Effect of weightlessness on colloidal particle transport and segregation in self-organising microtubule preparations

Weightlessness is known to effect cellular functions by as yet undetermined processes. Many experiments indicate a role of the cytoskeleton and microtubules. Under appropriate conditions in vitro microtubule preparations behave as a complex system that self-organises by a combination of reaction and diffusion. This process also results in the collective transport and organisation of any colloidal particles present. In large centimetre-sized samples, self-organisation does not occur when samples are exposed to a brief early period of weightlessness. Here, we report both space-flight and ground-based (clinorotation) experiments on the effect of weightlessness on the transport and segregation of colloidal particles and chromosomes. In centimetre-sized containers, both methods show that a brief initial period of weightlessness strongly inhibits particle transport. In miniature cell-sized containers under normal gravity conditions, the particle transport that self-organisation causes results in their accumulation into segregated regions of high and low particle density. The gravity dependence of this behaviour is strongly shape dependent. In square wells, neither self-organisation nor particle transport and segregation occur under conditions of weightlessness. On the contrary, in rectangular canals, both phenomena are largely unaffected by weightlessness. These observations suggest, depending on factors such as cell and embryo shape, that major biological functions associated with microtubule driven particle transport and organisation might be strongly perturbed by weightlessness.     

Ultrastructure of a novel tube-forming, intracellular parasite of dinoflagellates: Parvilucifera prorocentri sp. nov. (Alveolata, Myzozoa)

We have characterized the intracellular development and ultrastructure of a novel parasite that infected the marine benthic dinoflagellate Prorocentrum fukuyoi. The parasite possessed a combination of features described for perkinsids and syndineans, and also possessed novel characters associated with its parasitic life cycle. Reniform zoospores, about 4 μm long, possessed a transverse flagellum, alveoli, a refractile body, a mitochondrion with tubular cristae, a syndinean-like nucleus with condensed chromatin, micronemes, bipartite trichocysts with square profiles (absent in perkinsids) and oblong microbodies. Like Parvilucifera, the zoospores also possessed a shorter posterior flagellum, a heteromorphic pair of central microtubules in the anterior axoneme and a reduced pseudoconoid positioned directly above an orthogonal pair of basal bodies. Early developmental stages consisted of a sporangium about 5–15 μm in diam that contained spherical bodies and amorphous spaces. The undifferentiated sporangium increased to about 20–25 μm in diam before being enveloped by a wall with a convoluted mid-layer. The sporangium differentiated into an unordered mass of zoospores that escaped from the cyst through a pronounced germ tube about 4–5 μm in diam and 10–15 μm long. Weakly developed germ tubes have been described in Perkinsus but are absent altogether in Parvilucifera and syndineans. Comparison of these data with other myzozoans led us to classify the parasite as Parvilucifera prorocentri sp. nov., Myzozoa. Although we were hesitant to erect a new genus name in the absence of molecular sequence data, our ultrastructural data strongly indicated that this parasite is most closely related to perkinsids and syndineans, and represents an intriguing candidate for the cellular identity of a major subclade of Group I alveolates.     

Ground-based methods reproduce space-flight experiments and show that weak vibrations trigger microtubule self-organisation

The effect of weightlessness on physical and biological systems is frequently studied by experiments in space. However, on the ground, gravity effects may also be strongly attenuated using methods such as magnetic levitation and clinorotation. Under suitable conditions, in vitro preparations of microtubules, a major element of the cytoskeleton, self-organise by a process of reaction–diffusion: self-organisation is triggered by gravity and samples prepared in space do not self-organise. Here, we report experiments carried out with ground-based methods of clinorotation and magnetic levitation. The behaviour observed closely resembles that of the space-flight experiment and suggests that many space experiments could be carried out equally well on the ground. Using clinorotation, we find that weak vibrations also trigger microtubule self-organisation and have an effect similar to gravity. Thus, in some in vitro biological systems, vibrations are a countermeasure to weightlessness.     

Microspike-mediated particle transport towards the cell body during early spreading of 3T3 cells

Freshly trypsinized 3T3 cells send out microspikes of 0.2 μm diameter and up to 10 μm length within 20 min after attachment to a glass substratum. The microspikes move actively and eventually attach to the substratum. Subsequently, lamellae flow out between lines of attached microspikes. If, however, colloidal gold particles of 0.2–0.4 μm diameter and clusters of gold particles up to 4 μm in diameter are placed on the substratum and a microspike attaches to them, we observed two reactions of the microspikes to this contact. They either retract upon contact, transporting the attached particles to the cell surface at a speed of 0.2 μm/sec, or the particles flow toward the cell body while the microspike stays in place. This action results in the clearing of a circular area around each spreading cell before lamellae flow out. “Clearing” proceeds at serum concentrations between 1 and 20% and in concentrations of colchicine up to 20 μm/ml. In concentrations of cytochalasin B higher than 5 μg/ml, however, particle removal is completely inhibited, although the microspikes are still produced by the cell. Transmission electron microscopy shows that the microspikes contain mostly longitudinally oriented microfilaments and only a few microtubules, if any.     

Numerical simulations of microtubule self-organisation by reaction and diffusion

This article addresses the physical chemical processes underlying biological self-organisation by which a homogenous solution of reacting chemicals spontaneously self-organises. Theoreticians have predicted that self-organisation can arise from a coupling of reactive processes with molecular diffusion. In addition, the presence of an external field, such as gravity, at a critical moment early in the process may determine the morphology that subsequently develops. The formation, in-vitro, of microtubules, a constituent of the cellular skeleton, shows this type of behaviour. The preparations spontaneously self-organise by reaction-diffusion and the morphology that develops depends upon the presence of gravity at a critical bifurcation time early in the process. Here, we present numerical simulations of a population of microtubules that reproduce this behaviour. Microtubules can grow from one end whilst shrinking from the other. The shrinking end leaves behind a chemical trail of high tubulin concentration. Neighbouring microtubules preferentially grow into these regions, whilst avoiding regions of low tubulin concentration. The chemical trails produced by individual microtubules thus activate and inhibit the formation of neighbouring microtubules and this progressively leads to self-organisation. Gravity acts by way of its directional interaction with the macroscopic density fluctuations present in the solution arising from microtubule disassembly.     

Photoprotective potential of lycopene, β-carotene, vitamin E, vitamin C and carnosic acid in UVA-irradiated human skin fibroblasts

The photoprotective potential of the dietary antioxidants vitamin C, vitamin E, lycopene, β-carotene, and the rosemary polyphenol, carnosic acid, was tested in human dermal fibroblasts exposed to ultraviolet-A (UVA) light. The carotenoids were prepared in special nanoparticle formulations together with vitamin C and/or vitamin E. Nanoparticle formulations, in contrast to dimethylsulphoxide, stablized lycopene in the cell culture medium and allowed efficient cellular uptake. The presence of vitamin E in the formulation further increased the stability and cellular uptake of lycopene. UVA irradiation of the human skin fibroblasts led to a 10–15-fold rise in metalloproteinase 1 (MMP-1) mRNA. This rise was suppressed in the presence of low μM concentrations of vitamin E, vitamin C, or carnosic acid but not with β-carotene or lycopene. Indeed, in the presence of 0.5–1.0 μM β-carotene or lycopene, the UVA-induced MMP-1 mRNA was further increased by 1.5–2-fold. This increase was totally suppressed when vitamin E was included in the nanoparticle formulation. Heme-oxygenase 1 (HO-1) mRNA expression was strongly induced by UVA irradiation but none of the antioxidants inhibited this effect at the concentrations used in this study. Indeed, β-carotene or lycopene (0.5–1.0 μM) led to a further 1.5-fold rise in the UVA-induced HO-1 mRNA levels. In conclusion, vitamin C, vitamin E, and carnosic acid showed photoprotective potential. Lycopene and β-carotene did not protect on their own but in the presence of vitamin E, their stability in culture was improved and the rise in MMP-1 mRNA expression was suppressed, suggesting a requirement for antioxidant protection of the carotenoids against formation of oxidative derivatives that can influence the cellular and molecular responses.     

Catalase-positive particles (“microperoxisomes”) from rat preputial gland and bovine adrenal cortex

Catalase activity was detected histochemically within membrane-bound cell organelles in epithelial cells of rat preputial gland and bovine adrenal cortex. These particles are oval to worm-like in rat preputial gland, 0.08 – 0.15 μm thick and up to 1.0 μm long. In bovine adrenal cortex the shape of catalase-positive particles is rather spherical (diameter 0.1 to 0.3 μm). Particles of both organs lack crystalline or dense cores.Biochemical examination of cell fractions prepared from tissue homogenates by differential centrifugation revealed the presence of two typical peroxisomal oxidases, viz. α-hydroxy acid and -amino acid oxidase, with maximal relative specific activities in the ‘microsomal’ fraction (preputial gland) and in the ‘lysosomal’ fraction (adrenal cortex), respectively. Urate oxidase is absent in both tissues.The concomitant occurrence of catalase and hydrogen peroxide producing oxidases in the particles described characterizes them as true peroxisomal systems (‘microperoxisomes’).     

Optimal design of bio-hybrid systems with a hollow fiber scaffold: model analysis of oxygen diffusion/consumption

The oxygen distribution in various bio-hybrid systems composed of cellular tissue on an artificial scaffold was estimated by mathematically modeling the oxygen consumption and diffusion. Mathematical models were established for practical systems such as bio-hybrid artificial liver (BAL) and bio-hybrid blood vessels, and the calculated results were compared with corresponding experimental data. Analysis of a spherical organoid (“spheroid”) composed of hepatic cells suggested that the oxygen consumption rate in hepatocyte spheroids incubated in a BAL is one or two orders of magnitude larger than the total average value that had been calculated for various organs. A model was established for a BAL system on a scaffold of commercially available hollow fiber (typical inner and outer radii of 150 and 200 μm) to determine the optimal conditions under which the hepatocytes can be packed as closely as possible into the hollow fiber lumen while still maintaining viability without falling into oxygen deficiency. A model of bio-hybrid blood vessels formed by vascular endothelial cells incubated on the inner wall of a hollow fiber scaffold was used to estimate the maximum thickness of viable endothelial tissue under various conditions of outer partial oxygen pressure and different sizes and permeabilities of the hollow fiber scaffold. The model suggested that the oxygen supply becomes quite restricted when the hollow fiber membrane is thicker than 100 μm; the thickness of the endothelium in a 500 μm-thick hollow fiber membrane was estimated to be 7 μm at most, even when the membrane permeability was as large as that of the culture medium.     

Effects of nalidixic acid, chloramphenicol, cycloheximide, and anisomycin on structure and development of plastids and mitochondria in greening Euglena gracilis

The substructure of plastids and mitochondria and the alterations caused by the addition of antibiotics were investigated during light-induced proplastid-to-chloroplast transformation in Euglena gracilis. The organisms were grown in presence of the inhibitors up to 3 days (5 generations). Both 40 μg/ml nalidixic acid and 1–1.5 mg/ml chloramphenicol prevent the formation of chloroplasts of normal size and structure by blocking development during early stages. Under our conditions 2 to 5 straight thylakoids are formed beside 1 to 2 girdle-like thylakoids. The former rarely fuse into bands. Non-crystalline prolamellar bodies of considerable size are formed at the distended ends of the plastids in the presence of both drugs. Chloramphenicol also influences mitochondrial size, shape and internal structure. Giant mitochondria can be observed. Nalidixic acid does not change the size and shape of mitochondria, but the matrix frequently appears highly osmiophilic. Cycloheximide in sublethal doses (2–5 μg/ml) or 50 μg/ml anisomycin inhibits plastid development only in the early period after addition. In later culture periods chloroplasts are found enlarged in size with an increased number of thylakoids and bands per organelle. Insertions of new bands are noted at the inner membrane of the chloroplast envelope. The electronmicroscopic observations agree with the results of chloroplast-specific biochemical activities such as light-induced increase in chlorophyll synthesis and of two chloroplast-bound enzyme activities. The results are discussed with respect to metabolic and biogenetic correlations between the two types of organelles in E. gracilis cells.     

Passage of cell through membranes of Millipore diffusion chambers

Diffusion chambers assembled with Millipore filters previously soaked in water are penetrable by peritoneal exudate cells. Those constructed with dry Millipore filters of porosity 0.1 and 0.22 μm are not penetrable by such cells, but they become penetrable when pore size reaches 0.3 μm.     

Large-sliding contact elements accurately predict levels of bone-implant micromotion relevant to osseointegration

Primary stability is recognised as an important determinant in the aseptic loosening failure process of cementless implants. An accurate evaluation of the bone–implant relative micromotion is becoming important both in pre-clinical and clinical studies. If the biological threshold for micro-movements is in the range 100–200 μm then, in order to be discriminative, any method used to evaluate the primary stability should have an accuracy of 10–20 μm or better. Additionally, such method should also be able to report the relative micromotion at each point of the interface. None of the available experimental methods satisfies both requirements. Aim of the present study is to verify if any of the current finite element modelling techniques is sufficiently accurate in predicting the primary stability of a cementless prosthesis to be used to decide whether the micromotion may or may not jeopardise the implant osseointegration. The primary stability of an anatomic cementless stem, as measured in vitro, was used as a benchmark problem to comparatively evaluate different contact modelling techniques. Frictionless contact, frictional contact and press-fitted frictional contact conditions were modelled using alternatively node-to-node, node-to-face and face-to-face contact elements. The model based on face-to-face contact elements accounting for frictional contact and initial press-fit was able to predict the micromotion measured experimentally with an average (RMS) error of 10 μm and a peak error of 14 μm. All the other models presented errors higher than 20 μm assumed in the present study as an accuracy threshold.     

Replicon size and rate of fork movement in early S of higher plant cells (Pisum sativum)

Measurements of chromosomal DNA fiber replication of cells of cultured pea root meristems in early S via autoradiography showed a 3-fold increase in rate of fork movement in the first 2 h. The initial rate was 4.5–6 μm h⁻¹ but forks active after 90 min moved at nearly 18 μm h⁻¹. The faster movement was not characteristic of all replicons. Certain fibers consisted of replicons of a smaller mean size (38–42 μm) with slowly moving forks (4.5–6 μm h⁻¹ fork⁻¹) and others had replicons almost 50 μm long with forks that moved more rapidly.     

The presence of the potentially toxic genera Ostreopsis and Coolia (Dinophyceae) in the North Aegean Sea, Greece

The examination of macrophyte, water and sediment samples, collected at depths less than 1.5 m from 50 different sites along the North Aegean coasts, has revealed, for the first time in Greek coastal waters, the presence of two Ostreopsis species (O. ovata and O. cf. siamensis) and Coolia monotis in the majority of the sampling sites (94% and 100%, respectively). Other epiphytic dinoflagellates of the genera Prorocentrum and Amphidinium and diatoms were accompanying species in this epiphytic community. Morphometric features, plate formula and thecal ornamentation were used for species identification. O. ovata cells were smaller in dorsoventral (DV) diameter and width (W) (26.18–61.88 μm and 13.09–47.60 μm, respectively) in comparison with O. cf. siamensis (35.70–65.45 μm and 23.80–49.98 μm, respectively). In contrast, the anterioposterior (AP) diameter of O. cf. siamensis was smaller (14.28–26.18 μm) resulting in DV/AP ≈ 3, whereas the above ratio for O. ovata was less than 2 (AP ranging between 14.28–35.70 μm). Moreover, the theca of O. ovata cells was ornamented with scattered pores, which fluctuated in a wider range (0.07–0.32 μm) than those of O. cf. siamensis (0.23–0.29 μm). Coolia monotis cells were almost round with average DV diameter 26.88 μm, AP 25.66 μm and width 26.76 μm. Small and large cells were recorded in both field and culture populations of Ostreopsis spp. and C. monotis, while hyaline cysts were observed for O. ovata. The presence of O. ovata and O. cf. siamensis exhibited a clear seasonal pattern dominating (maximum abundance up to 4.05 × 10⁵ cells gr⁻¹ fwm) the period from midsummer to late autumn in years 2003 and 2004, while C. monotis was found also in winter and spring months.     

The last order of coiling in human chromosomes

The dimensions of chromatids in vivo and in fixed preparations of human chromosomes from cultured lymphocytes were compared. The relative variation in diameter in relation to length was the same in both conditions, but the lengths of the fixed chromosomes were about twice that of the chromosomes in vivo. The last order of coiling was studied in fixed chromosomes and in prematurely condensed chromosomes. The pitch of the coils in the fixed chromosomes, 0.6 μm, was independent of haploid length in the interval 90–220 μm. A clear indication of a spiralization of an underlying fibre was found throughout the haploid length interval of the prematurely condensed chromosomes, which ranged from 130 μm to more than 350 μm.     

Two new coccidia (Apicomplexa:Eimeriidae) from the green peacock (Pavo muticus) from Saudi Arabia

Two new species of Eimeria were found from faecal samples of ten green peacocks (Pavo muticus) collected at Al-Kharj area, a central region of Saudi Arabia. Sporulated oocysts of Eimeria mutica n.sp. are ellipsoidal 23.1×17.4 (22.4–25.0×16.7–18.9) μm, with a smooth bilayered wall. A micropyle and bilobed polar body are present, but without an oocyst residuum. The sporocyst is an elongated-ovoid 13.7×6.2 (12.0–14.2×5.4–6.7) μm, with a Stieda body and a residuum. Sporulated oocysts of E. kharjensis n.sp. are subspherical 20.3×17.7 (19.0–21.5×16.2–18.7) μm, with a two layered wall and a single polar body. The micropyle is covered by a dome-shaped cap and the sporocyst is an elongate-ovoid 12.7×6.3 (11.9–13.5×5.4–6.8) μm, with a Stieda body. The sporocyst residuum is present as several small granules.     

Paralygodium meckertii sp. nov. (Schizaeaceae) from the Upper Cretaceous (Coniacian) of Vancouver Island, British Columbia, Canada

More than 50 specimens of permineralized fertile pinnules with abaxially borne sporangia have been discovered in calcareous marine nodules from the Upper Cretaceous (Coniacian) Comox Formation from the Eden Main localities on Vancouver Island, British Columbia, Canada. Isolated pinnules 1.6–3.0 mm wide × 1.6–2.8 mm long are lobed and abaxially enrolled to form irregular globose structures. Pyriform sporangia 216–300 μm wide × 360–468 μm long occur in two rows on the abaxial surface of pinnule lobes. Sporangia have an apical annulus of 15–18 cells. Spores are tetrahedral and trilete, 33–42 μm in diameter, with straight to concave interradial sides, laesurae extending nearly to the equator, and a psilate exine. Spores are assignable to the sporae dispersae genus Deltoidospora. Fertile pinnules are compared to fossils of Anemia poolensis and two previously described species of Paralygodium, and show closest similarities to P. vancouverensis from the Eocene of British Columbia. The Cretaceous Eden Main specimens differ in number of pinnule lobes and their morphology and are described as a new taxon: P. meckertii sp. nov. This discovery extends the Cretaceous geographic range of Paralygodium from Japan to North America and adds to our knowledge of the diversity of extinct schizaeaceous ferns.     

Simultaneous determination of felbamate and three metabolites in rat and dog plasmas by high-performance liquid chromatography

An isocratic liquid chromatographic method employing one extraction step and a 150 mm × 4.6 mm I.D. Spherisorb ODS2, 3-μm HPLC column using UV-absorbance detection at 210 nm has been developed for the quantitation of felbamate and three felbamate metabolites in 0.100-ml aliquots of rat and dog plasmas. The linear quantitation range in rat plasma is 0.195–200 μg/ml for felbamate; 1.563–200 μg/ml for the p-hydroxy metabolite; 0.391–200 μg/ml for the 2-hydroxy metabolite; and 0.098–200 μg/ml for the monocarbamate metabolite. The linear quantitation range in dog plasma is 0.195–200 μg/ml for felbamate; 0.781–200 μg/ml for the p-hydroxy metabolite; 0.195–200 μg/ml for the 2-hydroxy metabolite; and 0.098–200 μg/ml for the monocarbamate metabolite.     

Inhibitory cholinergic control of endogenous GABA release from electrically stimulated cortical slices and K-depolarized synaptosomes

In the present study we characterize the optimal experimental conditions under which to investigate the cholinergic regulation of endogenous electrically evoked γ-aminobutyric acid (GABA) release from guinea pig cortical slices. Superfusion with the neuronal GABA reuptake inhibitor, SKF89976A (10 μM) caused cortical GABA release to be linearly correlated with the frequency of electrical stimulation (5, 10, 20 Hz). Electrically evoked GABA release (10 Hz) was tetrodotoxin-sensitive and Ca²⁺-dependent and was under GABA_B autoreceptor control. Under these experimental conditions, acetylcholine (0.1–10 μM) and physostigmine (30 μM) decreased the electrically evoked GABA release while the M₂ receptor antagonist AFDX-116 (0.01–0.1 μM) counteracted these effects. Similar results were also observed in a cortical synaptosomal preparation stimulated with K⁺ (10 mM). These findings demonstrate an inhibitory cholinergic regulation of electrically evoked GABA release via M₂ receptors located on cortical GABAergic terminals.     

A developmental study on the capacity of rabbit granulosa cells to respond to trophic hormones and secrete progesterone in vitro

To determine when undifferentiated rabbit granulosa cells first develop the capacity to secrete progesterone, pieces of intact ovaries from neonatal rabbits (newborn—30 days old) and pure granulosa cells from 150–1200 μm follicles at 60–600 days old were cultured in vitro for 6–10 days with human chorionic gonadotropin (HCG), Pergonal (LH/FSH), dibutyryl cyslic AMP (Bu₂CAMP), prostaglandin E₂, estradiol-17β, and as controls. The culture medium was collected every 2 days, and progesterone, estrone, and estradiol-17β were measured by radioimmunoassay.None of the neonatal ovaries or granulosa cell cultures secreted estrone or estradiol-17β spontaneously or in response to stimulation by gonadotropins, Bu₂CAMP, or prostaglandin E₂.Control cultures of newborn and 7-day-old ovaries did not secrete progesterone, but ovaries from 17- and 30-day-old rabbits did. Gonadotropins and Bu₂CAMP induced progesterone secretion in 7-day-old ovaries and stimulated its production 5-10-fold in ovaries at 17 and 30 days old, but prostaglandin E₂ and estradiol-17β were without effect.Granulosa cells from all antral follicles (200–1200 μm) secreted progesterone spontaneously, and its production was stimulated 100–1000-fold with gonadotropins and Bu₂CAMP, but not with estradiol-17β or prostaglandin E₂. In contrast, granulosa cells from 100–150 μm preantral follicles from 200-day-old animals did not secrete progesterone under these culture conditions.These results demonstrate that rabbit granulosa cells differentiate the capacity to secrete progesterone at the time the primary follicle develops an antrum, and suggest the differentiation process involves the acquisition of the capacity to respond to gonadotropins perhaps by the synthesis or unmasking of gonadotropin receptors.     

Cadmium removal from human plasma by Cibacron Blue F3GA and thionein incorporated into polymeric microspheres

Poly(2-hydroxyethylmethacrylate–ethyleneglycoldimethacrylate) [poly(HEMA–EGDMA)] microspheres carrying Cibacron Blue F3GA and/or thionein were prepared and used for the removal of cadmium ions Cd(II) from human plasma. The poly(HEMA–EGDMA) microspheres, in the size range of 150–200 μm in diameter, were produced by a modified suspension copolymerization of HEMA and EGDMA. The reactive triazinyl dye-ligand Cibacron Blue F3GA was then covalently incorporated into the microspheres. The maximum dye incorporation was 16.5 μmol/g. Then, thionein was bound onto the Cibacron Blue F3GA-incorporated microspheres under different conditions. The maximum amount of thionein bound was 14.3 mg/g. The maximum amounts of Cd(II) ions removed from human plasma by poly(HEMA–EGDMA)–Cibacron Blue F3GA and poly(HEMA–EGDMA)–Cibacron Blue F3GA–thionein were of 17.5 mg/g and 38.0 mg/g, respectively. Cd(II) ions could be repeatedly adsorbed and desorbed with both types of microspheres without significant loss in their adsorption capacity.