The Determination of Volume of Dunaliella Cells by Transmission Electron Microscopy and Image Analysis

A method has been developed to measure the cell volume of theunicellular, green alga Dunaliella bioculata 19/4 during saltstress conditions, where shape change in the alga becomes problematicand the cells can no longer be recognised as 'prolate ellipsoids',by using image analysis of transmission electron micrographs.The image analysis of the micrographs employs a specialisednumerical integration programme or 'variable frames analysis'for unicellular microorganisms which possess a single axis ofsymmetry. Basic mathematics was used to determine: (a) the functionaldependence of the calculated volume on the angle of the cutto the axis of symmetry and the distance of the origin of thecut from the centre of mass; (b) errors resulting from the orientationof the longest axis off-vertical for image analysis; (c) theuppermost range of calculated volumes obtained which representthe 'true' volumes within required confidence levels. The procedurewas applied to a series of experiments on the effects of saltstress on Dunaliella bioculata cells.Copyright 1994, 1999 AcademicPress Dunaliella, image analysis, TEM, volume, variable frames, numerical integration, salt stress     

Variation of Phosphoenolpyruvate Carboxylase Activity in Dunaliella Associated with Changes in Atmospheric CO2 Concentration

In Dunaliella tertiolecta, D. bioculata and D. viridis the activitiesof phosphoenolpyruvate carboxylase and carbonic anhydrase werehigher in the cells grown in ordinary air (low-CO₂ cells) thanin those grown in air enriched with 1–5% CO₂ (high-CO₂cells), whereas in Porphyridium cruentum R-1 there was no differencein phosphoenolpyruvate carboxylase activity between these twotypes of cells. Apparent K_m(NaHCO₃) values for photosynthesisin low-CO₂ cells of all species tested were smaller than thosein high-CO₂ cells. Most of the ¹⁴C was incorporated into 3-phosphoglycerate,sugar mono- and di-phosphates during the initial periods ofphotosynthetic NaH¹⁴CO₃ indicating that both types of cellsin D. tertiolecta are C₃ plants. (Received May 27, 1985; Accepted June 25, 1985)     

Use of the cell wall-less alga Dunaliella bioculata in herbicide screening tests

An assay was developed using Dunaliella bioculata, a cell wall-less, unicellular alga with two flagella, to determine whether it could supplement a programme of herbicide screening. Growth was monitored by measuring the optical density of the culture. Additional information was obtained by microscopic examination; lysis of cells, loss of flagella, immobilisation, and bleaching could be readily seen. Comparing 24 commercial herbicides with an untreated check, 20 compounds clearly affected D. bioculata. The assay with D. bioculata can be used as a primary indicator of mode of action; as an example, all members of the chemical class of dinitroanilines affected movement and shape of D. bioculata, probably by influencing microtubule structure.     

Observations on the Bionomics of the Freshwater Nematode Chromadorina bioculata

The biology and morphology of Chromadorina bioculata is presented. Tile nematode was abundant on the alga Cladophora of one lily pond, but absent from similar habitats in nearby ponds. The setae, caudal glands, high Iocomotory rates and positive photo response have been interpreted in relation to maintenance on, amongst and between algal filaments, suspended over large volumes of water.When placed in tap or distilled water, C. bioculata became inactive and died. The influence of pH buffers, tonicity, temperature and starvation on activity were investigated. C. bioculata survived longer in artificial sea water diluted 10 or 100 times with distilled water, or in NaCl isotonic with sea water diluted 100 or 1000 times, than in tap or distilled water. No evidence of wide osmotic toleration or osmoregulation was observed. Activity was influenced by temperature, with peak activities occurring at the seasonal normal temperature. These findings are discussed in terms of general hematology and habitat selection.     

An Examination of some Symbiotic Systems for Fixation of Nitrogen

By the use of ¹⁵N the occurrence of fixation of elemental nitrogenhas been demonstrated in the lichens Collema granosum and Leptogiumlichenoides (both of which contain Nostoc as a symbiont), andalso in the liverwort Blasia pusilla (which has Nostoc-containingcavities in the thallus). The circumstances indicate that thefixation in these instances should be attributed to the Nostoc. No evidence of fixation was obtained in similar isotopic testson the mycorrhizal roots of intact plants of Calluna vulgarisand of Pinus sylvestris, although these, more especially inthe case of Calluna, have been held by some previous authorsto be nitrogen-fixing.     

The Olfactory System as a Model for the Analysis of the Contribution of Gene Expression to Programmed Cell Death

The process of programmed cell death is frequently attenuatedby inhibitors of protein and RNA synthesis. This implies thatgene expression is necessary for the active elimination of somecell types. Genes such as bcl-2 and bax have been implicatedin the direct control of cell death, while cellular immediate-earlygenes (clEGs), such as c-fos and c-jun have been repeatedlyassociated with neuronal degeneration. We are using the olfactoryneuroepithelium as a model system to investigate the role thatexpression of such genes might play in cell death. The advantagesof this system is that even in the adult, there is spontaneousdegeneration of olfactory receptor neurons followed by theirreplacement by the division and differentiation of precursors.Futhermore, the receptor neurons can be induced to die synchronouslyby removal of the olfactory bulb or intranasal administrationof toxic agents. We have generated fos-lacZ and jun-lacZ transgenicmice that can be used to assess expression of c-fos and c-junfollowing these various manipulations. In addition, a line oftransgenic mice has been derived that express Bcl-2 under thecontrol of the olfactory receptor protein promoter. These micehave high levels of Bcl-2 selectively in receptor neurons ofthe primary neuro-epithelium and vomeronasal organ. Since insome circumstances, Bcl-2 can protect against programmed celldeath these mice are being assessed for neuronal turnover underbasal conditions and following olfactory bulbectomy.     

Xenopus oocyte plasma membrane sheets for FRET analysis

Plasma membrane sheets from Xenopus oocytes have been isolated for use in fluorescence resonance energy transfer (FRET) measurements. This system has the following advantages: 1) fluorescent recordings from a large surface area to maximize the signal-to-noise ratio, 2) reduction in background fluorescence from proteins retained in intracellular compartments, and 3) access to the cytoplasmic surface of the plasma membrane for rapid solution changes. To demonstrate the utility of this approach, we have examined a previously published FRET-based Ca²⁺ sensor, namely, the Cameleon-PM. This construct targets to the plasma membrane and, upon various Ca²⁺ additions to the cytoplasmic face of the membrane, shows ratiometric FRET changes. From the ratiometric changes recorded, an apparent Ca²⁺ affinity of 1.65 µM was determined. Thus preparation of Xenopus oocyte plasma membrane sheets and FRET measurements demonstrates all three of the advantages outlined above. fluorescence resonance energy transfer     

Transient Gene Expression in Plant Cells Mediated by Agrobacterium tumefaciens: Application for the Analysis of Virulence Loci

A simple system is described for detection of the transfer ofT-DNA from Agrobacterium cells to suspension-cultured tobaccoBY-2 cells. A modified reporter gene for rß-glucuronidase(GUS) that contained an intron sequence was introduced intothe T-DNA region such that the GUS protein could be synthesizedin plant cells only after transfer of the T-DNA to plant nuclei.When BY-2 cells were co-cultured with Agrobacterium cells thatcontained the modified reporter gene, transient synthesis ofGUS protein was observed between 36 and 48 h after the onsetof co-culture. The level of GUS activity reached a plateau withinas little as 48 h. This temporal profile of GUS activation suggeststhat the transient activity might have been due to expressionof the GUS gene in the T-DNA that had been transferred to theplant nuclei but had not yet been integrated into the plantchromosomes. Levels of transient GUS activity were also examinedwith various vir mutants of Agrobacterium and in a mutant withan altered chromosomal acvB gene, the gene for a protein thathas been postulated to function outside bacterial cells. Duringco-culture with virB, virD2, virD4 and acvB mutants, GUS activityremained at background levels, and the GUS activity in the caseof the virE2 mutant was thirty-fold lower than with the wildtype. On the basis of these results, we discuss the roles ofthese genes during infection by Agrobacterium of plant cells. ⁴Present address: Biochemistry Laboratory, Kanebo Ltd., 5-3-28Kotobuki-cho, Odawara, Kanagawa, 250 Japan     

A Rapid Method for the Determination of the Leaf Area of Oxalis corniculata L. and Tephrosia purpurea (Linn.) pers.

A rapid simple method has been described for the determinationof leaf area of Oxalis corniculata L. (Oxalidaceae) and Tephrosiapurpurea (Linn.) pers. (Leguminoseae) by evaluating area = a²x0.866and a²x0.335 respectively for the two plants, where a representsa single linear measurement. The relation has been derived afterintegrating the equation of the curve r² = a² cos 2 coveringthe area of each leaflet. Areas of a large number of leaflets have been determined withthe help of two methods (1) Planimeter method, (2) Evaluationmethod. The significance of the difference between the two valuesfor a particular leaf has been statistically examined and itappears that the former method can be replaced by the latterin the case of these plants.     

Energy-Level Model for Isothermal Seed Germination

We have been successful in building an energy-level model thatdescribes seed germination. We used the autocatalytic reactionrate equation to fit the germination rate for seed germination.The two parameters [A]₀ and [F]₀ were found by fitting the integratedgermination rate equation to the data. The values of [A]₀ and[F]₀ obey the Arrhenius equation and give activation energiesfor the second and third stage of the four-compartmental modelof seed germination. The thermodynamics of isothermal seed germinationis proposed and the enthalpy, entropy, and free energy are calculatedfor the transition from state A to state F. The time delay isa function of temperature and it leads to a rate constant thatcan be used to get the activation energy for the total germinationprocess. We believe the model is universal. It fits alfalfa(Medicago sativa), turnip (Brassica rapa), and lettuce (Lactucasativa) seeds. Key words: Seed germination, thermodynamics, kinetics, Arrhenius, energy-level model     

The Diatom Genus Tabellaria: II. Taxonomy and Morphology of the Plankton Varieties

Plankton samples of Tabellaria from all the lakes of the EnglishLake District have been examined and evidence of morphologicallydistinct populations has been found in samples from (a) a singlelake at one time, (b) different lakes, and (c) a single lakeat different times. Clone cultures have been used to study thenature of these differences. Fifteen characters have been foundto show quantitative or qualitative variation in plankton populationsof Tabellaria, and the morphological diversity of differentpopulations is largely due to different combinations of thesecharacters. All known plankton populations of Tabellaria arereferred to the species T. flocculosa.     

Wavelength options for monitoring leghaemoglobin oxygenation gradients in intact legume root nodules

Nodule oximetry, based on spectrophotometric measurements ofleghaemoglobin (Lb) oxygenation in intact nodules, has providednumerous insights into legume nodule physiology. Fractionaloxygenation of Lb (FOL) has been monitored at various wavelengths,but comparisons among wavelengths have not been published previously.Changes in transmittance were monitored simultaneously at 660nm and either 560 or 580 nm as FOL was manipulated by changingthe O₂ concentration around nodules of Medicago sativa L. orLotus comiculatus L. Video microscopy at 580 nm was used togenerate two-dimensional maps of FOL gradients in intact nodules.In general, all three wavelengths gave similar results. Smalldiscrepancies between 660 and 580 nm, sometimes seen in noduleswith high O₂ permeability, may indicate interference by theferric Lb peak at 625 nm. A slightly longer wavelength, forexample 670 nm, might be preferable. No significant discrepanciesamong wavelengths were seen in nodules whose O₂ permeabilityhad been reduced by a 48 h exposure to 10 mM nitrate. Minorgradients in FOL were seen in nodules of M. sativa and Trifoliumrepens L. under air and steeper gradients could be induced byvarious treatments. The existence of these gradients indicatesat least some restriction of longrange O₂ diffusion within theinfected zone. The FOL maps do not have enough spatial resolutionto measure gradients within infected cells. Key words: Leghaernoglobin oxygenation, nodules, spectrophotometry, nodule oximetry     

Studies on the Growth of Basidiomycetes in Soil

Fructifications of basidiomycetes have been collected at LakenheathWarren, and thirty-nine species have been obtained from fivegrassland soils. The different soils have been found to havedifferent species growing upon them and most species occur onthe alkaline-slightly acid grasslands. Highly acid grasslandat Lakenheath has a poor fungus flora. Studies have been made of the regions in the soil in which themycelia of certain of these basidiomycetes occur, and attemptshave been made to isolate these fungi from soil. Some specieshave been found to inhabit the litter zone rather than the minerallayers of the soil. A few form extensive rhizomorph systemswhich may penetrate deeply into the soil. A few species of ringfungi, such as Marasmius oreades, Psalliota arvensis, and Tricholomanudum, have been found to possess a well-defined mycelial zonein the soil, from which the fungus could be isolated. Profile studies have shown that the mycelial zones of thesespecies contain a restricted population of microfungi, bothfewer species and fewer colonies, as compared with the normalsoil around. Ascomycetes, such as Arachniotus, Chaetomium, Gymnoascus,and Penicillium, have been isolated more frequently from mycelialzones than from normal soil.     

Descriptions of some planktonic larvae of the Calappidae (Crustacea: Decapoda; Brachyura)

Some unidentified planktonic larval stages have been assignedto the Calappidae using Rice's key of Brachyura families. Zoeastage I from the eastern region of the Algerian coast (westernMediterranean Sea) has been attributed, according to Manningand Holthuis (1980), to Calappa granulata (L.), the only recordedrepresentative of the family from the Mediterranean Sea. Zoealstages 1-lV from the Jordanian coast of the Gulf of Aqaba, NorthernRed Sea, have been reported to the subfamily Matutinae. Comparisonsof larvae of Calappa and of Hepatus and then, of larvae of Hepatusand of Matuta, show that Hepatus larvae are more closely relatedto those of Calappa, while those of Matuta are clearly distinct.It s suggested to place the genus Hepatus with the Calappinae.Guinot (1966. 1978) placed some genera, including Hepatus. ina 'unit parthenoxystomienne, lea Aethrinae'. Larval developmentis unknown for Aethra, Actaeomorpha. Hepatella andOsachila.However, comparison of Hepatus larvae with those of Parthenopidaeshows that larval characters, particularly the setation of theendopod of the first maxilliped, are not conclusive for supportingsuch a regrouping. Moreover, the Calappidae have a combinationof primitive and advanced features which suggest that theirgrouping in the Oxystomata seems more likely to be an adaptationto a similar way of life. The morphotypal taxon Oxystornatahas been definitively rejected by Guinot (1978).     

Amplified Fragment Length Polymorphism (AFLP) Provides Molecular Markers for the Identification of Caladium bicolor Cultivars

Caladiums are popular ornamental plants that have not been wellstudied at the molecular level. Identification of species withinthe genus Caladium (Araceae) has been based primarily on morphology.However, the lack of comprehensive references makes identificationof Caladium cultivars extremely difficult. Amplified fragmentlength polymorphism (AFLP) analysis using 17 primer combinationswas carried out on two species of Caladium (C. bicolor and C.schomburgkii), including six cultivars of C. bicolor. Resultsshowed that AFLP can be used to distinguish these two speciesby their unique and different banding patterns. Unweighted PairGroup Method using Arithmetic Averages (UPGMA) permitted clusteranalysis of data from 17 selected primer combinations on sixcultivars of C. bicolor and one cultivar ofC. schomburgkii .It showed that closely related species can clearly be differentiatedand that genetic difference between cultivars can also be established.Unique AFLP molecular markers were detected for all the C. bicolorcultivars used. The use of AFLP has potential for preciselycharacterizing and identifying particular caladium cultivarsas well as for the registration of new cultivars. It will alsobe useful in future breeding programmes and systematics studies.Copyright 1999 Annals of Botany Company Araceae, Caladium species and cultivars, AFLP DNA fingerprinting, diversity, AFLP markers.     

Escherichia coli as an expression system for K(+) transport systems from plants

The value of theEscherichia coli expression system has long been establishedbecause of its effectiveness in characterizing the structure andfunction of exogenously expressed proteins. When eukaryotic membraneproteins are functionally expressed in E. coli, thisorganism can serve as an alternative to eukaryotic host cells. A fewexamples have been reported of functional expression of animal andplant membrane proteins in E. coli. This mini-review describes the following findings: 1) homologousK⁺ transporters exist in prokaryotic cells and ineukaryotic cells; 2) plant K⁺ transporters canfunctionally complement mutant K⁺ transporter genes inE. coli; and 3) membrane structures of plant K⁺ transporters can be elucidated in an E. colisystem. These experimental findings suggest the possibility ofutilizing the E. coli bacterium as an expression system forother eukaryotic membrane transport proteins.

     

Sulfated glycans on oral mucin as receptors for Helicobacter pylori

Helicobacter pylori is able to colonize gastric epithelia, causingchronic active gastritis, gastric and duodenal ulcers and presumablygastric malignancies. Attempts to identify the natural reservoirfor this microorganism other than the stomach have been unsuccessful.It is suspected that H.pylori can be transmitted orally, sincethe microorganism has been detected at various sites of theoral cavity. The aim of the present study was to determine whetherH.pylori can bind to salivary mucins, which in vivo coat theoral epithelia, and characterize further the interaction. Bindingof salivary mucins and of synthetic oligosaccharides was studiedIn ELISA and immunoblotting, using specific mono-and polyclonalantibodies, and synthetic neoglycoconjugates. H.pylori boundmost avidly to a highly sulfated sub-population of high molecularweight salivary mucins, secreted from the palatine salivaryglands, and with less avidity to mucin species secreted by thesublingual and submandibular salivary glands, which are lesssulfated. Binding was strongly enhanced upon decreasing pH from6.0 to 5.0. Using synthetic polyacrylamide coupled oligosaccharidesit was found that SO₃-3-Gal and the SO₃-3-Lewis^a blood groupantigen bound to H.pylori. In contrast, binding of sialylatedLewis^a and Lewis^b antigens was much weaker. This study indicatesthat sulfated oligosaccharides on salivary mucins may providereceptor structures for adhesion of H.pylori to oral surfaces. H.pylori saliva sulfomucin nickel oligosaccharide