Reflective properties of different eyespot types in dinoflagellates

The reflective properties of different types of dinoflagellate eyespots were investigated using confocal laser scanning microscopy in the epireflection contrast mode. Although the eyespots studied differed with respect to localization (cytosol or plastid) and organization of the globule layer(s), all types effectively absorbed and reflected blue-green laser light (principal lines of 488/514 nm). The relative orientation of the eyespot surface towards the light source strongly influenced the reflective properties. Maximal reflection occurred when the eyespot surface was approximately perpendicular to the light source and rapidly decreased at increasing angles of light incidence. Horizontal and vertical optical sectioning of live and fixed cells resolved differences in the reflection patterns. Focusing of reflected light on the basal portion of the longitudinal flagellum was observed for the cytosolic eyespot of Glenodinium sp. and the triple membrane-bounded eyespot of Peridinium foliaceum, presumably a vestige of a host plastid. This flagellum is thought to be mainly involved in mediating orientational movement responses. In contrast, the reflection patterns obtained from the eyespot of Woloszynskia pascheri, which represents the third and most commonly observed dinoflagellate eyespot type within a plastid, point to only minor focusing. Reflection signals could be followed a considerable distance into the sulcus in all cases, indicating that in dinoflagellate eyespots, irrespective of the presumed receptor location (plasma membrane overlying the eyespot and/or the basal part of the longitudinal flagellum), back reflection of non-absorbed light can enhance the excitation probability of the photoreceptor(s). Such a combined reflection/absorption screen allows maximal contrast modulation and will, in conjunction with the specialized geometry of the dinoflagellate eyespots, increase the directionality of these eyespot aparatuses considerably.     

Morphology and ultrastructure of spiracles in phlebotomine sandfly larvae

The morphology and ultrastructure of the larval spiracle system of three phlebotomine sandfly species, Phlebotomus perniciosus, P. perfiliewi and P. papatasi, were examined by scanning (SEM) and transmission (TEM) electron microscopy and by confocal scanning laser microscopy (CSLM). During larval development, thoracic and abdominal spiracles show considerable modifications. In fourth instar larvae, the spiracles consist of a plate with a sclerotized central portion and a peripheral circle of papillae. The latter is distinctive in the larvae of P. papatasi, which are readily distinguished from the other species. Opening clefts across the papillae communicate with an internal chamber that encircles an electrondense plug. Many cylindrical projections cross the chamber, uniting the central plug with the larval body, forming an air filter. Spiracular development in successive larval instars has both a taxonomic and adaptive value.     

Diversity of coexisting <Emphasis Type="Italic">Planktothrix</Emphasis> (Cyanobacteria) chemotypes deduced by mass spectral analysis of microystins and other oligopeptides

Cyanobacteria are reported to produce secondary metabolites of which toxic and bioactive peptides are of scientific and public interest. Many peptides are synthesized by the non-ribosomal peptide synthesis pathway and their presence is a stable feature of individual clones. We isolated 18 clonal strains of Planktothrix from a single water sample from lake Maxsee near Berlin and analyzed them by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, HPLC, and PCR for their production of peptides and the presence of microcystin synthetase genes. Microcystins could be detected in seven of the strains with considerable variability of contents and numbers of structural variants. Other known peptides like anabaenopeptins B and E/F, microviridin I, and prenylagaramide B and new variants of known peptide classes like aeruginosins and cyanopeptolins were detected in some strains while lacking in others. The 18 strains represented 15 chemotypes with respect to their peptide patterns. In contrast, all strains were morphologically very similar with respect to cell dimensions and pigmentation. Given the diversity of chemotypes among the randomly selected isolates, an immense diversity of chemotypes in the entire population can be assumed.     

Pollen dispersion, pollen viability and pistil receptivity in Leymus chinensis

BACKGROUND AND AIMS: Leymus chinensis is an economically and ecologically important grass that is widely distributed across eastern areas of the Eurasian steppe. A major problem facing its propagation by man is its low sexual reproductivity. The causes of low fecundity are uncertain, largely because many aspects of the reproductive biology of this species remained unknown or incomplete. This study aims to address some of these issues. METHODS: Pollen dispersion, pollen viability, pollen longevity and pistil receptivity were studied in a representative, natural population of L. chinensis growing in Inner Mongolia. KEY RESULTS: Flowering of L. chinensis occurred at the end of June and lasted for 5 d. Pollination peaked between 1600 h and 1700 h, and about 56.1 % of the total pollen grains were released at this time. Pollen density was highest towards the middle of flowering spikes and lowest at the bottom over the 5 d measurement period. Pollen viability (62.4 %) assessed using TTC was more accurate than using IKI (85.6 %); 50 % of pollen arriving on stigmas germinated. Pollen remained viable for only 3 h and the pollen : ovule ratio was 79 333 : 1. Pistil receptivity lasted for only 3 h and, overall, 86.7 % of pistils were pollinated. Within the spike, the relative fecundity of different positions was middle > lower > upper throughout the period of pollination; daily variation of fecundity was similar to that of the pollen flow. The spikes that opened on the day of highest pollen density exhibited the highest fecundity (36.0 %). No seeds were produced by self-pollination. CONCLUSIONS: The data suggest that low pollen viability, short pollen longevity and short pistil receptivity all appear to contribute to the low seed production typical of this important forage crop.     

Dipolar assisted rotational resonance NMR of tryptophan and tyrosine in rhodopsin

Two dimensional (2D) solid-state (13)C.(13)C dipolar recoupling experiments are performed on a series of model compounds and on the visual pigment rhodopsin to establish the most effective method for long range distance measurements in reconstituted membrane proteins. The effects of uniform labeling, inhomogeneous B(1) fields, relaxation and dipolar truncation on cross peak intensity are investigated through NMR measurements of simple amino acid and peptide model compounds. We first show that dipolar assisted rotational resonance (DARR) is more effective than RFDR in recoupling long-range dipolar interactions in these model systems. We then use DARR to establish (13)C-(13)C correlations in rhodopsin. In rhodopsin containing 4'-(13)C-Tyr and 8,19-(13)C retinal, we observe two distinct tyrosine-to-retinal correlations in the DARR spectrum. The most intense cross peak arises from a correlation between Tyr268 and the retinal 19-(13)CH(3), which are 4.8 A apart in the rhodopsin crystal structure. A second cross peak arises from a correlation between Tyr191 and the retinal 19-(13)CH(3), which are 5.5 A apart in the crystal structure. These data demonstrate that long range (13)C em leader (13)C correlations can be obtained in non-crystalline integral membrane proteins reconstituted into lipid membranes containing less than 150 nmoles of protein. In rhodopsin containing 2-(13)C Gly121 and U-(13)C Trp265, we do not observe a Trp-Gly cross peak in the DARR spectrum despite their close proximity (3.6 A) in the crystal structure. Based on model compounds, the absence of a (13)C em leader (13)C cross peak is due to loss of intensity in the diagonal Trp resonances rather than to dipolar truncation.     

Histochemical,ultrastructural, and three-dimensional observation of smooth muscle cells in human gastric mucosa

The present study was undertaken to clarify the histochemical and ultrastructural properties and the three-dimensional distribution of the smooth muscle cells (SMCs) located in the lamina propria (LP) of the human gastric mucosa. Standard paraffin sections obtained from stomachs surgically resected for gastric cancer were immunostained for alpha-smooth muscle actin (alpha-SMA), vimentin, desmin, laminin, and type IV collagen. In addition, 100-m-thick sections were fluorostained for alpha-SMA and CD34, while three-dimensional images were prepared by confocal laser scanning microscope. Ultrastructural studies were carried out using normal gastric biopsy specimens. The results indicated that SMCs in the LP differed between the upper and lower regions, SMCs in the lower LP being fairly typical SMCs, whereas those in the upper LP had apparently lost reactivity for desmin but gained that for vimentin. The basal lamina became sparser, but a fibronexus was occasionally seen in SMCs in the upper LP. Three-dimensional images revealed bundles of SMCs in the upper LP encircling several foveolae to form acinus-like structures and, in the upper LP, SMCs branching into fine fibrils with a brush-like (corpus) or besom-like (i.e., a twiggy witchs broom) appearance (antrum).     

Solvent effects on focused microwave assisted extraction of polyphenolic acids from Eucommia ulmodies

An open microwave-assisted extraction system was used to extract gallic acid, protocatechuic acid, chlorogenic acid and caffeic acid from Eucommia ulmodies. The effect of extraction variables, especially solvent, on the recoveries of these polyphenolic compounds was investigated using factorial design. As extracting solvent for these compounds, methanol produced a higher recovery than pure water. For straight chain alcohol solvents, the lower the carbon number, the higher the recoveries of the polyphenolic acids. The optimal ratio of methanol:water:glacial acetic acid in the solvent mixture used in microwave-assisted extraction was 2:8:0.3 (v/v) and this solvent could be directly used as the mobile phase in HPLC separation without additional intermittent treatment as reported in literature. The extraction under the condition of 50% microwave power and 30 s irradiation at a solvent:sample ratio of 10 (mL/g) was found to be the most advantageous. The repeatability test of extraction and chromatographic analysis was satisfactory for the analysis of these polyphenolic compounds.     

High-throughput peptide mass fingerprinting of soybean seed proteins: automated workflow and utility of UniGene expressed sequence tag databases for protein identification

Identification of anonymous proteins from two-dimensional (2-D) gels by peptide mass fingerprinting is one area of proteomics that can greatly benefit from a simple, automated workflow to minimize sample contamination and facilitate high-throughput sample processing. In this investigation we outline a workflow employing robotic automation at each step subsequent to 2-D gel electrophoresis. As proof-of-concept, 96 protein spots from a 2-D gel were analyzed using this approach. Whole protein (1 mg) from mature, dry soybean (Glycine max [L.] Merr.) cv. Jefferson seed was resolved by high resolution 2-D gel electrophoresis. Approximately 150 proteins were observed after staining with Coomassie Blue. The rather low number of detected proteins was due to the fact that the dynamic range of protein expression was greater than 100-fold. The most abundant proteins were seed storage proteins which in total represented over 60% of soybean seed protein. Using peptide mass fingerprinting 44 protein spots were identified. Identification of soybean proteins was greatly aided by the use of annotated, contiguous Expressed Sequence Tag (EST) databases which are available for public access (UniGene, ftp.ncbi.nih.gov/repository/UniGene/). Searches were orders of magnitude faster when compared to searches of unannotated EST databases and resulted in a higher frequency of valid, high-scoring matches. Some abundant, non seed storage proteins identified in this investigation include an isoelectric series of sucrose binding proteins, alcohol dehydrogenase and seed maturation proteins. This survey of anonymous seed proteins will serve as the basis for future comparative analysis of seed-filling in soybean as well as comparisons with other soybean varieties.     

Imaging plant cells by two-photon excitation

Summary. Along the past recent years, two-photon excitation (TPE) microscopy has moved from the realms of technical curiosity to be a standard application in many advanced cell biology laboratories. The growing body of literature covered in this review points out the obvious advantages of TPE over any other imaging method based on fluorescence, clearly improving signal-to-noise ratio and thick-tissue penetration and showing added potential for vital imaging. Like any new technology that has to gain its own space, TPE microscopy is still going through the growing pains in which reproducible protocols, probes, and applications are scarce. Yet, the published reports and unpublished results covered in this review point out that TPE can eventually accommodate most available protocols and probes, most of the times with evident advantages. Further, the potential for plant sciences is obvious, as plant cells possess many absorbing molecules and structures and are routinely more opaque than tissues of other organisms. Since prices make it one of the most expensive microscopies, TPE is coming slow to be a generalised technology, but enough data are emerging to establish it as a method with no alternative for some objectives.Correspondence and reprints: Instituto Gulbenkian de Ciência. 2780-156 Oeiras, Portugal.