Suitability of dried herbarium specimens for NMR metabolomics of mushrooms. A comparison of four species of the genera Kuehneromyces and Hypholoma (Strophariaceae)

Herbarium specimens are a treasure trove for biochemical studies. However, this implies understanding of the chemical changes during the drying and storage of the specimen. We compared herbarium specimens at different ages and fresh samples of four mushroom species (Kuehneromyces mutabilis, Hypholoma capnoides, Kuehneromyces lignicola, Hypholoma fasciculare) of two genera in the family Strophariaceae by using proton nuclear magnetic resonance (¹H NMR) spectroscopy combined with principal component analysis (PCA). 25 metabolites were identified. No significant alterations were found between herbarium samples at different ages, suggesting that they are stable enough for comparative studies. The most dominant differences between fresh and herbarium samples was that sugars such as α-α-trehalose, and fumaric and malic acids were more abundant in fresh fungi. Total contents of fatty and amino acids, uracil and γ-aminobutyric acid (GABA) were higher in herbarium specimens. In addition, pyroglutamic acid was observed only in Kuehneromyces mutabilis and fasciculic acid E in Hypholomafasciculare. Hence, based on results of the studied taxa, we conclude that NMR metabolomics can be used for both fresh and dried mushrooms when such alterations are properly addressed.     

Colony formation of mammalian cells on agar plates and its application to Lederberg's replica plating

In this paper we describe a new technique of cloning by use of agar plates and its application to replica plating. It was found that most cell lines form colonies on the surface of solid agar, although the plating efficiency and size of colony is dependent on specimens and concentrations of agar and agarose used. When 0.5% Noble-agar was used as substrate, plating efficiencies were obtained comparable to those of conventional cloning techniques in liquid medium and of agar suspension cultures. In some cases, including the primary culture of Yoshida sarcoma, the efficiency of plating was apparently higher than that obtained by the already established procedures. In an experiment with a series of BHK-21 cells, it was found that virally transformed cells could form colonies on agar plate, whereas untransformed and reverted cells could not divide, suggesting that agar plate culture, as well as agar suspension culture, can be used for a selective assay of transformation.Two methods of replica plating were employed. Method I is that devised by Lederberg in which colonies on the master plate are imprinted on pile fabrics and then transferred to the replica plates. With FM3A cells, the fidelity of replica plating was around 95%. Method II is inoculation of clones by applying a glass rod to the replica plates on which positions of inocula were identified by a grid. Fidelity of replica plating of FM3A, L5178Y and YSC cells was 99.7, 100 and 100% respectively.     

柚皮干制前后精油香气成分分析

为明确干制对柚皮中精油香气成分的影响,采用水蒸气蒸馏法提取干制前后柚皮中精油,通过气相色谱-质谱法（GC-MS）对精油进行成分分析.结果表明,从鲜柚皮中共检出74种香气成分,占精油总质量分数的99.61%;从干柚皮中共检出45种香气成分,占精油总质量分数的99.45%;两者共有香气成分44种,相对质量分数分别为98.16%和99.41%.干制后柚皮香气成分中烯烃类化合物的相对质量分数增加;醇类、酯类和酮类化合物的相对质量分数减少.干制对柚皮主体香气成分影响不大.     

Scanning Electron Microscopy of Selected Members of the Streptomyces hygroscopicus Group

Distinctive variations in whole spore morphology and spore surface morphology of Streptomyces hygroscopicus strains, which had been observed previously by the authors by the preshadowed carbon replica technique, were confirmed by observations with the scanning electron microscope.     

Individual phenolic response and peroxidase activity in peel of differently sun-exposed apples in the period favorable for sunburn occurrence

Extreme weather events like high solar radiation can cause stress in apple fruits (Malus domestica Borkh.). The aim of the study was to make a screening of individual phenols and peroxidase activity in apple peel as a response to sunburn and different sun-exposures in the period when weather conditions are suitable for sunburn occurrence. Apple fruits of ‘Golden Delicious’ and ‘Braeburn’ were sampled. Fruit temperature and color were measured prior HPLC–MS² and peroxidase activity analyses. Sunburned peel was darker and more yellow-red in comparison to healthy peel, which appeared yellow-green. Fruit temperature, total as well as individual flavonols and dihydrochalcones, total hydroxycinnamics and perixodase activity were highest in sunburned peel in comparison with healthy sun-exposed peel, furthermore both were different than shaded sides of both fruits and peel of apples inside the tree crown; moreover in sunburned peel dihydrochalcones were determined for the first time. Chlorogenic acid was up to 2.5 times higher, 3-hydroxy-phloretin-2′-O-xyloglucoside was up to 10 times higher and quercetin-3-galactoside was up to 33 times higher in sunburned peel, comparing to shaded sided peels. Flavanols did not show a distinct pattern. A deeper insight in phenolic response against environmental stress caused by high solar radiation and high air temperatures has been made.     

A replica plating technique for the study of plaque-forming centers

Mouse spleen cells were cultured on Millipore filters supported on the surface of Eagle's agarose medium. The secretory products of individual plaque-forming centers (PFC) revealed after diffusion into the agarose, were studied over an extended period of time by transferring the Millipore filters to fresh agarose surfaces repeatedly in a manner analogous to the replica plating technique employed with bacteria.     

Characterization of the Topography and Wettability of English Weed Leaves and Biomimetic Replicas

The topography and wettability of the underside of English weed （Oxalis pes-caprae） leaves and of their biomimetic replicas are investigated. Polyvinyl siloxane molds were cast from the leaves and then filled with an epoxy pre-polymer to produce replicas. The particular topographical structures of leaves and replicas were evaluated by optical microscopy and Scanning Electron Microscopy （SEM） analysis. The static wettability of leaves and replicas was assessed by contact angle measurements, while the dynamic wettability was characterized by estimating contact angle hysteresis and studying the dynamic behavior of impacting water droplets. A smooth glass slip and its replica were used as control surfaces. The replica moulding method used was able to transfer the characteristic pattern of irregular 100 μm - 200 μm × 60 μm convex papillae interspersed with stomata of the original leaf to the epoxy replicas. The static contact angle of 143°± 3° and the contact angle hysteresis of 2~ indicate that the underside of the English weed leaf is close to superhydrophobic. The lower contact angles （130° ± 4°） and higher hysteresis （31°） observed for the replica when compared with the original leaves were associated to an inaccurate replication of the chemistry and structures of the three-dimensional wax projections covering the plant surface. Also, trichomes in the original leaves could not be accurately reproduced due to their flexibility and fragility. Differences in wetting behavior were also evident from droplet impact experiments, with rebound regimes prevailing in the original leaves and regimes characterized by higher adhesion and larger dissipation predominating in the replicas. Nevertheless, the morphological features of the leaf transferred to the replica were sufficient to promote a clear hydrophobic behavior of the replica when compared with the smooth epoxy reference surface.     

ELECTRON MICROSCOPE REPLICAS FROM THE SURFACE OF A FRACTURE THROUGH FROZEN CELLS

A technique is described, which is new in certain details, for fracturing frozen cells and taking replicas from the fracture surface. This technique has been developed as a cytological preparative technique complementary to those currently used in electron microscopy. The present paper describes results for human red cells, chosen for preliminary work to facilitate recognition of artifacts. The cells are suspended in 20 per cent glycerol, 0.9 per cent NaCl solution, allowed time to equilibrate, and rapidly frozen in a small chamber plunged into propane at -196°C. The chamber is kept at -196°C., and fractured in vacuo, in such a way that the fracture plane runs through the frozen suspension, and through the cells in its path. After fracture, the chamber is brought to -105°C., still in vacuo, to etch the fracture surface, and a platinum-carbon replica is deposited on the surface from a carbon arc. The replica is subsequently cleaned in concentrated alkaline solution and examined in the electron microscope. The outlines of the fractured cells can be recognised. There are indications that in areas of the replica which correspond to the interior of a cell, individual haemoglobin molecules can be seen, and in favourable cases the arrangement of some of the four molecular subunits.     

An improved method for freeze-fracture radioautography of tissues and cells, as applied to duodenal epithelium and thymic lymphocytes

An improved method has been devised for the localization of radioactive substances to either one of the leaflets of cellular membranes. After tissue specimens are freeze-fractured and covered with a platinum-carbon replica, they are freeze-dried to allow coating with radioautographic emulsion at room temperature. After exposure at 4 degrees C and development, the emulsion is protected by layers of carbon and grease before the tissue underlying the replica is dissolved in sodium hypochlorite. The grease is removed in Freon 14 and the replica with its emulsion cover is mounted on a specimen grid for electron microscopic examination. The accuracy of radioactivity localization was demonstrated using 3H-thymidine-labeled liver by finding silver grains over the same sites after freeze-fracture as after thin section radioautography. Tests with 3H-methacrylate revealed that the interposition of a platinum-carbon replica decreased the radioautographic reaction by over 80%; hence, the need for long exposure. Only 67% of the silver grains came from radiation sources located beyond the upper 0.05 micron of the specimen and, therefore, the emulsion could be affected by radiation sources located not only within membrane leaflets but also in nearby cytoplasm. Thus, when 3H-fucose was injected into rats to locate newly formed glycoproteins within intestinal epithelium membranes, some of the silver grains found over E and P faces might be produced by radiation coming from the adjacent cytoplasm. To localize label within membrane leaflets in the absence of radiation sources in the cytoplasm, lymphocyte suspensions were incubated with 3H-concanavalin A at 0 degrees C. The plasmalemma radioactivity was then restricted to the two membrane leaflets, with 87-93% of the silver grains on the E leaflet and 7-13% on the P leaflet. It appears that, under these conditions, the technique provides adequate localization of radioactivity to the leaflets of the cell membrane.     

Carbohydrate Changes during Maturation of Cucumber Fruit : Implications for Sugar Metabolism and Transport

Changes in the carbohydrate profiles in the mesocarp, endocarp, and seeds of maturing cucumber (Cucumis sativus, L.) fruit were analyzed. Fruit maturity was measured by a decrease in endocarp pH, which was found to correlate with a loss in peel chlorophyll and an increase in citric acid content. Concentrations of glucose and fructose (8.6-10.3 milligrams per gram fresh weight, respectively) were found to be higher than the concentration of sucrose (0.3 milligrams per gram fresh weight) in both mesocarp and endocarp tissue. Neither raffinose nor stachyose were found in these tissues. The levels of glucose and fructose in seeds decreased during development, but sucrose, raffinose, and stachyose accumulated during the late stages of maturation. Both raffinose and stachyose were found in the seeds of six lines of Cucumis sativus L. This accumulation of raffinose saccharides coincided with an increase in galactinol synthase activity in the seeds. Funiculi from maturing fruit were found to be high in sucrose concentration (4.8 milligrams per gram fresh weight) but devoid of both raffinose and stachyose. The results indicated that sucrose is the transport sugar from the peduncle to seed, and that raffinose saccharide accumulation in the seed is the result of in situ biosynthesis and not from direct vascular transport of these oligosaccharides into the seeds.     

Comparison of the pick-and-smear and Saccomanno methods for sputum cytologic analysis

The two methods of preparing sputum specimens for cytologic study, the (fresh) pick-and-smear technique and the (blended) Saccomanno technique, were compared using 249 consecutive specimens. Two slides were prepared for each specimen by each technique. Of the specimens, 103 showed squamous metaplasia, carcinoma in situ or carcinoma. A semiquantitative rating system (0 to 4+) was used to determine the number of diagnostic cells for each method for those 103 cases. More diagnostic cells were found on the Saccomanno preparations (217) than on the fresh preparations (154). There were 121 diagnostic cells in the Saccomanno preparations versus 95 diagnostic cells in the fresh preparations from 63 squamous metaplasias; 7 versus 3 for the preparations from 5 carcinomas in situ; 64 versus 42 from 28 squamous cell carcinomas; 3 versus 1 from 1 large cell undiffernomas; and 12 diagnostic cells in Saccomanno preparations versus 5 in fresh preparations from 3 small cell cancers. Twelve squamous metaplasias, two carcinomas in situ, four squamous carcinomas, one adenocarcinoma and one small cell cancer had no diagnostic cells on the fresh preparations; four squamous metaplasias and one squamous carcinoma had no diagnostic cells on the Saccomanno preparations. More diagnostic information and fewer false-negative results were achieved with the Saccomanno technique.     

Spinal biomechanical properties are significantly altered with a novel embalming method

In vitro tests on the biomechanical properties of human spines are often performed using fresh frozen specimens. However, this carries the risk of pathogen transfer from specimen to the worker and the specimens can only be used for a limited amount of time. Human spinal specimens embalmed with formaldehyde carry an almost absent risk of transfer of pathogens and can be stored and used for a long time, but the tissue properties are strongly affected making this method inapplicable for biomechanical testing. In this study, a new embalming technique called Fix for Life (F4L), which claims to preserve the tissue properties, was tested. The range of motion (ROM) and stiffness of six fresh human spinal specimens was measured using a spinal motion simulator before and after F4L embalming. After F4L embalming, spinal stiffness increased in flexion-extension by 230%, in lateral bending by 284% and in axial rotation by 271%. ROM decreased by 46% in flexion-extension, 56% in lateral bending and 54% in axial rotation. In conclusion, based on this study, F4L does not maintain physiological spinal biomechanical properties, and we propose that this method should not be used for biomechanical studies. Nevertheless, the method may be an alternative to formaldehyde fixation in situations such as training and education because the effect on spinal biomechanics is less detrimental than formaldehyde and tissue color is maintained.     

Patterns of pigment changes in apple fruits during adaptation to high sunlight and sunscald development

Reflectance spectra of four apple (Malus domestica Borkh.) cultivars were studied and chlorophyll, carotenoid, anthocyanin and flavonoid content in sunlit and shaded peel was determined. In all cases sunlit peel accumulated high amounts of phenolics (flavonoid glycosides). Adaptation to strong sunlight of an apple cultivar with limited potential for anthocyanin biosynthesis (Antonovka) was accompanied by a decrease in chlorophyll and a significant increase in total carotenoid content. The increase in carotenoids also took place in sunlit sides of the Zhigulevskoye fruits, accumulating high amounts of anthocyanins, but chlorophyll content in sunlit peel was higher than that in shaded peel. Significant increases in carotenoids and anthocyanins were detected during fruit ripening when chlorophyll content fell below 1.5–1.8 nmol cm^–2. Chlorophyll in sunlit fruit surfaces of both cultivars was considerably more resistant to photobleaching than in shaded (especially of Zhigulevskoye) sides. Induced by sun irradiation, the photoadaptive responses were cultivar-dependent and expressed at different stages of fruit ripening even after storage in darkness. The development of sunscald symptoms in susceptible apple cultivars (Granny Smith and Renet Simirenko) led to a dramatic loss of chlorophylls and carotenoids, which was similar to that observed during artificial photobleaching. The results suggest that apple fruits exhibit a genetically determined strategy of adaptation of their photoprotective pigments to cope with mediated by reactive oxygen species photodynamic activity of chlorophyll under strong solar irradiation. This includes induction of synthesis and accumulation of flavonoids, anthocyanins and carotenoids that could be expressed, if necessary, at different stages of fruit development     

Rapid Mapping of Conditional and Auxotrophic Mutations in Escherichia coli K-12

The approximate genetic map locations of auxotrophic and conditional lethal mutations of Escherichia coli can be rapidly determined with replica plating techniques. A set of patches of 15 streptomycin-sensitive (Str^S) Hfr strains with points of origin distributed around the map is replica plated onto a recombinant-selective plate with a lawn of Str^R cells which carry an unmapped mutation. The map interval defined by the Hfr points of origin which are closest to the mutant locus is seen by the presence or absence of heavy patches of recombinants produced by transfer of early wild-type genes from the Hfrs. An alternative method is to replicate patches of different mutant strains (100 per plate) onto Hfr lawns; in this case more than 1,000 different mutants can be mapped in a single experiment in a few days. In this way, many types of mutations with similar phenotypes can be grouped as to approximate location on the genetic map. For ordering mutations within groups, the same replica plating methods can be used to cross F-prime derivatives of mutants with other mutants of the same group. Relative merits of these and other mapping methods of E. coli are discussed.     

Herbarium tale: the utility of dry specimens for DNA barcoding Juncaceae

Phylogenetic and barcoding studies usually use fresh plant tissues as sources of DNA and have successfully amplified DNA for various loci. The use of dried samples, however, is often necessary due to the frequent inaccessibility of fresh rare plants or their parts for genetic analyses or barcoding. The difficulty in obtaining amplifiable DNA is a major restriction of the use of herbarium specimens for DNA analyses. Recent study has highlighted the crucial issues for comparing herbarium and fresh plants for barcoding. We analysed the performance of samples of the family Juncaceae from various herbarium specimens of different ages with fresh plant material in PCRs and the sequences of seven loci (rbcL, rpoC1, trnL-F intergenic spacer, trnL intron, and psbA-trnH from chloroplast DNA; atp1 from mitochondrial DNA; and ITS1-5.8S-ITS2 from nuclear DNA) using a combination of 28 primers. The herbarium specimens amplified well and may thus be successfully applied for both phylogenetic analyses and barcoding for the Juncaceae family. Amplifying DNA was more difficult from dried herbarium specimens than fresh samples but could be successful in most cases when appropriate internal primers were designed or methods were optimised. Using the set of universal primers recommended by the Consortium for the Barcode of Life and designing specific primers for a particular group of interest were both useful. Specimen age and amplicon length had limited detrimental effects on amplification success for most of the Juncaceae loci tested.     

Comparison of Fluorescent Antibody with Cultural Technique for Isolation of Enteropathogenic Escherichia coli from Swine

In an investigation of hogs as possible reservoirs of human strains of enteropathogenic Escherichia coli (EEC), 92 six-month-old grain- and garbage-fed hogs were examined on the farm and again at the packing plant. Of the 331 specimens obtained by swabbing the rectum, cecum, and edible meat carcass of these hogs, 125 were presumptively positive for EEC when screened by the fluorescent-antibody (FA) technique. These “presumptive positive” specimens then underwent extensive bacteriological examination and complete serological typing. The FA technique proved to be an easier, simpler, and more economical procedure than culture when a large number of specimens were examined for possible EEC serogroups. It was found especially valuable for identification of multiple serogroups of EEC within a single specimen. It also appeared to be more sensitive than cultural examination, since results were not dependent on the presence of large numbers of organisms in the specimen, or even on their viability. However, the FA technique was found to be less specific than culture because of cross-reactivity with antigenically related Enterobacteriaceae when fluorescein-labeled antisera were used. Therefore, any specimen found positive on FA examination should be considered as presumptive positive until confirmed by bacteriological examination and complete serological study.     

Fluorescence-Based Bacterial Overlay Method for Simultaneous In Situ Quantification of Surface-Attached Bacteria

For quantification of bacterial adherence to biomaterial surfaces or to other surfaces prone to biofouling, there is a need for methods that allow a comparative analysis of small material specimens. A new method for quantification of surface-attached biotinylated bacteria was established by in situ detection with fluorescence-labeled avidin-D. This method was evaluated utilizing a silicon wafer model system to monitor the influences of surface wettability and roughness on bacterial adhesion. Furthermore, the effects of protein preadsorption from serum, saliva, human serum albumin, and fibronectin were investigated. Streptococcus gordonii, Streptococcus mitis, and Staphylococcus aureus were chosen as model organisms because of their differing adhesion properties and their clinical relevance. To verify the results obtained by this new technique, scanning electron microscopy and agar replica plating were employed. Oxidized and poly(ethylene glycol)-modified silicon wafers were found to be more resistant to bacterial adhesion than wafers coated with hydrocarbon and fluorocarbon moieties. Roughening of the chemically modified surfaces resulted in an overall increase in bacterial attachment. Preadsorption of proteins affected bacterial adherence but did not fully abolish the influence of the original surface chemistry. However, in certain instances, mostly with saliva or serum, masking of the underlying surface chemistry became evident. The new bacterial overlay method allowed a reliable quantification of surface-attached bacteria and could hence be employed for measuring bacterial adherence on material specimens in a variety of applications.     

Sucrose synthase and invertase in isolated vascular bundles

Vascular bundles were isolated from grapefruit (Citrus paradisi Macf.) during periods of rapid sucrose translocation into fruit. Invertase and sucrose synthase activities were assayed in these strands and compared with immediately adjacent tissues (inner most peel and segment epidermis) and phloem-free juice sacs during four growing seasons. Although sucrose synthase was present in sink cells, the significantly greater activity in vascular strands (per unit fresh weight and protein) indicated that the role of this enzyme in translocation may include a vascular function in addition to its proposed involvement in metabolism of importing cells.     

Usefulness of manufactured tomato extracts in the diagnosis of tomato sensitization: Comparison with the prick-prick method

Background

Commercial available skin prick test with fruits can be negative in sensitized or allergic patients due to a reduction in biological activity during the manufacturing process. Prick-prick tests with fresh foods are often preferred, but they are a non-standardized procedure. The usefulness of freeze-dried extracts of Canary Islands tomatoes, comparing the wheal sizes induced by prick test with the prick-prick method in the diagnosis of tomato sensitization has been analyzed. The objective of the study was to assess the potential diagnostic of freeze-dried extracts of Canary Islands tomatoes, comparing the wheal sizes induced by prick test with the prick-prick method.

Methods

Two groups of patients were analyzed: Group I: 26 individuals reporting clinical symptoms induced by tomato contact or ingestion. Group II: 71 control individuals with no symptoms induced by tomato: 12 of them were previously skin prick test positive to a tomato extract, 39 were atopic and 20 were non-atopic. All individuals underwent prick-prick with fresh ripe peel Canary tomatoes and skin prick tested with freeze-dried peel and pulp extracts obtained from peel and pulp of Canary tomatoes at 10 mg/ml. Wheal sizes and prick test positivity (≥ 7 mm²) were compared between groups.

Results

In group I, 21 (81%) out of 26 patients were prick-prick positive. Twenty patients (77%) had positive skin prick test to peel extracts and 12 (46%) to pulp extracts. Prick-prick induced a mean wheal size of 43.81 ± 40.19 mm² compared with 44.25 ± 36.68 mm² induced by the peel extract (Not significant), and 17.79 ± 9.39 mm² induced by the pulp extract (p < 0.01). In group II, 13 (18%) out of 71 control patients were prick-prick positive. Twelve patients (all of them previously positive to peel extract) had positive skin prick test to peel and 3 to pulp. Prick-prick induced a mean wheal size of 28.88 ± 13.12 mm² compared with 33.17 ± 17.55 mm² induced by peel extract (Not significant), and 13.33 ± 4.80 mm² induced by pulp extract (p < 0.05 with peel extract and prick-prick).

Conclusion

Canary peel tomato extract seems to be as efficient as prick-prick tests with ripe tomatoes to diagnose patients sensitized to tomato. The wheal sizes induced by prick-prick and peel extracts were very similar and showed a high correlation coefficient.