FURTHER SEM STUDY OF MARINE DINOFLAGELLATES: THE GENUS OSTREOPSIS (DINOPHYCEAE)1

This paper presents a comprehensive examination of the taxonomy of the genus Ostreopsis Schmidt. The morphology of six species of marine dinoflagellates, Ostreopsis siamensis Schmidt 1902. Ostreopsis lenticularis Fukuyo 1981, Ostreopsis ovata Fukuyo 1981, Ostreopsis heptagona Norris, Bomber, et Balech 1985, Ostreopsis mascarenensis Quod 1994, and Ostreopsis labens Faust et Morton 1995 from three geographical regions (Japan, Southwest Indian Ocean, and the Caribbean) and three marine habitats (sand, water column, and macroalgal surfaces) are described from scanning electron micrographs. Differences in the following morphological characteristics differentiated the species: cell shape and size, and ornamentation of the epitheca, cingulum, and hypotheca. The thecal plate formula of the six Ostreopsis species is P_o, 3′, 7″, 6C, 6S?, V_p, R_p, 5′″, 1p, 2″″, with differences in thecal plate size and shape. The cingulum in ventral view has two prominent structures: a ventral plate (V_p) with a ventral pore (V_o) and a ridged plate (R_p) that distinguishes Ostreopsis species from any other dinoflagellate taxa. This paper also includes ecological and toxicity information regarding the six Ostreopsis species.     

VARIABILITY OF THE HEPATOTOXIN MICROCYSTIN-LR IN HYPEREUTROPHIC DRINKING WATER LAKES1

The patterns of occurrence of the peptide hepatotoxin microcystin-LR (MC-LR) was studied in three hypereu-trophic hardwater lakes (Coal, Driedmeat, and Little Beaver) in central Alberta, Canada, over three open-water seasons. MC-LR concentration was based on high-performance liquid chromatography detection and expressed as μg.g^?1 of total plankton biomass, ng.L^?1 of lake water, and μg.g^?1 of Microcystis aeruginosa Kuetz. emend. Elenkin. MC-LR was highly variable temporally (differences up to 3 orders of magnitude) within each lake over an individual year, between years in an individual lake, and between lakes in any year. Seasonal (within-year) changes in MC-LR concentration (expressed in the preceding units) were positively correlated to the abundance and biomass Of the cyanobacterium M. aeruginosa (r =0.60–0.77), total and total dissolved phosphorus concentration (r =0.46–0.59), pH (r=0.38–0.58), and chlorophyll a (r=0.25–0.59). Surprisingly, there was no relationship between MC-LR concentration and water temperature (range: 7°-24°C, r =-0.13 to 0.02) and a negative correlation with nitrate concentration (r =–0.27 to -0.34). In two synoptic surveys examining spatial variability, MC-LR concentrations were quite variable (CV of 185 and 36% between sampling sites for Coal and Little Beaver lakes, respectively). Spatial distribution of MC-LR on any one day was correlated with the abundance and biomass of M. aeruginosa. Over a 24-h period, MC-LR concentration in M. aeruginosa decreased more than 6-fold at night relative to daytime concentrations. In general, analytical and within-site variation of MC-LR was relatively small (CV < 4 and 9%, respectively) but greatest both within and between years in a lake followed by diel and spatial variation.     

MORPHOLOGY AND ECOLOGY OF THE MARINE DINOFLAGELLATE OSTREOPSIS LABENS SP. NOV. (DINOPHYCEAE)1

A new species, Ostreopsis labens Faust et Morton sp. nov., is described from three marine habitats: lagoonal water and lagoonal sand from the barrier reef of Belize, and associated with macroalgae from coral reef habitats of Oshigaki and Iriomote Islands, Japan. Dimensions of Ostreopsis labens cells are 60–86 μm long, 70–80 μm wide, and 81–110 μm in dorsoventral depth. Cells are broadly ovoid, anterioposteriorly compressed bearing a spherical nucleus and many chloroplasts. The epitheca is convex and composed of three apical plates, seven precingular plates, and an apical pore plate. The cingulum is composed of six plates. The hypotheca is constructed of five postcingular plates, one posterior intercalary, and two antapical plates. The sulcus is small, recessed, and hidden and exhibits a ventral pore and a ridged, curved plate. The thecal arrangement of O. labens is P_o, 3′, 7″ 6C, 6S(?), V_p, R_p, 5″, 1p, 2″. Only one sulcal list is present. The thecal plates have a smooth surface with distinct round pores. The intercalary band between the thecal plates is smooth. A row of marginal pores line the lipped cingulum. Ostreopsis species are anteroposteriorly flattened, photosynthetic, benthic dinoflagellates that are more diverse in ecology than previously known. Ostreopsis labens is capable of living in three marine habitats: in the water column, in sand, and on macroalgal surfaces. It was most numerous in sand and less in lagoonal waters, and only a few cells were associated with macroalgae. Light and scanning electron microscopy studies revealed engulfed cells within O. labens, which indicates mixotrophic/phagotrophic behavior. A ventral opening situated in the cingulum of O. labens exhibits size variability; it may serve as an opening for engulfiing food particles because it varies in size. We propose that ingestion of prey by O. labens occurs through the ventral opening, the proposed feeding apparatus of this species, which is similar to the function of the peduncle-like structure of mixotrophic dinoflagellates. The behavior of O. labens appears similar to that previously described for Dinophysis species.     

The Rab Protein Family: Genetic Mapping of Six Rab Genes in the Mouse

Rab proteins constitute a family of GTP-binding proteins that are located in distinct intracellular compartments and play a role in the regulation of vesicular trafficking. Yeast mutations in Rab gene homologs cause defects in vesicular transport similar to those observed in beige (bg) mice. To investigate Rab genes as candidates for mouse mutations characterized by defects in vesicular trafficking, we utilized an intersubspecific backcross [C57BL/6J-bg^J × (C57BL/6J-bg^J × CAST/Ei)F₁] segregating for the bg locus. Restriction fragment length polymorphisms (RFLPs) were obtained through Southern hybridization of F₁ and C57BL/6J chromosomal DNA with the coding sequences of Rab genes. These RFLPs and 12 polymorphic microsatellites were used to determine the segregation of the Rab genes in 93 backcross mice. Rab4a, Rab4b, Rab7, Rab10, Rab22, and Rab24 were localized on mouse chromosomes 8, 7, 9, 12, 2, and 13, respectively. Although the results exclude these loci as candidates for bg , they demonstrate a wide dispersion of Rab genes throughout the mouse genome and reveal that Rab4b and Rab24 are possible candidates for the mouse mutations reduced pigmentation (rp) and purkinje cell degeneration (pcd), respectively.     

Preparation and antitumor activity of nontoxic lipid A

Summary Highly refined, disaggregated endotoxic glycolipids (B5) from heptose-less (Re) mutant Salmonella typhimurium quantitatively converted to nontoxic (lethality for chick embryos) and nonpyrogenic (fever in rabbits) lipid A by treatment with boiling 0.1 N HCl (B5-HC1). Nontoxic B5-HCl, like toxic B5, caused regression of line-10 tumors and elimination of lymph node metastasis in 27 of 32 (84%) syngeneic strain 2 guinea pigs at a dosage of 150 g. At this dosage, toxic B5 led to a cure in 54 of 67 (81%) tumor-bearing animals. All cured animals rejected a second line-10 tumor cell transplant. This activity depended on combining the toxic or nontoxic endotoxins with mycobacterial trehalose mycolate (P3) and an essentially nontoxic peptide-containing side-fraction (ACP) recovered during the isolation of B5. In contrast to toxic B5 or endotoxins in general, nontoxic B5-HCl did not cause endotoxic shock when combined with adjuvant dipeptide (MDP) and injected IV into guinea pigs. Chemical analysis showed that the phosphate content of nontoxic B5-HCl was about one-half that observed in toxic B5 or in toxic KDO-free lipid A, which was obtained by treating toxic B5 with sodium acetate at pH 4.5 at 100° C (B5-pH 4.5). The molar ratio of glucosamine: phosphorus: fatty acids was 2:1:4 for nontoxic B5-HCl and was 2:2:4 for toxic B5-pH 4.5. These results demonstrate that endotoxic extracts could be selectively detoxified while retaining antitumor properties. Thus, nontoxic B5-HCl may be a potential candidate for immunotherapy of human cancer.Presented at the 72nd Annual Meeting of the American Association for Cancer Research, 1981, and abstract no. 1123 published in the Proceedings of the American Association for Cancer Research, Vol. 22, 1981 Abbreviations used in this paper: ACP, a nontoxic acetone-chloroform precipitated side-fraction of endotoxin that contains (an) ingredient(s) necessary for tumor regression of line-10 tumors in strain 2 guinea pigs; ReGl, endotoxic glycolipids from Re mutant gram-negative bacteria; ReGl-PCP, ReGl extracted with phenol-chloroform-petroleum ether (PCP); B5, refined endotoxin, free of phospholipids, divalent cations and disaggregated; B5-HCl, nontoxic lipid A prepared from B5 by treatment with hydrochloric acid; B5-pH 4.5, toxic lipid A prepared from B5 by treatment with sodium acetate at pH 4.5; lipid A, hydrochloric acid or sodium acetate hydrolysate of ReGl-PCP or B5; MDP, N-acetyl-muramyl-l-seryl-d-isoglutamine; KDO, keto-3-deoxyoctonate     

Molecular cloning of the cDNA encoding a stress-inducible protein phosphatase 1 (PP1) catalytic subunit from French bean (Phaseolus vulgaris L.)

A cDNA showing high sequence similarity (>70%) to plant protein phosphatase 1 catalytic subunit variants from other species has been isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. The clone appears to be a near full-length 1431 bp with a 172 bp 5-untranslated region and a 317 bp 3-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with predicted M _r of 35552. Alternatively an ATG situated to the 5 end of the putative start site would increase the protein size by 6 amino acids.The mRNA for Pvpp1 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean. The cloned cDNA represents one of the few examples of a gene product that is probably involved in dephosphorylation events arising after the initial responses to biotic stress.Abbreviations PAL phenylalanine ammonia-lyase - PP1 protein phosphatase 1 - Pvpp1 Phaseolus vulgaris protein phosphatase 1     

Dissecting complex physiological functions through the use of molecular quantitative genetics

Testing possible associations between physiological and biochemicaltraits by comparing plant phenotypes and looking for correlationsbetween them is unreliable. The development of molecular markertechnologies offers powerful alternative methods to examinethe relationships between traits. This review describes thegenetical methods required to analyse possible associationsbetween traits that are inherited in a quantitative manner usingquantitative trait locus (QTL) analysis. The regulation of carbohydratemetabolism is chosen as an example of how QTL analysis can beused to identify key control factors in a series of processes,by identifying possible candidate genes for QTL effects on sucroseand starch metabolism. Methods are also described to study theassociation between physiological traits such as abscislc acidconcentrations and stomata1 conductance. Advantages and somelimitations of QTL analysis over other methods currently inuse by physiologists to test associations between traits arediscussed. Key words: Candidate genes, genetic maps, molecular markers, quantitative trait locus (QTL) analysis, physiological traits     

Skeletal muscle satellite cells cultured in simulated microgravity

Summary Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and, therefore, provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (∼ 200 gm) were used for all studies and were composed of greater than 75% satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D culture and 3-D HARV culture. Plating efficiency (cells attached ÷ cells plated ×100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and nonsatellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability because glucose levels in medium from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARV were joined together by cells into 3-D aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a 3-D level of organization that could provide a more suitable model to study postnatal muscle development than is currently available with standard culture methods.     

Effects of managanese salts on the AIDS-related pathogen,Cryptosporidium parvum in vitro and in vivo

The authors examined the effects of manganese salts on the interaction of the AIDS-related pathogen,Cryptosporidium parvum, with human ileoadenocarcinoma (HCT-8) cells in vitro. Manganese (Mn) inhibited binding ofC. parvum sporozoite membrane antigens to intact, fixed HCT-8 cells in a dose-dependent fashion, whereas Ca⁺⁺, Mg⁺⁺, and Zn⁺⁺ salts had no effect. Manganese was also found to affect sporozoite penetration of live HCT-8 cells, which resulted in a dose-dependent inhibition of parasite development. However, the levels of Mn⁺⁺ needed in the live cell assays was approx 10-fold greater than in the fixed-cell assays. This inhibition of parasite development was not reversible when Ca⁺⁺ or Mg⁺⁺ were used as competitors. Oral supplementation of suckling mice infected withC. parvum with MnSO₄ resulted in significant reductions and, in some cases, elimination of intestinally derived oocysts.     

Synergistic iron reduction and citrate dissimilation by Shewanella alga and Aeromonas veronii

Two bacterial isolates from Great Bay Estuary, New Hampshire, in co-culture carried out anaerobic dissimilation of citric acid with Fe(III) as the terminal electron acceptor. Neither isolate oxidized citrate with Fe(III) anaerobically in axenic culture. The Fe(III) reducer, Shewanella alga strain BrY, did not grow anaerobically with citrate as an energy source. The citrate utilizer, Aeromonas veronii, did not reduce iron axenically with a variety of electron donors including citrate. The onset of iron reduction by the co-culture occurred after initiation of citrate dissimilation and just prior to initiation of growth by either organism (as measured by viable plate counts). Anaerobic culture growth rates and final cell densities of each bacterial strain were greater in co-culture than in axenic cultures. By 48 h of growth, the co-culture had consumed 27 mM citrate as compared with 12 mM dissimilated by the axenic culture of A. veronii. By 48 h the co-culture produced half as much formate (6 mM) and twice as much acetate (40 mM) as did A. veronii grown axenically (12 mM and 20 mM, respectively). Formate produced from citrate by A. veronii appeared to have supported growth and Fe(III) reduction by S. alga.Although not obligatory, nutrient coupling between these two organisms illustrates that fermentative (A. veronii-type) organisms can convert organic compounds such as citrate to those used as substrates by dissimilatory Fe(III) reducers, including S. alga. This synergism broadens the range of substrates available for iron reduction, stimulates the extent and rate of organic electron donor degradation (and that of iron reduction) and enhances the growth of each participant. Received: 11 December 1995 / Accepted: 19 June 1996