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.     

Newly discovered linkages between the cortical (pellicular) ridges of Opalina

Samples of Opalina ranarum have been prepared for electron microscopy by ultra-rapid cryofixation followed by substitution fixation in a solvent containing tannic acid. This technique has made it possible to see that very thin linkages exist between the pleated ridges that form the surface of the cell. Between any two adjacent cortical ridges, the linkages, which are approximately 0.1 μm long, occur as a single row, 0.1 μm below the free edge, with an impressively regular spacing of 0.1 μm. The cortical ridges of the Opalinids are spaced with remarkable uniformity, even when thrown into undulating patterns. The linkages described here will inevitably stabilize the complex architecture of the cortex. Other possible functions are discussed.     

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.     

The directionality of locomotion of mouse fibroblasts : Role of cell adhesiveness

To evaluate the role of adhesiveness in cell locomotion we have compared the parameters of motion (rate and directionality index) of an adhesive-deficient mutant, AD6, and of its parental strain, BALB 3T3, by means of cinematography. The low adhesive cells, AD6, have the same migration rate (1 μm/min) as the parental cells, but they have lost the directionality of motion (directionality index is 6-fold lower in AD6). Similarly, the parental strain BALB 3T3 or the mutant biochemically reverted, cultured on a poorly adhesive substratum, showed a significant decrease in the persistence of direction of movement. The adhesiveness of AD6 cells is increased when cultured in presence of LETS (50 μg/ml). In these conditions the high directionality index of the wild-type cells is restored in the mutant. On the other hand, we have reported that N-acetyl glucosamine bypasses the enzymatic block of the AD6 cells, and restores to normal the cell surface carbohydrates and adhesiveness. We show here that the directionality of motion is also reverted by the amino-sugar. A good correlation was found between the loss of directionality and the absence of microfilament bundles. We conclude that adhesiveness to substratum controls the directionality of fibroblast locomotion as follows: (1) Cell-to-substrate adhesion is necessary to stabilize the leading edge and to promote the organization of microfilaments in bundles; (2) the stabilization of the leading edge and the axial organization provided by the actin cables are required for the persistence of direction of motion.     

Effects of geometry on transmission and sensing potential of tapered fiber sensors

Geometry of tapered fiber sensors critically affects the response of an evanescent field sensor to cell suspensions. Single-mode fibers (nominally at 1300 nm) were tapered to symmetric or asymmetric tapers with diameters in the range of 3–20 μm, and overall lengths of 1–7 mm. Their transmission characteristics in air, water and in the presence of Escherichia coli (JM101 strain) at concentrations of 100, 1000, 7000 and 7 million cells/mL were measured in the 400–800 nm range and gave rich spectral data that lead to the following conclusions. (1) No change in transmission was observed due to E. coli with tapers that showed no relative change in transmission in water compared to air. (2) Tapers that exhibited a significant difference in transmission in water compared to air gave weak response to the presence of the E. coli. Of these, tapers with low waist diameters (6 μm) showed sensitivity to E. coli at 7000 cells/mL and higher concentration. (3) Tapers that showed modest difference in water transmission compared to air, and those that had small waist diameters gave excellent response to E. coli at 100–7000 cells/mL. In addition, mathematical modeling showed that: (1) at low wavelength (470 nm) and small waist diameter (6 μm), transmission with water in the waist region is higher than in air. (2) Small changes in waist diameter (0.05 μm) can cause larger changes in transmission at 470 nm than at 550 nm at waist diameter of 6 μm. (3) For the same overall geometry, a 5.5 μm diameter taper showed larger refractive index sensitivity compared to a 6.25 μm taper at 470 nm.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

The effect of triisopropyl phosphate on the mobility of surface concanavalin a receptors and on the locomotion of polymorphonuclear leucocytes

The non-phosphorylating organophosphorus compound triisopropyl phosphate, which is known to inhibit rabbit leucocyte locomotion, can stimulate the locomotion of guinea pig leucocytes under certain conditions. Different methods of preparing guinea pig leucocyte monolayers can give preparations with different proportions of motile cells. With preparations that contain relatively slowly moving cells triisopropyl phosphate increases the number of stationary cells without significantly affecting the speed of the cells that remain motile. Most rabbit leucocytes labelled with fluorescein-labelled concanavalin A form caps within 5–10 min at 37 °C. In contrast the rate of cap formation in guinea pig leucocytes is much slower and after 20 min many cells have only random patches. Triisopropyl phosphate accelerates cap formation in guinea pig leucocytes but not in rabbit leucocytes. The local anaesthetic nupercaine inhibits cap and patch formation in rabbit and guinea pig leucocytes. Inhibition of rabbit leucocyte locomotion is induced by concanavalin A at 1 μg/ml. These results are briefly related to the known effects of triisopropyl phosphate on the isolated leucocyte plasma membrane.     

Pre-treatment of Caco-2 cells with zinc during the differentiation phase alters the kinetics of zinc uptake and transport(2)

The Caco-2 cell model was used to study the efficiency of absorption and endogenous excretion of zinc (Zn) regulated by dietary Zn concentration. Cells were seeded onto high pore-density membranes and maintained in medium supplemented with 10% FBS. After confluence, cells were treated with 5 or 25 μmol Zn/L for 7 d, and Zn uptake and transport were measured in both apical (AP) and basolateral (BL) directions by using ⁶⁵Zn. Similar cells were labeled with ⁶⁵Zn and the release of Zn to the AP and BL sides was measured. The AP uptake of Zn in cells exposed to 25 μmol Zn/L was slower (p < 0.05) than that in cells exposed to 5 μmol Zn/L. The AP to BL transport rate in the 25 μmol Zn/L group was only 40% (p < 0.05) of that in the 5 μM group. In contrast, the rate of BL Zn uptake was 4-fold higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). The BL to AP transport rate was 2-fold higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). Basolateral uptake was 6 to 25 times greater (p < 0.05) than AP uptake for cells treated with 5 and 25 μmol Zn/L, respectively. The rate of Zn release was enhanced about 4-fold (p < 0.05) by 25 μmol Zn/L treatment. Release to the BL side was 10 times greater than to the AP side. Zn-induced metallothionein (MT), thought to down-regulate AP to BL Zn transport, was 4-fold higher (p < 0.001) in the 25 μmol Zn/L group than in the 5 μM group, but the rate of BL Zn release was higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). Induced changes in transport rates by media Zn concentrations could involve the up- and/or down-regulation of Zn influx and efflux proteins such as the ZIP and ZnT families of Zn transporters.     

Cells in focus: endothelial cell

The endothelial cell is thought to arise from the splanchnopleuric mesoderm. Endothelial cells form the inner lining of a blood vessel and provides an anticoagulant barrier between the vessel wall and blood. In addition to its role as a selective permeability barrier, the endothelial cell is a unique multifunctional cell with critical basal and inducible metabolic and synthetic functions. The endothelial cell reacts with physical and chemical stimuli within the circulation and regulates hemostasis, vasomotor tone, and immune and inflammatory responses. In addition, the endothelial cell is pivotal in angiogenesis and vasculogenesis. Endothelial cell injury, activation or dysfunction is a hallmark of many pathologic states including atherosclerosis, loss of semi-permeable membrane function, and thrombosis.

Cell facts: (1) Endothelium consists of approximately (1–6)×10¹³ endothelial cells forming an almost 1 kg organ. (2) They uniquely contain Weibel–Palade bodies, 0.1 μm wide, 3 μm long membrane-bound structures that represent the storage organelle for von Willebrand factor (vWF). (3) The endothelial cell is not only a permeability barrier but also a multifunctional paracrine and endocrine organ. It is involved in the immune response, coagulation, growth regulation, production of extracellular matrix components, and is a modulator of blood flow and blood vessel tone.     

Protection by tropolones against H2O2-induced DNA damage and apoptosis in cultured Jurkat cells

Tropolones, the naturally occurring compounds responsible for the durability of heartwood of several cupressaceous trees, have been shown to possess both metal chelating and antioxidant properties. However, little is known about the ability of tropolone and its derivatives to protect cultured cells from oxidative stress-mediated damage. In this study, the effect of tropolones on hydrogen peroxide-induced DNA damage and apoptosis was investigated in cultured Jurkat cells. Tropolone, added to the cells 15 min before the addition of glucose oxidase, provided a dose dependent protection against hydrogen peroxide induced DNA damage. The IC₅₀ value observed was about 15 μM for tropolone. Similar dose dependent protection was also observed with three other tropolone derivatives such as trimethylcolchicinic acid, purpurogallin and β-thujaplicin (the IC₅₀ values were 34, 70 and 74 μM, respectively), but not with colchicine and tetramethyl purpurogallin ester. Hydrogen peroxide-induced apoptosis was also inhibited by tropolone. However, in the absence of exogenous H₂O₂ but in the presence of non-toxic concentrations of exogenous iron (100 μM Fe³⁺), tropolone dramatically increased the formation of single strand breaks at molar ratios of tropolone to iron lower than 3 to 1, while, when the ratio increased over 3, no toxicity was observed. In conclusion, the results presented in this study indicate that the protection offered by tropolone against hydrogen peroxide-induced DNA damage and apoptosis was due to formation of a redox-inactive iron complex, while its enhancement of iron-mediated DNA damage at ratios of [tropolone]/[Fe³⁺] lower than 3, was due to formation of a lipophilic iron complex which facilitates iron transport through cell membrane in a redox-active form.     

Taxonomic characterization of two marine peritrichous ciliates, Epicarchesium corlissi n. sp. and Pseudovorticella jiangi n. sp. (Ciliophora: Peritrichia), from northern China

Two new marine peritrich ciliates, Epicarchesium corlissi n. sp. and Pseudovorticella jiangi n. sp., were discovered in mariculture waters on the coast of northern China near Qingdao. Their morphology, infraciliature and silverline system were investigated based on both living and silver-impregnated specimens. E. corlissi is characterized as follows: marine Epicarchesium with dichotomously branched stalk; zooids elongate, approximately 60–70×25–35 μm in vivo; peristomial collar double-folded; macronucleus J-shaped; single, small contractile vacuole ventrally positioned; more than 60 striations between peristome and aboral trochal band, 13–18 from aboral trochal band to scopula; abstomal end of row 1 of infundibular polykinety 3 terminating at same level as rows 2 and 3 of infundibular polykinety 3; rows 2 and 3 of infundibular polykinety 3 much longer than row 1 and converging adstomally with infundibular polykinety 1. The new species P. jiangi is diagnosed as follows: marine Pseudovorticella; zooid inverted bell-shaped, approximately 80×60 μm in vivo and with a broad, flat, thin peristomial collar that measures approximately 90 μm across; pellicle with transparent cortical vesicles; macronucleus J-shaped; number of silverlines between peristome and aboral trochal band 20–24, from aboral trochal band to scopula 9–11; abstomal end of row 1 of infundibular polykinety 3 diverges from the other two rows of this polykinety and ends alongside row 3 of infundibular polykinety 2.     

Galanin-induced relaxation in gastric smooth muscle cells is mediated by cyclic AMP

Galanin has numerous effects on gastrointestinal motility in different species; however, its cellular basis of action in mediating these effects is unclear. Dispersed gastric smooth muscle cells have been shown to possess high-affinity galanin receptors that increase cAMP and cause relaxation. Recent studies show some smooth muscle relaxants such as VIP cause relaxation by both cAMP-dependent and -independent mechanisms. It is unknown if galanin's cellular basis of relaxation is similar or different from that of VIP. To investigate galanin's relaxant effect and compare it to VIP's effect, dispersed smooth muscle cells from guinea pig stomach were prepared by collagenase digestion. The mean length in resting cells was 110 ± 2 μm and, with carbachol treatment, contracted to 89 ± 2 μm. VIP and galanin alone had no effect on cell length, but each caused a dose-dependent inhibition of carbachol-induced contraction and both had an EC₅₀ of 3–7 nM. Galanin (1 μM) and VIP (1 μM) increased cellular cAMP from 118 ± 10 pmol/10⁶ cells in control to 212 ± 14 and 214 ± 12 pmol/10⁶ cells, respectively. The protein kinase A inhibitor, Rp-cAMPS, at 100 μM, completely inhibited the relaxant effect of an EC₅₀ concentration of galanin (3 nM), but only inhibited that by VIP by 80% (p < 0.05). Adding the nitric oxide inhibitor, -NNA ( ), at 100 μM did not alter the length of resting cells or inhibit carbachol-induced contraction. However, -NNA (100 μM) decreased VIP-induced relaxation by 45%, whereas it had no effect on galanin-induced relaxation. To determine the ability of each peptide to activate nitric oxide, the incorporation of [³H]arginine into [³H]citrulline was determined. Galanin (1 μM) did not cause nitric oxide generation whereas VIP (1 μM) increased nitric oxide generation above the control by 97 ± 14% (p < 0.01). These results demonstrated that with galanin, in contrast to VIP, nitric oxide is not involved in its ability to cause gastric smooth muscle cell relaxation. The relaxant action of galanin can be accounted for completely by its ability to activate protein kinase A and therefore resembles recent results with β-adrenergic agents.     

Effect of U-50,488H on the contractile response of cardiomyopathic hamster ventricular myocytes

We examined the effects of a selective κ opioid receptor agonist (U-50,488H) on the contractile properties of single ventricular myocytes from 127 day old control (F1B) and cardiomyopathic (BIO 14.6) hamsters. Myocytes in bicarbonate buffered solution with 1.5 mM [Ca²⁺] were electrically stimulated with field electrodes in the bath. Length changes were monitored via myocyte edge tracking. Twitch amplitude and the velocity of cell shortening were less in the cardiomyopathic hamster myocytes than in age-matched hamsters (P≤0.05). There was a concentration-dependent effect of U-50,488H (0.1–20 μM) to decrease twitch amplitude and shortening velocity in both control and cardiomyopathic myocytes (P≤0.001). In cells loaded with the Ca²⁺ indicator indo-1 the negative inotropic action of U-50,488H was associated with a decreased indo-1 fluorescence transient amplitude. There was no difference in the negative inotropic effect of U-50,488H on control and cardiomyopathic cells. Thus, the CM hamster does not demonstrate a different contractile response to U-50,488H.     

Chromosomes of caryophyllidean cestodes: Diploidy, triploidy, and parthenogenesis in Glaridacris catostomi

Mitotic and meiotic chromosomes were studied in Giemsa post-stained Feulgen squashes of testicular, ovarian, and vitelline cells. G. catostomi is 2n = 20 with eight pairs of metacentrics and two pairs of submetacentrics in its karyotype. The fundamental number of chromosome arms is FN = 40. At mitotic metaphase the mean total chromosome length (TCL) of the complement was 111·4 μm; the largest chromosomes were 8 μm (7% TCL) and the smallest 3 μm (3% TCL). Karyotype variation was not found among five diploid populations from New York State and Canada. Meiotic activity was abundant in spermatogenesis which proceeds as usual with sperm developing after two meiotic divisions. Chiasma frequency at diakinesis was 37/cell; as many as five chiasmata were observed in a single bivalent. A triploid ‘race’ (3n = 30) of G. catostomi was discovered in Bozenkill Creek near Albany, New York. Karyotype analysis confirms the triploid nature of these variants. Spermatogenesis in triploids is abnormal to the point of complete failure. The presence of an extra set of chromosomes has resulted in a breakdown of meiosis with subsequent sterility of the male system. Eggs remain unfertilized and parthenogenetic reproduction is presumed to occur. The possible origins of this unusual condition and its evolutionary implications are discussed. Seventeen figures, two tables of measurements, and a system of chromosome nomenclature supplement the paper.     

A new floating sensor array to detect electric near fields of beating heart preparations

A new flexible sensor for in vitro experiments was developed to measure the surface potential, Φ, and its gradient, E (electric near field), at given sites of the heart. During depolarisation, E describes a vector loop from which direction and magnitude of local conduction velocity θ can be computed. Four recording silver electrodes (14 μm × 14 μm) separated by 50 μm, conducting leads, and solderable pads were patterned on a 50 μm thick polyimide film. The conductive structures, except the electrodes, were isolated with polyimide, and electrodes were chlorided. Spacer pillars mounted on the tip fulfil two functions: they keep the electrodes 70 μm from the tissue allowing non-contact recording of Φ and prevent lateral slipping. The low mass (9.1 mg) and flexibility (6.33 N/m) of the sensor let it easily follow the movement of the beating heart without notable displacement. We examined the electrodes on criteria like rms-noise of Φ, signal-to-noise ratio of Φ and E, maximum peak-slope recording dΦ/dt, and deviation of local activation time (LAT) from a common signal and obtained values of 24–28 μV, 46 and 41 dB, 497–561 V/s and no differences, respectively. With appropriate data acquisition (sampling rate 100 kHz, 24-bit), we were able to record Φ and to monitor E and θ on-line from beat-to-beat even at heart rates of 600 beats/min. Moreover, this technique can discriminate between uncoupled cardiac activations (as occur in fibrotic tissue) separated by less than 1 mm and 1 ms.     

Structural and viral association comparisons of bovine zonae pellucidae from follicular oocytes, Day-7 embryos and Day-7 degenerated ova

Bovine zonae pellucidae (ZP) from follicular oocytes and from embryos and degenerated ova collected on Day 7 from superovulated cows were examined by scanning electron microscopy, by dimensional measurement, and by total protein determination. The number of plaque-forming units (PFU) of infectious bovine rhinotracheitis virus (IBRV) that were associated with ZP-intact embryos/ova from each of the 3 sources after in vitro exposure was also determined.

Scanning electron microscopy revealed that the surfaces of Day-7 embryos and degenerated ova were smoother than those of follicular oocytes. Mean dimensional measurements of the diameter/thickness of the ZP from follicular oocytes, Day-7 embryos, and degenerated Day-7 ova were 156.7 μm/12.3μm, 161.3μm/12.6μm, and 158.9μm/12.8μm, respectively. The mean total protein per ZP of follicular oocytes, embryos, and degenerated ova was 0.331 μg, 0.349 μg, and 0.254 μg, respectively. Considerable variability existed within groups, but significantly greater quantities of IBRV were associated with follicular oocytes (mean PFU/oocyte = 68.1) than with Day-7 embryos (mean PFU/embryo = 43.0; P<0.05) or with Day-7 ova (mean PFU/ovum = 31.9; P<0.01).

The reliability of using an assay for IBRV associated with nontransferable ova/embryos as an indicator of the presence or absence of the virus in transferable embryos from the same collection (Day 7) was supported. Although structural differences between the ZPs of follicular oocytes and Day-7 embryos were observed in this study, further investigation is needed to determine if there are differences in the protective function of the respective ZPs.     

Cytochrome P-450 distribution in rat liver and the effect of sodium phenobarbitone administration

A microspectrophotometric method for assaying cytochrome P-450 in fresh 24 μm unfixed cryostat sections of rat liver has been developed. When used to assay this cytochrome in sections of microsomal preparations it has yielded results equivalent to those obtained by the conventional spectrophotometric assay of the same preparations. Random measurements made throughout sections of liver have given mean values for cytochrome P-450 concentrations which are twice those measured in microsomes prepared from the livers of the same animals (not corrected for the yield in the homogenate).

Measurements of the cytochrome P-450 content of liver cells by the microspectrophotometric method show that in liver from male Wistar rats, cells nearer to the central veins contain up to twice as much cytochrome P-450 as those nearer to the portal tract (mean cell concentrations of 26.4 (±4.4) μmol/l and 17.5 (±3.0) μmol/l respectively). In the livers from similar rats, killed at the same time, but which had received 1 mg/ml sodium phenobarbitone in their drinking water for one week, the cells near the central vein contained up to five times as much cytochrome P-450 as those near the portal tract (mean cell concentrations of 77.3 (±25.0) μmol/l and 28.3 (±9.6) μmol/l respectively).

The results show a selective increase in cytochrome P-450 content by the cells in the centrilobular region after treatment with sodium phenobarbitone and a smaller increase by some of the cells in the periportal region.     

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.     

The neurotoxic effect of sickle cell hemoglobin

A growing body of experimental evidence suggests that the oxidative neurotoxicity of hemoglobin A may contribute to neuronal loss after CNS hemorrhage. Several hemoglobin variants, including hemoglobin S, are more potent oxidants in cell-free systems. However, despite the increased incidence of hemorrhagic stroke associated with sickle cell disease, little is known of the effect of hemoglobin S on cells of neural origin. In the present study, its toxicity was quantified and directly compared with that of hemoglobin A in murine cortical cell cultures. Reactive oxygen species production, as assessed by cellular fluorescence after treatment with dihydrorhodamine 123, was significantly increased by exposure to 10 μM hemoglobin S for 2-4 h. Neuronal death, as measured by propidium iodide staining and lactate dehydrogenase release, commenced at 4 h; for a 20-h exposure, the EC₅₀ was approximately 0.71 μm. Glial cells were not injured. Cell death was completely blocked by iron chelation with deferoxamine or phenanthroline. Direct comparison of sister cultures exposed to either hemoglobin A or hemoglobin S revealed a similar amount of cell injury in both groups. A significant difference was consistently observed only after treatment with 1 μM hemoglobin for 20 h, which resulted in death of approximately one third more neurons with hemoglobin S than with hemoglobin A. The results of this study suggest that sickle cell hemoglobin is neurotoxic at physiologically relevant concentrations. This toxicity is iron-dependent, oxidative, and quantitatively similar to that produced by hemoglobin A.     

Hyperbaric and normobaric reoxygenation of hypoxic rat brain slices--impact on purine nucleotides and cell viability

Hyperbaric oxygen treatment has been suggested as able to reduce hypoxia induced neuronal damage. The aim of the study was to compare the impact of different reoxygenation strategies on early metabolical (purine nucleotide content determined by HPLC) and morphological changes (index of cell injury after celestine blue/acid fuchsin staining) of hypoxically damaged rat neocortical brain slices. For this purpose slices (300 μm and 900 μm) were subjected to either 5 or 30 min of hypoxia by gassing the incubation medium with nitrogen. During the following reoxygenation period treatment groups were administered either 100% oxygen (O) or room air (A) at normobaric (1 atm absolute, NB-O; NB-A) or hyperbaric (2.5 atm absolute, HB-O; HB-A) conditions. After 5 min of hypoxia, both HB-O and NB-O led to a complete nucleotide status restoration (ATP/ADP; GTP/GDP) in 300 μm slices. However, reoxygenation after 30 min of hypoxia was less effective, irrespective of the oxygen pressure. Furthermore, administering hyperbaric room air resulted in no significant posthypoxic nucleotide recovery. In 900 μm slices, both control incubation as well as 30 min of hypoxia resulted in significantly lower trinucleotide and higher dinucleotide levels compared to 300 μm slices. While there was no significant difference between HB-O and NB-O on the nucleotide status, morphological evaluation revealed a better recovery of the index of cell injury (profoundly injured/intact cell-ratio) in the HB-O group. Conclusively, the posthypoxic recovery of metabolical characteristics was dependent on the duration of hypoxia and slice thickness, but not on the reoxygenation pressure. A clear restorative effect on purine nucleotides was found only in early-administered HB-O as well as NB-O in contrast to room air treated slices. However, these pressure independent metabolic changes were morphologically accompanied by a significantly improved index of cell injury, indicating a possible neuroprotective role of HB-O in early posthypoxic reoxygenation.