A unique pattern of F-actin organization supports cytokinesis in vacuolated cells of Macrocystis pyrifera (Phaeophyceae) gametophytes

Summary. The organization of actin filaments and their role in cytokinesis was studied in regenerating protoplasts and thallus cells of gametophytes of the brown alga Macrocystis pyrifera. Before the onset of cytokinesis, a ring of actin filaments appeared on the putative cytokinetic plane just under the plasmalemma. Light and electron microscopy of cytokinetic cells revealed that large vacuoles occupy the space between the daughter nuclei, which very often are eccentrically positioned at the cell cortex. By the progress of cytokinesis, actin filament bundles emanating from the cytokinetic ring tend to form an actin plate that enters cytoplasmic pockets in which the cytokinetic diaphragm develops. The mechanism of this cytokinetic pattern that has not been reported so far for brown algae is discussed. Correspondence and reprints: Department of Botany, Faculty of Biology, University of Athens, Athens 157 84, Greece.     

Structural and ultrastructure changes show an increase in amoeboid forms in Heterosigma akashiwo (Raphidophyceae), during recovery after nutrient depletion

Heterosigma akashiwo shows remarkable ultrastructural changes during the recovery from a late stationary phase (“aged” culture) induced by nutrient depletion. H. akashiwo cells showed different morphological types in “aged” cultures, with an increase in irregular cells and cell fragments. The irregular cells mostly corresponded to an amoeboid shape of the cell. Many of these cells showed chloroplasts with a homogeneous matrix of medium electron density lacking most thylakoids and condensed nucleus, probably as a result of cyst/resting cells germination. In other cells, we observed nuclear blebbing without chromatin condensation and changes in mitochondrion ultrastructure. Some vegetative cells in active phase (“young” culture) were connected to each other, apparently phagocytizing cytoplasmic fragments and intact chloroplasts in the medium. An explanation for the phenomenon may reside in the need of acquiring organic material after nutrient reduction for a faster recovery. On the basis of our observations, we conclude that some ultrastructural features, normally used to distinguish between different species and strains of Raphidophyceae, may be related to different physiological states and should be used with caution for systematic purposes.     

The drosophila protein asp is involved in microtubule organization during spindle formation and cytokinesis

Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been suggested to be involved in microtubule nucleation from the centrosome. Here, we show that Asp is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules. Localization to these structures is independent of a functional centrosome. Moreover, colchicine treatment disrupts Asp localization to the centrosome, indicating that Asp is not an integral centrosomal protein. In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and gamma-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, a gene required for centrosome function, we have determined the role of Asp in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. Our data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis.     

Pollen morphology of Campanula (Campanulaceae) and allied genera in Iran with special focus on its systematic implication

Pollen grains of 47 taxa of Campanulaceae including 35 taxa of Campanula that represent its five subgenera and nine sections are investigated. Moreover, five species and three subspecies representing three sections in Asyneuma and one species of each genera Legousia, Michauxia, Zeugandra and Theodorovia were studied by light and scanning electron microscopy. The basic shape of the pollen grains was spheroidal. The apertures vary from tri- to hexa-porate. The sculpturing pattern of exine was rugulate-echinate, rugulate-microechinate or in few species rugulate-microreticulate and microechinate. The most valuable characters for subgeneric classification were the length and density of echini. The length of echini were significantly long (> 2 μm) in C. sclerotricha, Legousia falcata and Michauxia laevigata. Pollen grains show low variation in different species of subgen. Rapunculus, but were variable among different species of some groups, such as sect. Rupestres, probably indicating their non-monophyly despite homogeneity with respect to other morphological characters. Pollen morphology does not support recognition of Asyneuma, Legousia, Michauxia, Symphyandra, Theodorovia, and Zeugandra as separated from Campanula, since none of them exhibit any unique feature.     

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 26.87 h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3 weeks post inoculation. The highest level of spore production was at 4 weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34 ± 0.9 × 10⁶ spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.     

Evaporative water loss and energy metabolic in two small mammals,voles (Eothenomys miletus) and mice (Apodemus chevrieri), in Hengduan mountains region

Evaporative water loss (EWL) and energy metabolism were measured at different temperatures in Eothenomys miletus and Apodemus chevrieri in dry air. The thermal neutral zone (TNZ) of E. miletus was 22.5–30 °C and that of A. chevrieri was 20–27.5 °C. Mean body temperatures of the two species were 35.75±0.5 and 36.54±0.61 °C. Basal metabolic rates (BMR) were 1.92±0.17 and 2.7±0.5 ml O₂/g h, respectively. Average minimum thermal conductance (C_m) were 0.23±0.08 and 0.25±0.06 ml O₂/g h °C. EWL in E. miletus and A. chevrieri increased with the increase in temperature; the maximal EWL at 35 °C was 4.78±0.6 mg H₂O/g h in E. miletus, and 5.92±0.43 mg H₂O/g h in A. chevrieri. Percentage of evaporative heat loss to total heat production (EHL/HP) increased with the increase in temperature; the maximal EHL/HP was 22.45% at 30 °C in E. miletus, and in A. chevrieri it was 19.96% at 27.5 °C. The results may reflect features of small rodents in the Hengduan mountains region: both E. miletus and A. chevrieri have high levels of BMR and high levels of total thermal conductance, compared with the predicted values based on their body masses, while their body temperatures are relatively low. EWL plays an important role in temperature regulation.     

The expression of the proximodistal axis patterning genes Distal-less and dachshund in the appendages of Glomeris marginata (Myriapoda: Diplopoda) suggests a special role of these genes in patterning the head appendages

The genes Distal-less, dachshund, extradenticle, and homothorax have been shown in Drosophila to be among the earliest genes that define positional values along the proximal-distal (PD) axis of the developing legs. In order to study PD axis formation in the appendages of the pill millipede Glomeris marginata, we have isolated homologues of these four genes and have studied their expression patterns. In the trunk legs, there are several differences to Drosophila, but the patterns are nevertheless compatible with a conserved role in defining positional values along the PD axis. However, their role in the head appendages is apparently more complex. Distal-less in the mandible and maxilla is expressed in the forming sensory organs and, thus, does not seem to be involved in PD axis patterning. We could not identify in the mouthparts components that are homologous to the distal parts of the trunk legs and antennnae. Interestingly, there is also a transient premorphogenetic expression of Distal-less in the second antennal and second maxillary segment, although no appendages are eventually formed in these segments. The dachshund gene is apparently involved both in PD patterning as well as in sensory organ development in the antenna, maxilla, and mandible. Strong dachshund expression is specifically correlated with the tooth-like part of the mandible, a feature that is shared with other mandibulate arthropods. homothorax is expressed in the proximal and medial parts of the legs, while extradenticle RNA is only seen in the proximal region. This overlap of expression corresponds to the functional overlap between extradenticle and homothorax in Drosophila.     

Extension of the breeding season and its effects on fertilization and development in two species of lugworm (Arenicola marina and A. defodiens)

In the autumn/winter breeding polychaetes, Arenicola marina and A. defodiens, spawning can be advanced or delayed by a number of months through temperature manipulation of the adults. However, this manipulation may have significant consequences for fertilization rates and embryo developmental success and so in vitro fertilizations were performed to assess the impact of manipulation. Firstly, we used oocytes and sperm obtained from advanced or delayed individuals. For both species, using gametes from 4 weeks advanced individuals did not result in a significant reduction in development, however, gametes from individuals advanced (A. marina only) or delayed by 8 weeks resulted in significantly fewer embryos developing normally. Reciprocal crosses of temperature-manipulated A. marina gametes (from 4 weeks advanced and 4 weeks delayed individuals) with those at the natural spawning time confirmed that the reduction in developmental success in both was attributable to the oocytes. After 5 h post-fertilization, the majority of oocytes from advanced individuals had fertilized, but by 24 h most were abnormal. For fertilizations with gametes from delayed individuals, nearly 100% of the embryos were developing normally after 24 h, but after 144 h significantly more were abnormal in crosses involving oocytes from delayed females. Although both species have reproductive plasticity to extend their breeding season, the significant reduction in the numbers of competent larvae produced as the spawning is delayed or advanced further may be a significant bottleneck in aquaculture and it may also have considerable implications for the long-term reproductive success of a population in response to environmental change.Sympatric populations of the species exist in many locations and the inherent variability in the breeding seasons could allow spawning times to overlap. Artificially delaying A. marina individuals enabled fertilizations to be performed with A. defodiens at the natural spawning time in the laboratory. Both conspecific fertilizations produced 100% trochophore larvae after 120 h, but A. defodiens oocytes failed to fertilize after incubation with A. marina sperm, in comparison to the A. marina oocytes incubated with A. defodiens sperm where 40% developed to the trochophore stage. This asymmetric gamete incompatibility may be one of a suite of mechanisms to minimise hybridisation.     

The progression of the intra-erythrocytic cell cycle of Plasmodium falciparum and the role of the centriolar plaques in asynchronous mitotic division during schizogony

The cell division cycle and mitosis of intra-erythrocytic (IE) Plasmodium falciparum are poorly understood aspects of parasite development which affect malaria molecular pathogenesis. Specifically, the timing of the multiple gap (G), DNA synthesis (S) and chromosome separation (M) phases of parasite mitosis are not well defined, nor whether genome divisions are immediately followed by cleavage of the nuclear envelope. Curiously, daughter merozoite numbers do not follow the geometric expansion expected from equal numbers of binary divisions, an outcome difficult to explain using the standard model of cell cycle regulation. Using controlled synchronisation techniques, confocal microscopy to visualise key organelles and fluorescence in situ hybridization (FISH) to follow the movements and replication of genes and telomeres, we have re-analysed the timing and progression of mitotic events. The asynchronous duplications of the P. falciparum centrosome equivalents, the centriolar plaques, are established and these are correlated with chromosome and nuclear divisions in a new model of P. falciparum schizogony. Our results improve the resolution of the cell cycle and its phases during P. falciparum IE development, showing that asynchronous, independent nuclear division occurs during schizogony, with the centriolar plaques playing a major role in regulating mitotic progression.     

Morphological and genetic differentiation of two loliginid squids, Uroteuthis (Photololigo) chinensis and Uroteuthis (Photololigo) edulis (Cephalopoda: Loliginidae), in Asia

The squids Uroteuthis (Photololigo) edulis and Uroteuthis (Photololigo) chinensis (family Loliginidae) are commercially important fishery species in many coastal regions of Asia. The morphologies of these two squids are very similar, and identification based on morphology has been inadequate. The occurrence of cryptic species in the family Loliginidae has been reported. The widely distributed U. (P.) chinensis and U. (P.) edulis are believed to comprise several cryptic species. In this study, the taxonomic status of the two species in East Asia was elucidated by morphological and genetic analyses. Analysis of U. (P.) chinensis from Hong Kong and Xiamen (China) and U. (P.) edulis from Yamaguchi (Japan) and Shanghai (China) was performed in order to determine the effectiveness of different morphometric variables in discriminating between the two species. Multivariate analysis of 27 morphometric indices revealed no new morphological characters for the taxonomic identification of the two taxa, which can be distinguished by the teeth shape and number on arm sucker rings, and the percentage of hectocotylized part of left arm IV of males. The morphometric differences between U. (P.) edulis individuals from the two localities is most probably due to differences in the maturity stages of the sampled individuals between the two localities. Genetic analysis based on the mitochondrial COI and 16S rRNA genes revealed a high divergence of 15.5% and 7.5% respectively, indicating that U. (P.) edulis and U. (P.) chinensis are distinct species.     

Light-harvesting complex II (LHCII) and its supramolecular organization in Chlamydomonas reinhardtii

LHCII is the most abundant membrane protein on earth. It participates in the first steps of photosynthesis by harvesting sunlight and transferring excitation energy to the core complex. Here we have analyzed the LHCII complex of the green alga Chlamydomonas reinhardtii and its association with the core of Photosystem II (PSII) to form multiprotein complexes. Several PSII supercomplexes with different antenna sizes have been purified, the largest of which contains three LHCII trimers (named S, M and N) per monomeric core. A projection map at a 13 Å resolution was obtained allowing the reconstruction of the 3D structure of the supercomplex. The position and orientation of the S trimer are the same as in plants; trimer M is rotated by 45° and the additional trimer (named here as LHCII-N), which is taking the position occupied in plants by CP24, is directly associated with the core. The analysis of supercomplexes with different antenna sizes suggests that LhcbM1, LhcbM2/7 and LhcbM3 are the major components of the trimers in the PSII supercomplex, while LhcbM5 is part of the “extra” LHCII pool not directly associated with the supercomplex. It is also shown that Chlamydomonas LHCII has a slightly lower Chlorophyll a/b ratio than the complex from plants and a blue shifted absorption spectrum. Finally the data indicate that there are at least six LHCII trimers per dimeric core in the thylakoid membranes, meaning that the antenna size of PSII of C. reinhardtii is larger than that of plants.     

Presumptive neurosecretion in Leucochloridiomorpha constantiae (Trematoda) and its possible role in governing maturation

A review of literature dealing with neurosecretion in the Platyhelminthes is presented and presumptive neurosecretory cells associated with the cerebral ganglia and longitudinal nerve cords ofLeucochloridiomorpha constantiae metacercariae and adults are described. Both the mean number of aldehyde-fuchsin stained presumptive neurosecretory cells and the intensity of staining are less in adults than in metacercariae. This suggests that maturation in this trematode may be directly or indirectly associated with a reduction in neurosecretion as in nereid annelids. Results of cytochemical studies indicate the neurosecretory product contains a glycoprotein.     

Helicobacter pylori releases a factor(s) inhibiting cell cycle progression of human gastric cell lines by affecting cyclin E/cdk2 kinase activity and Rb protein phosphorylation through enhanced p27(KIP1) protein expression

Helicobacter pylori, the main cause of chronic gastritis, plays a central role in the etiology of peptic ulcer disease and gastric cancer. In vitro studies have shown that H. pylori increases gastric epithelial cell turnover, thus increasing the risk for the development of neoplastic clones. The mechanisms by which H. pylori promotes perturbation of cell proliferation are not yet elucidated. To investigate whether products released by H. pylori in culture media interfere with cell cycle progression of human gastric epithelial cells, four cell lines (MKN 28, MKN 7, MKN 74, and AGS) were incubated in the presence of H. pylori broth culture filtrate. Cell cycle analysis showed that a H. pylori-released factor(s) significantly inhibited the G1- to S-phase progression of MKN 28 and MKN 7 cell lines, with a reversible, nonlethal mechanism, independent of the expression of VacA, CagA, and/or urease. The cell cycle inhibition occurred concomitantly with an increase in p27(KIP1) protein levels, a reduction in Rb protein phosphorylation on serine residues 807-811, and a significant decrease in cyclin E-associated cdk2 activity. In contrast, the cell cycle progression of MKN 74 and AGS cell lines was not affected by the H. pylori-released factor(s). In normal human fibroblasts, G1-phase cell accumulation was concomitant with the reduction in Rb protein phosphorylation; that, however, appeared to be dependent on p21(WAF1/CIP1) rather than on p27(KIP1) protein. A preliminary characterization showed that the molecular mass of the partially purified cell cycle inhibitory factor(s) was approximately 40 kDa. These results suggest that H. pylori releases a soluble factor(s) that may affect cell cycle progression of gastric epithelial cells through elevated levels of cdk inhibitor p27(KIP1). This factor(s) might act in vivo on noncolonized distant cells, the most proliferating cells of human gastric mucosa.     

Vitellogenin polypeptide levels in one susceptible and one cadmium-resistant strain of Oncopeltus fasciatus (Heteroptera: Lygaeidae), and its role in cadmium resistance

The heavy metal Cd has recently been shown to have endocrine disrupting activity in different vertebrate species. In the insect Oncopeltus fasciatus, Cd exposure delays ovarian maturation and inhibits vitellogenesis, probably through a reduction in vitellogenin (Vg) polypeptide synthesis. An O. fasciatus strain was selected in the laboratory for Cd resistance with gradually increasing Cd concentrations for 50 generations. Cd-selected (OCdR) insects were 2.7-2.3 times more resistant to chronic exposure to Cd than susceptible (OS) insects. OCdR females showed increased (up to 90%) Vg polypeptide levels in hemolymph. Vg polypeptide synthesis was equally decreased in OCdR and OS females after acute Cd exposure, indicating that resistance in the OCdR strain was not due to a reduced sensitivity of the target of Cd. Vg polypeptide concentrations and ovarian maturation in acutely exposed OCdR females, however, were equal to those of control OS females. OCdR females chronically exposed to Cd showed a 30% decrease in Vg polypeptide concentrations, relative to control OS females. It is concluded that increased Vg polypeptide levels play an important role in resistance to the toxic effects of Cd on reproduction in the OCdR strain, and that this novel Cd resistance mechanism has the potential to evolve in vertebrate species in which Cd inhibits Vg synthesis by disruption of the endocrine system.     

Acridine Orange based platinum(II) complexes inducing cytotoxicity and cell cycle perturbation in spite of GSTP1 up-regulation

A series of new ionic Pt(II) complexes of general formula [Pt(II)(A)n(Cl)(AO)]X (A=en, NH3; n=1, 2; X-=BF4-, NO3-, PF6-, CF3SO3-), 1-5, containing Acridine Orange (AO) bound to the metal atom through the endocyclic N atom, have been tested in human melanoma cells (M14, JR8 and PLF2), human neuroblastoma cell line SH-SY5Y and its cis-platin resistant subline SH-SY5Yres. The Pt(II) compounds, and in particular complexes 1 and 4, exhibit higher cytotoxic activity at lower concentration compared to cis-DDP in melanoma cells, affecting cell growth behavior and causing cell cycle perturbation. Moreover, M14 and JR8 cell lines were not able to rescue the impairment due to the new Pt(II) complexes since perturbation of cell cycle phases and cell proliferation inhibition were found after 72 h of recovery time. In order to evaluate whether GSTP1 may play a role in chemo-resistance of our melanoma model, we investigated the effect of the treatment with these Pt(II) compounds on GSTP1 gene expression. Up-regulation of GSTP1, evaluated by Qreal-time PCR was observed after treatment with complexes 1 and 4, showing that the effect of these Pt(II) compounds is GSTP1 indipendent. The lack of resistance of the new Pt(II)-AO complexes and their cytotoxicity, cell growth and cell cycle recovery in melanoma cells provide the basis for the development of new platinum anticancer compounds, directed to those tumors that over express GSTs enzymes.     

Pollen tube reallocation in two preanthesis cleistogamous species, Ranalisma rostratum and Sagittaria guyanensis ssp. lappula (Alismataceae)

Pollen deposition on stigmas and pollen tube growth in two apocarpous species, Ranalisma rostratum and Sagittaria guyanensis ssp. lappula (Alismataceae), were examined with fluorescence microscopy. The reallocation of pollen tubes among pistils was observed in both species. The percentage of pollinated stigmas per flower was only 22.0% in R. rostratum and 51.0% in S. guyanensis, though the seed/ovule ratios are higher than 65% in both species. The number of pollen grains on each single stigma ranged from 0 to 96 in R. rostratum, and from 0 to 125 in S. guyanensis. When more than one pollen grain deposited on a stigma, all pollen tubes grew to the ovary, but only one of them turned towards the ovule and finally entered the nucleus. The other tubes grew through the receptacle tissue into ovules of adjacent carpels whose stigmas were unpollinated or pollinated later. The intercarpellary growth of pollen tubes could be a mechanism to increase the efficiency of sexual reproduction in an apocarpous gynoecium with low pollination on the pistils.     

p66(Shc) gene has a pro-apoptotic role in human cell lines and it is activated by a p53-independent pathway

p66(Shc) protein has been proposed to be an indispensable factor for p53-dependent, mitochondria-mediated apoptosis in mice. Here, we show that p66(Shc) plays a pro-apoptotic role also in cell lines of human origin such as SaOs-2 and HeLa, where p53 is either absent or inactivated, thus, suggesting that p66(Shc) pro-apoptotic role is independent from the presence of a functional form of p53. The active form of p66(Shc) is phosphorylated in Serine 36. We confirm the importance of Serine 36 phosphorylation for p66(Shc) pro-apoptotic role, and our results suggest that the kinase involved in this process is activated independently from p53.     

Comparison of diet and feeding activity of two polychaetes, Nephtys caeca (Fabricius) and Nereis virens (Sars), in an estuarine intertidal environment in Québec, Canada

It is crucial to study how food resources are allocated in order to understand how invertebrate species of soft-bottomed benthic communities can live in the same environment, especially when belonging to the same trophic level. On the southern shore of the Lower St. Lawrence Estuary, the polychaetes Nereis virens (Nereidae) and Nephtys caeca (Nephtyidae) represent the main infaunal predators and are known to regulate (by predation) other populations within their community. For these animal populations, there is a high degree of spatial overlap in the horizontal dimension as well as vertically in the sediment column. The purpose of this study was to describe and compare the feeding activity of N. virens and N. caeca in relation to several environmental factors. The feeding activity of N. caeca was further studied under natural and experimental conditions and a feeding experiment was carried out to evaluate how the food resource is subdivided in conditions of high spatial overlap. Finally, the prospecting activity of N. virens was described under natural conditions. N. virens and N. caeca exhibited a high degree of dietary overlap, particularly at the adult stage. This food resource overlap can easily influence the regulation of these species, even though they display different feeding responses to environmental stimuli. Tides and storm events can also influence the feeding behaviour. We conclude that different behavioural feeding responses likely favour the occurrence of these two species in the same trophic environment.