Self-recognition of high-mannose type glycans mediating adhesion of embryonal fibroblasts

High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.     

Molecular phylogeny and character evolution of the chthamaloid barnacles (Cirripedia: Thoracica)

The Chthamaloidea (Balanomorpha) present the most plesiomorphic characters in shell plates and cirri, mouthparts, and oral cone within the acorn barnacles (Thoracica: Sessilia). Due to their importance in understanding both the origin and diversification of the Balanomorpha, the evolution of the Chthamaloidea has been debated since Darwin's seminal monographs. Theories of morphological and ontogenetic evolution suggest that the group could have evolved multiple times from pedunculated relatives and that shell plate number diminished gradually (8→6→4) from an ancestral state with eight wall plates surrounded by whorls of small imbricating plates; but this hypothesis has never been subjected to a rigorous phylogenetic test. Here we used multilocus sequence data and extensive taxon sampling to build a comprehensive phylogeny of the Chthamaloidea as a basis for understanding their morphological evolution. Our maximum likelihood and Bayesian analyses separate the Catophragmidae (eight shell plates and imbricating plates) from the Chthamalidae (8-4 shell plates and no imbricating plates), but do no support a gradual reduction in shell plates (8→6→4). This suggests that evolution at the base of the Balanomorpha involved a considerable amount of homoplasy.     

An appraisal of methods used in coral recruitment studies

 A new method for attaching individual artificial settlement plates directly to the reef surface using small stainless steel base plates is described. Recruitment of corals to settlement plates attached to the reef substratum and to steel mesh racks is compared. The effects of differences in depth, settlement plate angle, and local topography on recruitment of corals were also investigated. No significant difference in mean recruit density was found between settlement plates deployed using the two attachment methods. Small differences in depth and plate angle among replicate plates explained less than 6% of the variability in coral recruitment on replicate settlement plates. The direct-attachment method is less obtrusive, more cost and time efficient, and settlement plates can be deployed at precise locations. Additionally, because settlement plates are deployed individually rather than grouped on racks or frames, the direct-attachment method avoids complications associated with assumptions of independence implicit in most statistical procedures. Accepted: 24 November 1999     

鹅掌楸属(木兰科)次生木质部导管穿孔板的比较研究

利用扫描电子显微镜对鹅掌楸属仅存的2个自然种鹅掌楸和北美鹅掌楸的次生木质部导管穿孔板特征进行了详细的研究。结果显示,鹅掌楸属的2个种均以梯状穿孔板为主,同时存在网状-梯状混合穿孔板。鹅掌楸和北美鹅掌楸的导管穿孔板具有明显差异:(1)鹅掌楸具网状穿孔板,而北美鹅掌楸没有观察到;(2)北美鹅掌楸具单穿孔板,而鹅掌楸在该实验中未发现;(3)北美鹅掌楸具有横隔较粗的梯状穿孔板且横隔数目较多;(4)鹅掌楸的导管穿孔板多数横隔较少;(5)北美鹅掌楸的穿孔板倾斜角度较大;(6)北美鹅掌楸具麻黄式穿孔板的存在,且有纹孔膜残留存在。研究认为,北美鹅掌楸导管分子穿孔板分化较鹅掌楸更为剧烈。     

NEW TAXONOMIC CRITERIA IN CLASSIFICATION OF CHLOROCOCCUM SPECIES. II. PYRENOID FINE STRUCTURE2

The pyrenoids of 18 Chlorococcum species (including 3 now transferred to Tetracystis) have been investigated at the ultrastructural level. Three categories of pyrenoids were observed, including pyrenoids with unfragmented perforate starch plates in 11 species; those with many separate fragmented starch plates in 6 species; and those with hemispherical starch plates in 1 species. Pyrenoids were further distinguishable on the basis of the number of thylakoid disks or tubules which penetrate the ground substance. Stability and diversity of pyrenoid fine structure as a taxonomic criterion at the specific level have been demonstrated.     

Modified Microbiological Assay for Rapid Estimation of Antibiotic Concentrations in Human Sera

Antibiotic concentrations in human sera were estimated in 5 to 6 hr by a modified microbiological assay. By using Staphylococcus aureus and Streptococcus pyogenes as the assay organisms, the seeded assay plates were preincubated for 2 to 6 hr and then were stored at 4 C until used for assay. Paper discs saturated with the specimen were placed on the preincubated assay plates with reference discs saturated with known concentrations of antibiotic. After 5 to 6 hr of incubation, zones of antibacterial activity were measured and compared with a standard curve for estimation of antibiotic concentration. Results from this rapid assay method compared favorably with those from the commonly used 24-hr assay.     

Oriented Immobilization of Anti-Pneumolysin Tagged Recombinant Antibody Fragments

Recombinant antibodies such as Fab and scFv are monovalent and small in size, although their functional affinity can be improved through tag-specific immobilization. In order to find the optimum candidate for oriented immobilization, we generated Fab and scFv fragments derived from an anti-pneumolysin monoclonal antibody PLY-7, with histidine and cysteine residues added in diverse arrangements. Tagged antibody fragments scFv-Cys7-His6, His6-scFv-Cys7, and Fab-Cys7 lost considerable affinity for the antigen; however, Fab-His6, Fab-Cys1, and scFv-His6-Cys1 were able to detect immobilized antigen, revealing that the position and number of histidine and cysteine residues are involved differently in the reactivity of antibody fragments. Random and orientated immobilizations were carried out using conventional polystyrene and commercial surface-pretreated ELISA plates. The best orientation performance was obtained with Fab-Cys1-biotin on streptavidin-coated plates with increased signal levels of 62%, while oriented immobilization of Fab-His6 and scFv-His6-Cys1 on nickel- and maleimide-coated plates failed to improve the ELISA sensitivity.     

A simplified in vitro model of oxidant injury using vascular endothelial cells

Summary Oxidant injury of the vascular endothelium is considered an early event in the pathogenesis of atherosclerosis. The model of oxidant injury is crucial to the investigation of antioxidants. In the present study, a convenient in vitro model of oxidant injury induced by hydrogen peroxide (H₂O₂) was developed using bovine pulmonary artery endothelial cells (PAEC). Viability of PAEC grown in 96-well culture plates was determined with methylthiazol tetrazolium (MTT) colorimetric assay. Cell membrane integrity was measured by lactate dehydrogenase (LDH) release from PAEC grown in 24-well plates. Malondialdehyde (MDA, a product of lipid peroxidation) in PAEC grown in 6-well plates was detected by a thiobarbituric acid fluorometric assay. Incubation of H₂O₂ with PAEC caused a dose-dependent decrease of cell viability, an increase of LDH release, and an elevation of MDA production. MTT assay was convenient, quantitative, non-radioactive, and suitable for testing a large number of samples. The fluorometric assay for measuring MDA production in endothelial cells used 6-well plates instead of 80-cm² flasks employed by previous investigators. The use of multiwell culture plates in these assays made it possible for more samples to be tested in any single experiment. The three assays are reproducible with low intraplate and interplate coefficients of variation. This in vitro model is suitable for screening antioxidants and for studying pharmacodynamics at the cellular level.     

Improved differentiation of β-gal+ colonies on X-gal plates containing mycological peptone

Summary Inclusion of mycological peptone in the base medium of X-gal plates facilitates the detection of isolates of Saccharomyces cerevisiae producing -galactosidase due to improved colour development. Substitution of tryptone with mycological peptone in a standard Escherichia coli medium (LB) also facilitates colour development on X-gal plates and obviates the need for an inducer. The addition of mycological peptone to the base medium of X-gal plates offers a cost-effective method to improve the detection of the -galactosidase gene in recombinant strains of S. cerevisiae and E.coli.     

A 6 x 6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli

A protocol was developed using 96-well plates and multichannel pipettes for serial dilutions, followed by drop plating on agar in a 6 x 6 format. This protocol permits simultaneous plating of six dilutions which greatly decreases the number of plates utilized thereby saving incubator space for organisms such as Campylobacter which require unique environmental conditions.     

Pseudoalteromonas spp. Serve as Initial Bacterial Attractants in Mesocosms of Coastal Waters but Have Subsequent Antifouling Capacity in Mesocosms and when Embedded in Paint

The purpose of the present study was to determine if the monoculture antifouling effect of several pigmented pseudoalteromonads was retained in in vitro mesocosm systems using natural coastal seawater and when the bacteria were embedded in paint used on surfaces submerged in coastal waters. Pseudoalteromonas piscicida survived on a steel surface and retained antifouling activity for at least 53 days in sterile seawater, whereas P. tunicata survived and had antifouling activity for only 1 week. However, during the first week, all Pseudoalteromonas strains facilitated rather than prevented bacterial attachment when used to coat stainless steel surfaces and submerged in mesocosms with natural seawater. The bacterial density on surfaces coated with sterile growth medium was 10⁵ cells/cm² after 7 days, whereas counts on surfaces precoated with Pseudoalteromonas were significantly higher, at 10⁶ to 10⁸ cells/cm². However, after 53 days, seven of eight Pseudoalteromonas strains had reduced total bacterial adhesion compared to the control. P. piscicida, P. antarctica, and P. ulvae remained on the surface, at levels similar to those in the initial coating, whereas P. tunicata could not be detected. Larger fouling organisms were observed on all plates precoated with Pseudoalteromonas; however, plates coated only with sterile growth medium were dominated by a bacterial biofilm. Suspensions of a P. piscicida strain and a P. tunicata strain were incorporated into ship paints (Hempasil x3 87500 and Hempasil 77500) used on plates that were placed at the Hempel A/S test site in Jyllinge Harbor. For the first 4 months, no differences were observed between control plates and treated plates, but after 5 to 6 months, the control plates were more fouled than the plates with pseudoalteromonad-based paint. Our study demonstrates that no single laboratory assay can predict antifouling effects and that a combination of laboratory and real-life methods must be used to determine the potential antifouling capability of new agents or organisms.     

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.     

High biological activity of a recombinant protein immobilized onto polystyrene

The adsorption characteristics of glutathione S-transferases (GST) genetically fused with polystyrene (PS)-binding peptides (PS-tags) on PS plates with increase in hydrophilicity were studied to clarify the mechanisms of the specific interaction between the PS-tag-fused protein and PS plates. GST fused with the PS-tag PS19 (RAFIASRRIKRP) preferentially interacted with hydrophilic PS plates, even in the presence of high concentrations of competitors such as Tween 20 and BSA. Both basic and aliphatic amino acids in the PS-tags were involved in the specific interaction of PS-tags with the surface of the hydrophilic PS plate. Genetic fusion of the PS19 variants, PS19-4 (RAIARRIRR) and PS19-6 (RIIIRRIRR), further improved the immobilization yield of GST in the presence of a high concentration of the competitor BSA (50 mg/mL). The PS19-6 peptide specifically interacted with the surfaces of various hydrophilic PS plates, especially in the presence of Tween 20. Higher remaining activity was detected on all of the hydrophilic PS plates immobilized with GST-PS19-6 in comparison with those with wild-type GST and GST-PS19, and the remaining activity was further increased by the addition of Tween 20 in the adsorption state. The PS19-6 peptide developed in this study is therefore very useful as an affinity tag that can immobilize a target protein directly onto various hydrophilic PS supports with high remaining activity.     

AXENIC TISSUE CULTURES FROM THE SPOROPHYTES OF LAMINARIA DIGITATA AND LAMINARIA HYPERBOREA (PHAEOPHYTA)

Starting material for the tissue cultures was the meristematic basal zone of the blade. Pieces treated 30–60 sec in hypochlorite solution were rinsed and placed on agar plates made from the artificial seawater ASP6 F2 solidified with 6 g agar l^?1. After 6 weeks colorless callus-like tissue grew out from some pieces. Treatment with activated charcoal removed some inhibiting substances from the agar medium as numbers of callus developing pieces increased on such plates. A combination of 10^?5 M NAA and 5 · 10^?7 M kinetin gave a yellow-brown tissue. A differentiation in the tissue from L. hyperborea was observed as well as the formation of meiospores, which grew out into male and female plants. Thalli of sporophytes were observed but they never reached a length of more than one mm before they died or changed to an irregular pattern of growth.     

The Practical Significance in the Microbiological Examination of Cold Stored Foods of the Allegedly Low Heat Resistance among Psychrotrophic Organisms

Summary: The significance in food microbiology of the observation of Stapert, Sokolski & Northam (1962), that some bacteria occurring in water have such low heat resistances that they would be affected by the warm agar used for pouring plates, was tested. Forty-two samples of 6 different foods stored for 7 days at 3° to enrich their psychrotrophic microflora, were examined using the same medium, but in thin poured plates and on spread plates, respectively, incubated at 14°. The logarithmic average count of the foods was c. 10⁸/g and included about 60% Gram-negative rods and 30% cocci. In 90% of the samples no differences were observed between the results from the two methods of counting. The slightly higher spread plate counts in the remaining instances could be accounted for by the increased disruption of bacterial agglomerations always observed in this procedure. Hence there is no reason to query the results of poured plate counts in general. Where it is yet probably safer to use spread plates, rigorous asepsis in pouring and storing should be observed to prevent the development of colonies of contaminants too small to be detected when the plates are inoculated, but leading to erroneous counts after incubation.     

A survey of thecal fine structure in the Dinophyceae

The structure of the theca of some 30 marine and freshwater members of the Dinophyceae has been examined by light and electron microscopy. The basic arrangement of membranes, an outer continuous plasma-membrane beneath which lies a single layer of flattened vesicles, is the same in all flagellated forms. The periplast of coccoid and encysted forms is somewhat different. Thecae have been found to fall into eight reasonably distinct categories as follows: (1) Outer membrane underlain by irregular vesicles. (2) Membrane underlain by close-packed vesicles. (3) As Group 2 but with plug-like structures attached to the inner membrane of the vesicles. (4) As Group 2 but with thin plates within the vesicles and all plates of more or less similar shape. (5) As Group 4 but plates have slight overlap. (6) Similar to Group 5 but plates are reduced in number and can be individually recognized. (7) As Group 6 but plates have elaborate flanges and their surfaces are covered by reticulations. (8) Theca mainly consisting of only two plates. It is suggested that the thecal types could from the basis for some revision of the taxonomy of the class.     

Reverse-phase purification and silica gel thin-layer chromatography of porphyrin carboxylic acids

Thin-layer chromatography on silica gel 60 plates in the solvent N,N-dimethylformamide/methanol/ethylene glycol/glacial acetic acid/1-chlorobutane/chloroform (4/35/6/0.4/18/20 by volume) separates porphyrin carboxylic acids by the number of free carboxyl groups. Coproporphyrins I and III and isocoproporphyrin are separated in 30 min, other porphyrins in 15 min. The N,N-dimethylformamide and acetic acid in the solvent strongly increase porphyrin fluorescence on the plates. Fading and diffusion of the fluorescent patterns is prevented by storage of the plates in the cold and dark without oxygen and with desiccant. In a preliminary step, porphyrins are purified in high yields, concentrated, and deacidified rapidly (2 min) by reversephase chromatography on cartridges containing a C₁₈ spacer or on Amberlite XAD-2 columns. The methods are applied to urines of porphyria patients and for following porphyrin ester hydrolysis.     

大量可降解锌合金板钉体内埋植的早期生物安全性研究

目的:可降解锌合金材料有适中的降解速率,良好的机械性能。目前对于锌合金的体内生物安全性研究多集中于生物体内植入适量的锌合金材料。对于体内植入大量的可降解锌合金材料是否有不良影响,还未见文献报道。本实验从局部和全身反应来研究埋植过量可降解锌合金的早期生物安全性。方法:选取18只新西兰大白兔分为三组,于皮下植入锌合金内固定板及钉各4、6、8块,于术后3月、6月行大体观察,血常规、血生化、血液微量元素检查,内脏和材料周围组织的组织学检查和ICP-OES定量检测内脏锌含量观察锌的脏器蓄积情况,材料称重计算每日释放锌含量。结果:可吸收锌合金材料表面附着的白色粉末状物质随时间增加而增多,去掉表面白色物质后,材料表面愈加粗糙,术后3月、6月的白细胞计数(WBC),红细胞计数(RBC),谷丙转氨酶(ALT),谷草转氨酶(AST),总蛋白(TP),白蛋白(ALB),尿素氮(BUN),肌酐(Cr),血锌,血镁,血钙、血铜与术前相比无统计学差异。术后6月实验动物材料周围组织,心脏、肝脏、脾脏、肺脏、肾脏、性腺未检出异常。术后3月、6月肝脏、肾脏、脾脏的锌离子含量与术前相比无统计学差异。综合计算得到术后3月可降解锌合金内固定板的降解率为9.77±1.64%,术后6月为11.82±1.91%,螺钉的降解率术后3月为0.79±0.66%,术后6月为2.09±1.00%。结论:大量可降解锌合金植入体内的早期生物相容性良好。     

AN UNUSUAL EPICUTICULAR WAX ULTRASTRUCTURE ON LEAVES OF PROSOPIS TAMARUGO (LEGUMINOSAE)

Studies of Prosopis tamarugo Phil. leaflet surfaces by scanning and transmission electron microscopy disclosed an aggregate coating of basic wax structures of various shapes along with large semi-vertical plates. The basic structures, with some degree of variation, were also present on P. velutina Woot. and on several other Prosopis species which had been previously investigated. However, the plates, about 3 μm in size, were observed only on P. tamarugo. The basic wax structures were first observed on very young leaflets, about 6 h after emergence from the apical bud. The plates appeared after 24 to 72 h. Both plates and basic structures were larger on seedlings grown in a low (20–25%) relative humidity as compared to high (70%) humidity. When grown side-by-side in the greenhouse, leaflets of P. tamarugo seedlings were more isolateral in structure than those of P. velutina, suggesting a greater xeromorphic adaptation. The unusual epicuticular configuration described could be a mechanism that enhances the foliar absorption of atmospheric moisture, or at least minimizes cuticular transpiration, thereby aiding survival of this unique tree in its native habitat of northern Chile.