorescence microscopy; magnetization did not affect microtubule distribution. However, both vimentin intermediate filaments and f-actin reorganized after magnetization. These data demonstrate that magnetometry of isolated phagocytic cells can probe organelle movements, rheology, and physical properties of the cytoskeleton in living cells.     

Isolation,Characterization, and Stability of Discretely-Sized Nanolipoprotein Particles Assembled with Apolipophorin-III

Background

Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs.

Methodology

Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to >25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4°C was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes.

Conclusions

ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utility for biotechnological applications.     

腹部恒磁场作用对大鼠药物性胃损伤的治疗机制探讨

目的：观察腹部恒磁场治疗急性药物性胃损伤模型的同时,大鼠胃粘膜组织内皮素1（endothelin—1,ET-1）、一氧化氮（nitric oxide,NO）、谷胱甘肽过氧化物酶（glutathione peroxidase,GSH—Px）及超氧化物岐化酶（superoxide dismutase,SOD）的水平,探讨磁场治疗胃损伤的作用机制。方法：10只健康SD大鼠,在Indomethacin胃灌注法复制急性胃损伤模型后,以钡铁氧体恒磁场（表面磁强度为1300—1600GS）作用大鼠腹部3小时,观察胃损伤指数及病理损伤积分,同时对胃粘膜组织中ET-1、NO、GSH—Px及SOD水平进行比较。结果：腹部磁场作用3小时后,大鼠胃损伤指数及病理损伤积分均显著减轻（P均〈0．05）;胃粘膜组织内ET-1、NO及GSH—Px水平均无显著改变（p均〉0．05）,SOD含量较对照组均明显升高（P〈0．05）。结论：恒磁场（1300—1600CS）腹部作用3小时,大鼠急性胃损伤程度可明显减轻,磁场对胃粘膜组织内自由基的影响可能参与其作用机制。     

Association of ribosomes with in vitro assembled microtubules

Microtubules were purified from unfertilized eggs of the sea urchins Arbacia punctulata, Lytechinus pictus, Lytechinus variegatus, and Strongylocentrotus purpuratus. Numerous densely stained particles (24 x 26 nm) are associated with microtubules isolated from each of these sea urchins. The most striking aspect of this structure is an extended, slightly curved arm that appears to attach the particles to the microtubule. Morphologically similar particles are associated with microtubules of the isolated first cleavage mitotic apparatus. The particles are attached to the microtubules by ionic interactions and contain large amounts of extractable RNA. Based upon their size and density, RNA and protein composition, and sedimentation in sucrose gradients, the microtubule-associated particles are identified as ribosomes.     

The effect of size of polystyrene particles on their retention within the rat peritoneal compartment, and on their interaction with rat peritoneal macrophages in vitro

Polystyrene particles (size range 300 nm-3 microns diameter) were radioiodinated and their capture by rat peritoneal macrophages measured in vitro. For unmodified particles, most efficient accumulation was observed using a diameter of 600 nm (Endocytic Index (E.I.) = 16.4 +/- 2.9 microliters/10(6) cells/h). Particles (3 microns diameter) which had been modified to become more hydrophilic by hydroxymethylation showed an increased rate of capture (E.I. = 136.6 +/- 91.2 microliters/10(6) cells/h). Following intraperitoneal administration to rats, unmodified 3 micron particles showed selective accumulation in the omentum (18.4% injected dose/g), and this was increased for the hydroxymethylated bead (35.3% dose/g). The smaller (800 nm) particles were better able to leave the peritoneal compartment. Radiolabelled particles isolated from a peritoneal wash after 5 h were mostly cell-associated (72-86%, depending on the type of particle).     

Morphology of mouse egg cylinder development in vitro: a light and electron microscopic study.

The endocrine control of yolk deposition in Drosophila melanogaster was studied by ligation and transplantation techniques. Endocrine events associated with the initiation of vitellogenesis were found to be synchronized with eclosion rather than the completion fo adult development. Decapitation experiments showed that a cephalic event occurring at about the time of eclosion is necessary for each animal to initiate vitellogenesis. The morphogenetic effect of the head could be replaced by a juvenile hormone analog (JHA). In addition to the cephalic event, a thoracic factor is required for each follicle to initiate vitellogenesis, since preparation of isolated abdomens before 16 hours after eclosion prevented vitellogenesis. In abdomens isolated after this time, no early vitellogenic stages were formed. The suppression of vitellogenesis in isolated abdomens was reversed by implanting corpora allata or by treating these preparations with JHA, but not by implanting corpora cardiaca. Ovaries that were artificially induced to mature by treating isolated abdomens with JHA still displayed the normal complement of ovarian proteins after electrophoresis in polyacrylamide gels. These results show that a circadian clock triggers vitellogenesis via a cephalic signal at eclosion, which in turn triggers events in the thorax or abdomen. The cephalic signal can be superseded by juvenile hormone, whose presence is necessary for each follicle to become vitellogenic.     

Iron-rich particles in European eel (Anguilla anguilla L.)

Magnetic material in the European eel (Anguilla anguilla L.) was investigated by a combination of magnetic susceptibility measurements, energy dispersive X-ray fluorescence analysis and transmission electron microscopy. It was shown that the magnetic material is associated with iron. The main part of the iron is present in the form of iron-rich particles with irregular shapes about 100-3000 A large. The structures of magnetite (Fe3O4), hematite (alpha-Fe2O3) and alpha-iron (bcc structure) were identified. The particles are composed of more than one of these phases with magnetite being a minority phase when present. The iron-rich particles found in the eel are different from the materials reported for bacteria or bees.     

Biomolecular reactions studied using changes in Brownian rotation dynamics of magnetic particles

We have used magnetic particles to study specific binding of prostate specific antigen (PSA) to the surfaces of the bioparticles. The used particles have a mean diameter of about 130 nm and are placed in phosphate buffer saline (PBS). Each particle is composed of clusters of magnetic single domains of magnetite, which are covered with dextran. Changes in surface chemistry of the particles give rise to a change in the hydrodynamic volume of the particles. The later is mirrored by the changed frequency response of the complex magnetic susceptibility of a fluid containing these particles. Using ordinary induction coils and the lock-in amplifier technique it is possible to measure the complex magnetic susceptibility of the particle solution in a frequency range from about 10 Hz up to 10 kHz. From the measurement of the complex susceptibility versus the excitation frequency (both at the excitation frequency as well as at higher harmonics) we have shown that it is possible to quantitatively study the binding of PSA to the surfaces of the magnetic particles and thus to determine the PSA concentration in solution containing known concentration of nanoparticles functionalised with a monoclonal PSA antibody. Our method allows to perform an immunoassay in a single step and is much faster and cheaper compared to conventional ELISA procedures.     

Morphology and molecular composition of isolated postsynaptic junctional structures.

The solubilization of isolated brain synaptosomal plasma membranes by various detergents was studied and in each case found to depend upon detergent concentration. By using conditions sufficient to extract maximally protein and phospholipid from the membranes, postsynaptic junctional particles were isolated with each of four detergents and their ultrastructural appearances and protein contents compared. Two basic structural forms were identified. One, isolated with Triton X-100, consists of a planar array of dense-staining particles ca. 20 nm in diameter. It resembles the postsynaptic density seen in undigested synaptosomal plasma membranes. The other, isolated with sodium deoxycholate, contains less protein. It has the same overall shape and dimensions as the postsynaptic density, but consists of a branching network of short 5 nm fibres (the postsynaptic junctional lattice) making up an array of contiguous polygons, each ca. 20 nm across. The interior of these polygonal elements seems to be hydrophobic since it cannot be penetrated by metallic salts used for negative staining. It is suggested that the dense-staining 20 nm subunits observed at the postsynaptic junctional site may be composed of hydrophobic proteins inserted into the hollow cores of the lattice polygons. Electrophoretic analysis of the proteins present in the various postsynaptic junctional preparations identified two major common components with molecular masses of 275000 and 47500. The latter is tentatively identified as actin. Components comigrating respectively with alpha- and beta-tubulin are present, and the relation of the lattice structure to subjacent microtubules is discussed.     

Factors affecting change in sensitivity of prophoracic glands to juvenile hormone in Mamestra brassicae

The prothoracic glands of the early last-instar larva of Mamestra brassicae (day 0–3) were found previously to be insensitive to stimulation by juvenile hormone, whereas those later in the instar (from day 4 on) were activated by this hormone. When neck-ligatured young larvae (day-1, day-2 and day-3) were given juvenile hormone 5–10 days after ligation, pupation was induced. Similarly, juvenile hormone induced pupation of isolated abdomens which contained prothoracic glands taken from neck-ligatured day-3 larvae 5 days after ligation. If the glands were exposed to prothoracicotropic hormone (PTTH) from implanted brains before they were transplanted to isolated abdomens, their sensitivity to juvenile hormone activation was enhanced. Ecdysone but not 20-hydroxyecdysone given every 3 hr for 12 hr also slightly enhanced sensitivity. These results suggest that prothoracic glands from either day-1, day-2 or day-3 larvae can slowly acquire a sensitivity to juvenile hormone activation by prolonged incubation in the absence of factors from the head. The acquisition of sensitivity occurs more rapidly in the presence of both a factor from the brain, presumably PTTH, and ecdysone released from the prothoracic glands themselves.     

固体平板磁泳分离细菌新方法的研究

氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans）能够在胞内形成电子致密的磁性颗粒,它的这种特性使利用氧化亚铁硫杆菌合成生物纳米磁性材料成为了可能。本课题组为了筛选出合成磁性颗粒能力强的菌株,对原有的液体磁泳进行了改进,采用了新的固体平板磁泳方法来筛选纯化目的菌株。经过磁泳分离后,细菌中含磁性颗粒的细胞比例由原始菌群的30％上升到90％,胞内含有的磁颗粒数目也由1~2颗增加至2~5颗,筛选得到的细菌在人工磁场下会进行趋磁运动。实验结果表明,氧化亚铁硫杆菌具有较弱的趋磁性,在人工磁场下会进行趋磁运动,但仅在地磁场作用下不能定向运动,利用固体平板磁泳筛选纯化含有磁性颗粒的氧化亚铁硫杆菌的方法是切实可行的,磁泳分离技术的进一步完善和改进为传统的微生物菌种分离提供了新的途径,为研究纯氧化亚铁硫杆菌菌株胞内磁性颗粒的形成条件及机理提供了前提条件,也为今后从浸矿细菌中分离筛选更多的含有磁性颗粒的菌株打下基础。     

Dendritic cell uptake of iron-based magnetic nanoparticles

We have investigated the internalization of magnetic nanoparticles (NPs) into dendritic cells (DCs) in order to assess both the final location of the particles and the viability of the cultured cells. The particles, consisting of a metallic iron core covered with carbon, showed no toxic effects on the DCs and had no effect in their viability. We found that mature DCs are able to incorporate magnetic nanoparticles in a range of size from 10 nm to ca. 200 nm, after 24 h of incubation. We describe a method to separate cells loaded with NPs, and analyze the resulting material by electron microscopy and magnetic measurements. It is found that NPs are internalized in lysosomes, providing a large magnetic signal. Our results suggest that loading DCs with properly functionalized magnetic NPs could be a promising strategy for improved vectorization in cancer diagnosis and treatment.     

Magnetic properties of human liver and brain ferritin

Human brain (globus pallidus) and liver tissues were investigated by means of electron microscopy (EM), Mössbauer spectroscopy (MS) and SQUID magnetometry techniques. Based on MS measurements, the iron present was identified to be in the ferritin-like form (61–88%) and in the form of a low-spin iron species (the balance). Its overall concentration was estimated as 1.5(3) mg in the brain and 2.4(5) mg in the liver, per gram of lyophilized tissue. The average core diameter was determined by EM measurements to be equal to 7.5(1.3) nm for the liver and 3.3(5) nm for the brain. Magnetization measurements carried out between 5 and 300 K yielded an estimation of an average blocking temperature, KT _BL, as equal to 6.7 K and 8.5 K for the liver and the brain, respectively. From the dependence of KT _BL on the external magnetic field it was concluded that the ferritin-like cores in the studied samples can be regarded as non-interacting particles. Finally, the uniaxial magnetic anisotropy constant was determined to be 6×10³ J/m³ for the liver and 4×10⁴ J/m³ for the brain.