CHARACTERIZATION AND TOPOGRAPHY OF THE GLYCOPROTEINS OF ADRENAL CHROMAFFIN GRANULES

The glycoproteins of the membranes of bovine chromaffin granules were characterized by two polyacrylamide gel electrophoresis systems. Five components (I-V) were demonstrated with apparent molecular weights ranging in the unreduced form from 45,000 to 150,000. Glycoprotein I was identified as the enzyme dopamine β-hydroxylase. Four of these glycoproteins (with the exception of component IV) were apparently also present in the membranes of pig and horse chromaffin granules. The soluble proteins of chromaffin granules contained at least three glycoproteins. Only glycoprotein I (dopamine β-hydroxylase) was present both in the soluble content and in the membranes of chromaffin granules. Affinity chromatography with lectins demonstrated that from the soluble proteins only dopamine β-hydroxylase was adsorbed by concanavalin A, whereas none of these proteins reacted with wheat germ lectin and Ricinus communis agglutinin. Three membrane proteins including dopamine β-hydroxylase and glycoprotein II as major components were adsorbed by concanavalin A, whereas wheat germ lectin bound only component II and a small amount of component III. By electron microscopy it was demonstrated that concanavalin A did not bind to intact chromaffin granules whereas ruthenium red and cationized ferritin did. Isotope labelling after galactose oxidase treatment revealed that at least the carbohydrate portion of the major glycoproteins is present on the inner side of the granule membranes facing the content.     

DEVELOPMENT OF THE ENDOPARASITIC DINOFLAGELLATE AMOEBOPHRYA CERATII WITHIN HOST DINOFLAGELLATE SPECIES1

The endoparasitic dinoflagellate Amoebophrya ceratii Koeppen occurs in coastal waters of Nova Scotia within cells of two dinoflagellate hosts, a Scrippsiella species (probably S. trochoidea (Stein) Loeb. III) and Dino-physis norvegica Claparede & Lachman. We describe the development of the endoparasitic stage (the trophont) of A. ceratii within host cells using light and electron microscopy. After entry into the host, the trophont grows and expands until most of the host cell is occupied by the parasite. Growth is marked by a proliferation of trophont nuclei and flagella and by the formation of numerous lobes, each of which possesses a characteristic dinoflagellate amphiesma. The mature endoparasitic trophont is recognized at the light microscopic level as a beehive-shaped structure that consists of numerous lobes of the developing motile sporont cells and a mastigocoel cavity containing the sporont flagella.     

THE CHARACTERIZATION OF SPHINGOLIPIDS OF OLIGODENDROGLIA FROM CALF BRAIN

Abstract— Purified oligodendroglia isolated from bovine brain white matter were found to contain, in addition to galactosylceramide, sulfatide and sphingomyelin, significant quantities of glucosylcerai-mide, dihexosylceramide and esterified galactosylceramide. These sphingolipids were isolated and quan-titated and their fatty acid and long chain base patterns compared with those from sphingolipids isolated from bovine myelin, white matter and gray matter.
The minor glycosphingolipids, glucosylceramide, dihexosylceramide and esterified galactosylceramide, constituted a higher percentage of glial lipids than of myelin lipids. Glucosylceramide accounted for 12% of the total glial monohexosylceramide fraction and 0.8% of total lipids; dihexosylceramide was 0.9% of total glial lipids. Both of these lipids had small quantities of α-hydroxy fatty acids. The unsubstituted fatty acids of glucosylceramide were mostly short chain (16 and 18 carbons) and were different from those of the dihexosylceramides which were a mixture of short and long chain. The hydroxy acids of each of these lipids were, however, similar and resembled those of galactosylceramide.
The fatty acid patterns of galactosylceramide, sulfatide and sphingomyelin from glial cells resembled those of the corresponding lipids from myelin and white matter. The amide-linked acids of esterified galactosylceramide contained both unsubstituted and α-hydroxy chains. Their patterns were not identical to those of galactosylceramide, but were similar in all brain fractions.
With the exception of sphingomyelin and dihexosylceramide, which contained small amounts of C₂₀-sphingosine, all sphingolipids analyzed contained mostly sphingosine and dihydrosphingosine.
We conclude that the distribution of sphingolipids in the oligodendroglia is characteristic, but the lipophilic residues of these lipids are not cell-specific.     

A DINOFLAGELLATE CYST FROM ANTARCTIC SEA ICE1

The small (< 15 μm) hypnozygote of an autotrophic athecate dinofiagellate found in association with Antarctic sea ice had an external covering composed of approximately 60 plates, each of which was bounded by sutural ridging and possessed an intratabular process. A cingulum and sulcus were also evident. The ultrastructure of the cyst was increasingly dominated by storage bodies as the cyst matured, and the cell wall thickened from 0.2 to 0.8 μm over 2 months. This cyst has been encountered often but usually at low abundances (10³?10⁴ cells·L^?1); however, the maximum abundances observed (10⁶ cells·L^?1) indicate that the formation of this cyst may play an important part in the ecology of sea ice communities.     

SCANNING ELECTRON MICROSCOPY OF THECAE OF THE DINOFLAGELLATE GENUS ORNITHOCERCUS1

Members of the genus Ornithocercus are all tropical oceanic species in which the theca is extended in the form of elaborate wing-like projections, the lists, supported by ribs. This paper illustrates the topography of 6 of the species. The theca is penetrated by numerous simple pores opening flush with the outer surface. On the inner side of the thecae the pores have a raised rim. The hypotheca of all species examined except O. splendidus have shallow depressions, the areolae, over most of the surface. Secondary thickening of mature cell walls deepens the appearance of the areolae, and increases their extent over the hypotheca in O. quadratus. The number of pores is not directly correlated with the areolae but seems rather to be a function of cell size. A comparison of the surface features confirms views that O. splendidus occupies a relatively isolated phenetic position. It also confirms the close similarity of O. steinii with O. thumii. Unexpectedly it suggested a phenetic closeness between O. magnificus and O. quadratus on the basis of hypothecal structure and rib features of the left sulcal list. O. heteroporus could not be subjected to the same degree of study and its position cannot be commented on. Some theoretical hydrodynamic and morphogenetic problems in Ornithocercus are discussed.     

THECAL ULTRASTRUCTURE OF THE TOXIC MARINE DINOFLAGELLATE GONYAULAX CATENELLA1

The toxic marine dinoflagellate Gonyaulax catenella Whedon & Kofoid was studied with scanning and transmission electron microscopy to describe the thecal morphology and to accurately define the taxonomic characters of the species. The closing platelet which lies in a U-shaped apical pore was revealed to be disassociable from a partly obscured apical platelet. Two previously unreported sulcal plates were charaterized and described. The entire complement of thecal plates numbered 33.     

VEGETATIVE REPRODUCTION AND SEXUAL LIFE CYCLE OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM FROM TASMANIA,AUSTRALIA1

The toxic, chain-forming dinoflagellate Gymnodinium catenatum Graham was cultured from vegetative cells and benthic resting cysts isolated from estuarine waters in Tasmania, Australia. Rapidly dividing, log phase cultures formed long chains of up to 64 cells whereas stationary phase cultures were composed primarily of single cells (23-41 pm long, 27-36 pm wide). Vegetative growth (mean doubling time 3-4 days) was optimal at temperatures from 14.5-20° C, salinities of 23-34% and light irradiances of 50-300 μE·m^?2·s^?1. The sexual life cycle of G. catenatum was easily induced in a nutrient-deficient medium, provided compatible opposite mating types were combined (heterothallism). Gamete fusion produced a large (59-73 μm long, 50-59 μm wide) biconical, posteriorly biflagellate planozygote (double longitudinal flagellum) which after several days lost one longitudinal flagellum and gradually became subspherical in shape. This older planozygote stage persisted for up to two weeks before encysting into a round, brown resting cyst (42-52 μm diam; hypnozygote) with microreticulate surface ornamentation. Resting cysts germinated after a dormancy period as short as two weeks under our culture conditions, resulting in a single, posteriorly biflagellate germling cell (planomeiocyte). This divided to form a chain of two cells, which subsequently re-established a vegetative population. Implications for the bloom dynamics of this toxic dinoflagellate, a causative organism of paralytic shellfish poisoning, are discussed.     

THE UNIQUE,MICRORETICULATE CYST OF THE NAKED DINOFLAGELLATE GYMNODINIUM CATENATUM1

Gymnodinium catenatum Graham is an unarmored dinoflagellate responsible for episodes of paralytic shellfish poisoning. This species forms a resting cyst that is unique in several ways. The outer surface of the spherical, brownish cyst is microreticulate and composed of hundreds of 1-3 μm polygons. In several regions, these polygons are smaller, more uniform in shape, and oriented in distinct bands that define morphological features. These features on the cyst reflect the cingulum, sulcus, flagellar pore complex, and acrobase of the motile stage precursor to the cyst. The archeopyle is irregularly but extensively developed. Its margin is generally smooth and extends almost completely around the circumference of the cyst, though not consistently in the plane of the equator. The cyst wall is resistant to acetolysis and standard palynological preparation techniques. Gymnodinium catenatum Graham is emended to include the details of the cyst stage. The significance of this cyst is that it is the first described cyst of a naked dinoflagellate that bears oriented surface ornamentation reflecting features of the motile dinoflagellate. Its microreticulate surface ornamentation is unique to dinocysts, naked or armored, living or fossilized. Resistance of the cyst wall to harsh processing techniques suggests the presence of sporopollenin-like material commonly associated with cysts of armored dinoflagellates. From an ecological standpoint, the existence of a G. catenatum cyst has important implications with respect to species bloom dynamics and geographic distribution. In addition, the distinct differences between this cyst and those of the armored saxitoxin-producing gonyaulacoid species argues against a proposed evolutionary linkage.     

PARTIAL CHARACTERIZATION OF THE CHLOROPLAST GENOME FROM THE CHROMOPHYTIC ALGA SYNURA PETERSENII (SYNUROPHYCEAE)1

Hoechst dye 33258-CsCl density gradients were used to isolate two satellite DNA species from Synura petersenii Korsh. sensu lato, a member of the Synurophyceae. One satellite DNA was identified as the chloroplast genome. The chloroplast genome is the smallest (91.5 kb) published for any chromophyte and approximates the size of the smallest functional chlorophyte chloroplast genome (Codium fragile, 89 kb). The second satellite DNA was small (34.5 kb), and its origin is undetermined. The potential of using the S. petersenii chloroplast genome in comparative studies for evaluating organellar evolution and algal systematics is discussed.     

EFFECTS OF TURBULENCE ON THE MARINE DINOFLAGELLATE GYMNODINIUM NELSONII1

Laboratory experiments were conducted to study the effects of agitation on growth, cell division, and nucleic acid dynamics of the dinoflagellate Gymnodinium nelsonii Martin. When cultures were placed on an orbital shaker at 100 rpm, cell division was prevented, cellular volume increased up to 1.5 times that of the nonperturbed cells, the form and location of the cell nucleus were modified, and the RNA and DNA concentrations per cell increased up to 10 times those of the controls. When shaking was stopped after 10 days, cells divided immediately at about 2/3 of the division rate of the unshaken populations, and all the altered parameters were restored. If the agitation continued for more than 20 days, total cell death and disintegration occurred. Several cellular types differing in size and shape were observed in the control and shaken cultures. One possible hypothesis for these results is that failure of the cell to divide results from physical disturbance of the microtubule assemblage associated with chromosome separation during mitosis. My study suggests that small-scale oceanic turbulence of sufficient intensity may inhibit growth of individual dinoflagellate cells, but immediate development of the population may continue when calm weather follows the active mixing period.     

INTRACELLULAR LOCALIZATION OF SAXITOXINS IN THE DINOFLAGELLATE GONYAULAX TAMARENSIS1

The potent neurotoxin saxitoxin, and possibly several of its derivatives, are localized in two types of sites within the marine dinoflagellate Gonyaulax tamarensis Lebour. Immunocytochemical techniques using a polyclonal antibody and epifluorescence microscopy demonstrate toxin localization within the nucleus as well as on the periphery of small granules thought to be starch grains. In the nuclear region, the labelling occurred on or close to the permanently-condensed chromosomes as well as in an area within the two arms of the nucleus in the vicinity of the nucleous. No binding was observed in a closely-related, non-toxic dinoflagellate. Different binding affinities were observed between the nucleus and the grains at high and low antibody dilutions. This may relate to the polyclonal nature of the antiserum and to the presence of multiple toxins within the G. tamarensis isolate studied. Mechanistic interpretations of these labelling patterns remain speculative, especially the localization of the antigen at the outer edge of starch grains, but the distinct labelling in the nuclear region suggests that saxitoxin, with its two positively charged guanidinium groups, may bind to nucleic acids or nuclear proteins in a manner analogous to the polyamines and other cations. The labelling patterns reported here suggest that the saxitoxins may not simply be secondary metabolites but instead could be important compounds involved in the structure and function of the G. tamarensis genome.     

PURIFICATION AND CHARACTERIZATION OF TWO FORMS OF PHOSPHOGLYCERATE KINASE FROM THE GREEN ALGA SELENASTRUM MINUTUM1

We report the first purification and characterization of a eukaryotic algal phosphoglycerate kinase (PGK), Two forms of PGK (PGK₁ and PGK₂) from the green alga Selenastrum minutum (Naeg.) Collins were purified to electrophoretic homogeneity with specific activities of 1100 and 1069 units · mg^?1 protein, respectively. The portion of PGK₁ and PGK₂ (probably the cytosolic and chloroplastic forms, respectively) in this organism was estimated as 32 and 68%, respectively. PGK₁ was more heat-stable than PGK₂. The M_r estimation for PGK₁ and PGK₂ by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration indicated that they both were monomeric with a similar M_r of approximately 44 kDa. Antibodies raised against S. minutum PGK₁ cross-reacted with PGK₂ as well as PGKs from prokaryotic and eukaryotic sources, suggesting that PGK₁ was structurally and immunologically closely related to PGK₂ and other PGKs, which was consistent with NH₂-terminal sequence analysis. Comparative kinetic and regulatory properties of PGK₁ and PGK₂ from S. minutum were investigated, Both forms exhibited hyperbolic kinetics with respect to both 3-phosphoglycerate (3-PGA) and Mg-adenosine triphosphate^2- (MgATP^2-) under the conditions tested and had similar K_m values for each substrate (PGK₁; K_m (MgATP_2-) = 0.37 mM, K_m(3-PGA) = 0.59 mM; PGK₂; K_m(MgATP^2-) = 0.32 mM, K_m(3-PGA) = 0.46 mM). PGK₁ and PGK₂, however, differed significantly in several other kinetic properties. PGK₂ had a broad pH optimum between 7.3 and 7.8, as compared to PGK₁, with a pH optimum of 7.3 Mg²⁺ was the most efficient cofactor for both forms; it inhibited PGK₁ but not PGK₂ at higher concentrations (>10 mM). Other divalent cations (Mn²⁺, Zn²⁺, Co²⁺, Cd²⁺, and Ca²⁺) only partially replaced Mg²⁺ and were more effective for PGK₁ than for PGK₂, A wide range of metabolites was examined for regulatory properties. Energy charge was the most important factor in regulating the two forms of S. minutum PGK. These results were interpreted in light of the regulation of this kinase in response to the cell energy requirement and the need for glycolytic carbon flow to provide carbon skeletons for amino acid biosynthesis.     

SYNCHRONIZATION OF THE MARINE DINOFLAGELLATE AMPHIDINIUM CARTERI IN DENSE CULTURES1

Under improved conditions, Amphidinium carteri Hulburt can be obtained as dense cultures (5–9 × 10⁵ cells · ml^?1) 5 days after inoculation. The possible association of a marine bacterium with the alga is discussed. A method to synchronize this photosynthetic organism is described which is based on the endogenous rhythm of division and the effect of light-dark alternating periods.     

THE CHEMICAL NATURE OF KERATOHYALIN GRANULES OF THE EPIDERMIS

Keratohyalin granules were isolated in the native form from the epidermis of newborn rats by the use of citric acid and a detergent. The isolated granules revealed a fine granular substructure in the electron microscope similar to that seen in situ. Analyses of amino acids by automated column-chromatography showed that proline and cystine are present in large proportions whereas histidine is present in a small amount. Accordingly, it was concluded that keratohyalin represents a sulfur-rich amorphous precursor of the horny cell content, rather than a sulfur-poor side product of the keratinization process, or a unique histidine-rich protein as proposed by in situ histochemical and radioautographic studies.     

RECYCLING OF METABOLIZED IRON BY THE MARINE DINOFLAGELLATE AMPHIDINIUM CARTERAE1

Senescent, iron-limited cultures of Amphidinium carterae Hulburt were used to detect the presence of iron made available for phytoplankton growth by recycling of metabolized iron from disrupted cells. Both physical disruption of cells and physical disruption plus exposure to a low pH were tested. The test cultures remained viable over long periods of time, and were stimulated to full growth by the addition of Fe, but not by the addition of any other nutrients. Simple physical disruption of cells caused only a very slow release of available Fe, while disruption of cells plus exposure to a low pH resulted in a rapid release of available Fe. It is suggested that the digestive processes of herbivores are instrumental in the rapid regeneration of Fe as a nutrient available for phytoplankton growth.     

小定鞭藻毒素的分离与鉴定

从大量死鱼的鱼池中收集分离出小定鞭藻Ｐｒｙｍｎｅｓｉｕｍｐｏｒｖｕｍ的毒株,并在实验室成功地进行了单种培养,当温度２３℃,光照６００－８００ｌｘ盐度约１２—１６‰左右时,该藻在海水及人工海水培养基中均生长良好,在对数生长末期到平衡期溶血毒素活性最高。从藻细胞及浓缩的培养液中提取出二种毒素：溶血毒素（Ｈａｅｍｏｌｙｔｉｃｔｏｘｉｎｓ）和鱼毒素（Ｉｃｈｔｈｙｏｔｏｘｉｎｓ）。用新鲜牛血球测定了溶血毒素活性;用孔雀鱼测定了鱼毒素活性。用部分纯化的溶血毒素经元素分析、红外光谱、核磁共振及ＦＡＢ质谱测定,结果显示该藻溶血毒素可能是一个糖脂。     

PROTEIN SEQUENCE ANALYSIS OF THE HISTONE-LIKE PROTEIN HCC FROM THE HET-EROTROPHIC DINOFLAGELLATE CRYPTHECODINIUM COHNII (PYRROPHYTA)

Morris, R. L. & Rizzo P. J. Department of Biology, Texas A&M University, College Station, TX 77843 USA The major histone-like protein HCc was extracted from chromatin of the dinoflagellate Crypthecodinium cohnii, purified by carboxymethylcellulose (CMC) chromato-graphy and high performance liquid chromatography (HPLC), for protein sequencing. Four fractions were identified by HPLC fractionation of the CMC 400 mM NaCl peak, which proved to be very similar in amino acid composition, differing by only several amino acids. These differences are of the same level as the differences in histone variants of typical eukaryotes. The fractions were analyzed by peptide mapping using V8 protease, which also showed very close similarity between the four proteins. Protein sequence information was obtained by sequencing overlapping peptides, to yield approximately 80% of the protein sequence for two of the variants. Sequence comparisons with HCc1 and HCc2 from C,cohnii as reported by Sala-Rovira et al. (Chromosoma 100, 510) suggest that these variants are similar, but not identical to HCc1 and HCc2.     

ULTRASTRUCTURE OF THE FEEDING APPARATUS AND MYONEMAL SYSTEM OF THE HETEROTROPHIC DINOFLAGELLATE PROTOPERIDINIUM SPINULOSUM1

The feeding veil or pallium of the thecate heterotrophic dinoflagellate Protoperidinium spinulosum Schiller is a highly vesiculate membranous sac containing several arched, sometimes bifurcated microtubular ribbons. It originates from an internal microtubular basket, passes through a sphincter-like osmiophilic ring located inside the posterior flagellar pore, and emerges from the cell at that pore. The osmiophilic ring is part of an interconnected myonemal system (composed of two striated collars and several striated connectives) that is anchored to the pore plate and to two inward protrusions composed of minute sulcal plates. A related species, Protoperidinium punctulatum (Paulsen) Balech, also possesses a microtubular basket/osmiophilic ring complex. Elongate electron-dense bodies within the basket resemble digestive secretory granules found in other protists. Granular, electron-lucent microbodies clustered at the anterior end of the basket may also have a role in prey digestion. Dense membranous whorls observed within a P. spinulosum cell presented as it was preparing to initiate feeding indicate a condensed storage site for pallium membranes. A narrow microtubule-strengthened pseudopodal appendage found in two non-feeding cells constitutes the tow filament that serves as the initial linkage between the dinoflagellate and its food. The structures that constitute the pallium and pallium precursors, described here for the first time, are unlike those of other known protists, although some similarities with the dinoflagellate peduncle are evident. The existence of this unique system of organelles may have important ramifications in the search for evolutionary relationships among protists.     

红曲菌代谢产物中低极性组分的分离及表征

以石油醚∶醋酸乙酯 =4∶1(V/V)作为洗脱剂 ,采用柱层层析粗分离醇溶性红曲菌代谢产物中的低极性组分。经浓缩、结晶除去白色结晶后的浓缩液 ,用正己烷∶醋酸乙酯 =9∶1(V/V)作为展开剂进行薄层层析分离 ,在紫外灯下观察 ,从低极性组分中分离出六个组分 ,分别为 :具荧光组分、两个相隔较近的黄色组分、淡黄色具荧光组分、具浅蓝绿色荧光组分、具荧光组分。各组分的Rf 值分别为 :0 2 9、0 15、0 12、0 0 9、0 0 6、0 0 4。MS测定结果表明 ,Rf 值最大的具荧光组分可能为含有 OH及Br的共轭烯烃或脂肪酮 ,而在紫外灯下呈淡黄色组分为含有 OH的环状化合物。     

CAROTENOID INTERCONVERSION IN LIGHT-DARK CULTURES OF THE DINOFLAGELLATE AMPHIDINIUM KLEBSII1

The carotenoid pigments of Amphidinium klebsii cultures grown in LD 12:12 light regimes were determined in cells harvested during the log phase growth in the dark and light photoperiods. The analysis of the individual pigments revealed the presence of an endogenous redox carotenoid system involving the epoxide carotenoid diadinoxanthin and an unidentified carotenoid with the properties of a dihydroxy xanthophyll.