THE MITOTIC APPARATUS : Fine Structure of the Isolated Unit

The fine structure of the mitotic apparatus isolated from the sea urchin egg has been investigated. The isolation was accomplished by lysis of metaphase eggs in a 1 M solution of hexanediol, buffered at pH 6. The fine structure of the isolated apparatus was studied after fixation with osmium tetroxide directly in the isolation medium. The spindle is composed of fine fibrils, approximately 20 mµ in diameter, which appear tubular. Similar fibrils, radially oriented, are found in the aster. If the isolated mitotic apparatus is exposed to water at pH 6 before fixation, the structure is considerably modified. The most pronounced effects are an increase in the number of large membrane-bounded vesicles and in the amount of free granular material present. The conditions necessary for the fixation of the mitotic apparatus in dividing cells are discussed in the light of these observations on the isolated unit.     

氨基酸生产和海洋生物的氨基酸资源开发

氨基酸在医药、食品、饲料等领域有着极为重要和广泛的用途,世界上氨基酸总需求量以５～１０％递增,市场竞争十分激烈。生物资源提取、化学合成、生物合成和综合法是生产氨基酸的４种技术,目前的发展趋势为生物合成和综合法,特别是将现代生物工程技术应用于氨基酸生产。另外,氨基酸生产领域另一个新的倾向是海洋生物氨基酸资源的开发和应用,尤其是海洋生物所产生的特殊氨基酸、肽及其衍生物的开发,同时,综合利用海产品加工后的废弃物来生产氨基酸也受到重视。     

Allelochemistry in marine macroalgae

Allelochemistry refers to the effect of an organic compound released from one organism upon an organism separated from its source. When the donor and receptor are plants (or microorganisms placed in the plant kingdom), allelopathy is described whether the effect is harmful or beneficial. In the aquatic environment, water disperses any water‐soluble allelochemical from its point of release, and rapid dilution along with lack of contact between competing organisms reduces potential encounter. This review centers on macroalgae as the source of allelochemicals. In all examples, the releasor organism is a macroalga, but receptor organisms include algae, invertebrates, fish, and microbes. Direct evidence in the sea is scanty, and there is a need for appropriate experiments in the laboratory and field. The compounds that are released by macrolagae (e.g., polyphenolics, halogenated phenols, and terpenoids) may be fortuitous byproducts of metabolism. But where they alter colonization, growth, or reproduction in a target organism, it is conceivable that they influence community structure as is known for terrestrial systems. The potential for allelochemistry is maximized in sites where water is poorly mixed, allowing released algal products to concentrate (e.g., tide pools and backbays) and where the receptor organism is adjacent to the releasor (e.g., surfaces of thalli and seaweed farms). In combination with restricting environmental conditions (e.g., critical temperature, light, salinity, pH, or oxygen), the effect of allelopathy can be synergistic. Combinations of allelochemicals, each at a concentration too low to be physiologically effective, could have a pronounced impact.     

Transposon mutagenesis in a marine synechococcus strain: isolation of swimming motility mutants

Certain marine unicellular cyanobacteria of the genus Synechococcus exhibit a unique type of swimming motility characterized by the absence of flagella or any other obvious organelles of motility. While the abundant cell surface-associated 130-kDa glycoprotein SwmA is known to be required for the generation of thrust, identification of other components of the motility apparatus has, until recently, been unsuccessful. Here we report on the development of a transposon mutagenesis system for use with marine Synechococcus sp. strain WH8102, a model organism for which the genome has been sequenced. Utilizing this mutagenesis technique, we have isolated 17 independent mutants impaired in swimming motility. These 17 transposon insertions are located in nine open reading frames, which cluster in three separate regions of the genome. Included within these clusters are several multicomponent transport systems as well as a number of glycosyltransferases.     

An Odontogriphid from the Upper Permian of Australia

A problematic organism, Bowengriphus perphlegis gen. et sp. nov., is described based on two specimens from the Late Permian Rangal Coal Measures of eastern central Queensland. It displays a double-looped feeding apparatus bearing small conical elements, considered homologous with that of the supposed lophophorate Odontogriphus omalus Conway Morris, 1976, from the Middle Cambrian Burgess Shale of British Columbia, Canada. Odontogriphids are thus interpreted as a group that survived through the Palaeozoic and made the transition from marine to freshwater environments. Recent proposals that odontogriphids are stem-group chordates are not well established.     

Potentiality of artificial sea water salts for the production of carrageenase by a marine Cytophaga sp

Production of an extracellular enzyme complex (carrageenase) was studied by examining cell-free fluids from cultures of a marine Cytophaga, 1k-C783, growing on different media. Among artificial sea water salts, only NaCl and MgCl2 were utilized by the organism to produce carrageenase. The minimal concentrations of suitable combinations of NaCl and MgCl2 were found to be 0.05 M NaCl plus 0.25 M MgCl2, and 0.15 M NaCl plus 0.15 M MgCl2. KCl and CaCl2 did not have any role in carrageenase production in ZoBell 2216 E broth medium. Carrageenase was synthesized continuously within the resting cells and was released from the cells as well as in the growing cells, when nutrient had been supplied.     

Pathways of macroinvertebrate invasions in the Ob River basin (West Siberia)

Water bodies of West Siberia mainly belong to the Ob River basin, which is not connected to other basins by shipping canals. The relative isolation of the basin predetermined the major role of industrial fisheries and aquarium trade in alien invertebrate transfer into the basin reservoirs. About 60% of the alien species were introduced into water bodies of West Siberia through industrial fisheries, and about 40% through aquarium fisheries. This paper is the first attempt to supplement a list of alien aquatic invertebrates from the Ob River basin. The data on the spread of 21 known non-indigenous species of invertebrates in this area are available. It should be noted that 7 species were deliberately introduced to enhance food reserves for fish, 8 were released by aquarists, 5 were inadvertently released during fish and forage organism introduction, and 1 (a crayfish) was imported for human consumption. For species with a known establishment period, there was a prolonged lag time in cases of both intentional and inadvertent introduction. The high invasion rate in the relatively isolated water bodies of the Ob River basin is comparable to that in Europe; hence, establishment of control over fishery-induced movement of alien invertebrates is required.     

Estimating P-coverage of biosynthetic pathways in DNA libraries and screening by genetic selection: biotin biosynthesis in the marine microorganism Chromohalobacter

Traditional approaches to natural product discovery involve cell-based screening of natural product extracts followed by compound isolation and characterization. Their importance notwithstanding, continued mining leads to depletion of natural resources and the reisolation of previously identified metabolites. Metagenomic strategies aimed at localizing the biosynthetic cluster genes and expressing them in surrogate hosts offers one possible alternative. A fundamental question that naturally arises when pursuing such a strategy is, how large must the genomic library be to effectively represent the genome of an organism(s) and the biosynthetic gene clusters they harbor? Such an issue is certainly augmented in the absence of expensive robotics to expedite colony picking and/or screening of clones. We have developed an algorism, named BPC (biosynthetic pathway coverage), supported by molecular simulations to deduce the number of BAC clones required to achieve proper coverage of the genome and their respective biosynthetic pathways. The strategy has been applied to the construction of a large-insert BAC library from a marine microorganism, Hon6 (isolated from Honokohau, Maui) thought to represent a new species. The genomic library is constructed with a BAC yeast shuttle vector pClasper lacZ paving the way for the culturing of libraries in both prokaryotic and eukaryotic hosts. Flow cytometric methods are utilized to estimate the genome size of the organism and BPC implemented to assess P-coverage or percent coverage. A genetic selection strategy is illustrated, applications of which could expedite screening efforts in the identification and localization of biosynthetic pathways from marine microbial consortia, offering a powerful complement to genome sequencing and degenerate probe strategies. Implementing this approach, we report on the biotin biosynthetic pathway from the marine microorganism Hon6.     

Method of temperature-gradient incubation and its application to microbiological examinations

Nakae, Toshitaka (Tohoku University, Sendai, Japan). Method of temperature-gradient incubation and its application to microbiological examinations. J. Bacteriol. 91:1730-1735. 1966.-An apparatus for incubating organisms by means of a temperature-gradient system under steady-state conduction of liquid was devised. This "temperature-gradient bath" consists of a cylindrical temperature-gradient compartment mounted between two circulating water compartments which are regulated separately at constant temperature, the upper compartment being warmer than the lower compartment. A series of different, constant temperatures in the temperature-gradient compartment can be established simultaneously. Water is a suitable heat-transfer liquid for this purpose. Incubation tubes containing solid media are inserted vertically into the temperature-gradient bath and incubated. Visible growth of the organism appears in a definite portion of the agar column, corresponding to its temperature range for growth. The apparatus was successfully applied to the rapid and simultaneous estimation of maximal, minimal, and optimal temperatures for microbial growth, as well as for the simultaneous isolation of psychrophiles, mesophiles, and thermophiles.     

Pheromones in marine algae: A technical approach

It is now well known that many marine organisms use low-molecular volatile substances as signals, in order to coordinate activities between different individuals. The study of such pheromones requires the isolation and enrichment of the secretions from undisturbed living cells or organisms over extended periods of time. The Grob-Hersch extraction device, which we describe here, avoids adverse factors for the biological materials such as strong water currents, rising gas bubbles or chemical solvents. Furthermore, the formation of sea-water spray is greatly reduced. The application of this technique for the isolation of pheromones of marine algae and animals is described.     

The occurrence of Eubothrium fragile (Cestoda: Pseudophyllidae) in twaite shad, Alosa fallax (Lacépède) in the River Severn

The occurrence, size and maturity changes of Eubothrium fragile have been studied in postlarvae, juveniles and adult twaìte shad, Alosa fallax , from several locations in the River Severn. Parasites were only found in adult shad and not in post-larvae or juveniles. No juvenile or recently acquired cestodes were identified as such, but adults were present in shad throughout the whole period of their spawning migration. A large proportion of the parasites were gravid upon arrival in the river and, although eggs were subsequently released into fresh water, there was no loss of cestodes from the fish. It was concluded that E. fragile is a marine species, that the parasites found in adult shad in fresh water were the residue of a marine life cycle and that the eggs released in fresh water were part of the parasite's natural reproductive wastage. The distribution and biology of E. fragile were discussed and it is considered that it is typical of the marine species of the genus.     

Evidence of a human-mediated invasion of the tropical western Atlantic by the 'world's most common brittlestar'

Approximately three million years ago the Isthmus of Panama formed an impenetrable land barrier between the tropical eastern Pacific Ocean and the tropical western Atlantic Ocean. Since this time, isolated geminate species have evolved from once contiguous populations, either side of the barrier. One such organism whose distribution is divided by the Isthmus is the tropical brittlestar Ophiactis savignyi, once suggested to be the most common brittlestar in the world. Rather than showing a genetic pattern consistent with a history of isolation, we show that this species has recently dispersed between the Pacific Ocean and the western Atlantic Ocean. This conclusion is based upon a phylogenetic analysis using sequences of the COI mitochondrial DNA gene from these populations. Identical haplotypes between oceans, and a genetic signature of population expansion, provide compelling evidence that the western Atlantic contains at least one cluster of haplotypes recently derived from the Indo-Pacific. Inadvertent human-aided translocation of individuals, presumably in ballast water or fouling communities, is strongly implicated as a mechanism for dispersal between oceans. We believe that cryptic marine invasions are likely to be common and our awareness of them will rapidly increase as systematic and phylogeographic knowledge of marine taxa grow.     

Growth Rates of Marine Bacterial Isolates on Particulate Organic Substrates Solubilized by Freely Released Extracellular Enzymes

Abstract Growth rates of marine bacterial isolates on particulate organic substrates were measured using a novel apparatus which restricts bacterial cells to the uptake of hydrolysate produced from particulate substrates only by enzymes that are actively released from the bacterium into the culture medium. Significant, varying growth rates were measured for four different marine bacteria, using three different, ecologically significant particulate organic substrates (preparations of amylopectin, chitin, and animal hide). Growth rates sometimes approached but were usually lower than rates that have been reported in laboratory experiments using dissolved organic growth substrates. These results are consistent with recent model predictions and have important implications for microbial ecology and material cycling in diverse liquid-bathed environments. Received: 26 June 1998; Accepted: 20 October 1998     

Golgi fractions from livers of control and ethanol-intoxicated rats. Enzymic and morphologic properties following rapid isolation

Following rapid isolation, it has been found that Golgi apparatus from ethanol-intoxicated animals contain high levels of galactosyltransferase but also detectable glucose-6-phosphatase and microsomal esterase, as well as 5′-nucleotidase activity. In experiments carried out in parallel on littermate animals but without intoxication, similar recoveries and specific activities of the four enzymes were observed. Morphologic analysis of Golgi fractions isolated from control animals demonstrated no striking morphologic difference to those from the ethanol-intoxicated animals. Indeed, using galloyl glucoselead staining techniques to mark the lipoprotein particles in situ, it was found that all Golgi apparatus of hepatocytes from control animals were marked by very low density lipoprotein particles. It is therefore concluded that within the limits of the present analyses, Golgi fractions isolated from control animals are as valid as those isolated from ethanol-intoxicated rats.     

Golgi fractions from livers of control and ethanol-intoxicated rats. Enzymic and morphologic properties following rapid isolation.

Following rapid isolation, it has been found that Golgi apparatus from ethanol-intoxicated animals contain high levels of galactosyltransferase but also detectable glucose-6-phosphatase and microsomal esterase, as well as 5'-nucleotidase activity. In experiments carried out in parallel on littermate animals but without intoxication, similar recoveries and specific activities of the four enzymes were observed. Morphologic analysis of Golgi fractions isolated from control animals demonstrated no striking morphologic difference to those from the ethanol-intoxicated animals. Indeed, using galloyl glucose-lead staining techniques to mark the lipoprotein particles in situ, it was found that all Golgi apparatus of hepatocytes from control animals were marked by very low density lipoprotein particles. It is therefore concluded that within the limits of the present analyses, Golgi fractions isolated from control animals are as valid as those isolated from ethanol-intoxicated rats.     

MabCent: Arctic marine bioprospecting in Norway

The deep waters surrounding the coastline of the northern parts of Norway represent an exciting biotope for marine exploration. Dark and cold Arctic water generates a hostile environment where the ability to adapt is crucial to survival. These waters are nonetheless bountiful and a diverse plethora of marine organisms thrive in these extreme conditions, many with the help of specialised chemical compounds. In comparison to warmer, perhaps more inviting shallower tropical waters, the Arctic region has not been as thoroughly investigated. MabCent is a Norwegian initiative based in Tromsø that aims to change this. Since 2007, scientists within MabCent have focussed their efforts on the study of marine organisms inhabiting the Arctic waters with the long term goal of novel drug discovery and development. The activities of MabCent are diverse and range from sampling the Arctic ice shelf to the chemical synthesis of promising secondary metabolites discovered during the screening process. The current review will present the MabCent pipeline from isolation to identification of new bioactive marine compounds via an extensive screening process. An overview of the main activities will be given with particular focus on isolation strategies, bioactivity screening and structure determination. Pitfalls, hard earned lessons and the results so far are also discussed.     

Experimental study of vibrio parahaemolyticus (biotype 2) transfer from water and sediments to benthic marine food chain organisms]

Transfer of Vibrio parahaemolyticus (biotype 2) from sediments to water and from water to benthic marine organisms was studied experimentally using a streptomycin-resistant strain. Transmission by trophic pathways was also studied using reconstituted marine food chains (Mytilus edulis, Nereis diversicolor, Carcinus maenas, Scorpaena porcus, Mus musculus). Water colonization by sediments could be observed only at temperatures above 16 degrees C. Sediments could well constitute a disseminating reservoir for these germs, their cycle in water being dependent of the cycle followed in the sediments. Contamination of animal organisms is essentially effected by a direct mean, either water or sediments; transfer by trophic pathways being negligible. Infection of land consumers (mice) is linked quantitatively to the nature of the last marine organism of the food chain since bacteria can flourish in the digestive tract of certain animals (Carcinus maenas).     

Culture isolation and culture-independent clone libraries reveal new marine Synechococcus ecotypes with distinctive light and N physiologies

Marine microbial communities often contain multiple closely related phylogenetic clades, but in many cases, it is still unclear what physiological traits differentiate these putative ecotypes. The numerically abundant marine cyanobacterium Synechococcus can be divided into at least 14 clades. In order to better understand ecotype differentiation in this genus, we assessed the diversity of a Synechococcus community from a well-mixed water column in the Sargasso Sea during March 2002, a time of year when this genus typically reaches its annual peak in abundance. Diversity was estimated from water sampled at three depths (approximately 5, 70, and 170 m) using both culture isolation and construction of cyanobacterial 16S-23S rRNA internal transcribed sequence clone libraries. Clonal isolates were obtained by enrichment with ammonium, nitrite, or nitrate as the sole N source, followed by pour plating. Each method sampled the in situ diversity differently. The combined methods revealed a total of seven Synechococcus phylotypes including two new putative ecotypes, labeled XV and XVI. Although most other isolates grow on nitrate, clade XV exhibited a reduced efficiency in nitrate utilization, and both clade XV and XVI are capable of chromatic adaptation, demonstrating that this trait is more widely distributed among Synechococcus strains than previously known. Thus, as in its sister genus Prochlorococcus, light and nitrogen utilization are important factors in ecotype differentiation in the marine Synechococcus lineage.     

A simple method for the release of asparagine-linked oligosaccharides from a glycoprotein purified by SDS-polyacrylamide gel electrophoresis.

A simple method for the release of oligosaccharides from glycoproteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) has been developed. Asialo-alpha 1-acid glycoprotein, which was tritiated at the nonreducing terminal D-galactopyranosyl residue by reduction with sodium borotritide after incubation with D-galactose oxidase, was used as a model compound. After electrophoretic separation of the glycoprotein, oligosaccharides were released by the use of a gas-phase hydrazinolysis apparatus. In the first method, the gel was stained with Coomassie Blue and the glycoprotein together with the gel was directly subjected to gas-phase hydrazinolysis after removal of water in a P2O5 desiccator. The recovery of released oligosaccharides was 25.9 +/- 2.4%, based on the amount of the glycoprotein loaded on the gel within the range of 3.5-28.5 micrograms. In the second method, the glycoprotein was electroblotted onto an Immobilon transfer membrane and was visualized by staining with Coomassie Blue. A small piece of the membrane with the corresponding band was cut out, dried in a desiccator and subjected to gas-phase hydrazinolysis. In this case, the recovery of released oligosaccharides was 15.2 +/- 1.0%. These procedures, particularly the first one, should be widely applicable for the isolation of oligosaccharides from glycoproteins separated by SDS-PAGE.     

The physiological determinants of the parallelism (unity) of histological structures

The theory of structural parallelism put forward by A. A. Zavarzin (Senior) has been supported by the analysis of cytological specificity of effector organs involved in water-salt homeostasis, and of the excretory system of vertebrates and invertebrates. The similarity in morphofunctional organization of different excretory organs (i.e. the presence of ultrafiltration apparatus and cells which make it possible to absorb all vitally important substances) is likely to result from the fact that the excretory organ should excrete not only the final products of metabolism, but also any exogenic substances in addition to those which although important, are excessive for the organism. The brush border of asymmetrical epithelial cells of excretory organs is presumably a morphological expression of the structure which accounts for the inward transport of all physiologically important substances. Specificity of the membrane mechanism of water and sodium transport accounts for the identical principles in the structure of cells and areas of cell contacts of epithelia in different organs involved in the formation of hypotonic (saliva glands, renal tubules) or hypertonic fluids (salt glands, marine teleost gills).