Fluorescence in situ hybridization on vibratome sections of plant tissues

This protocol describes the application of fluorescence in situ hybridization (FISH) to three-dimensionally (3D) preserved tissue sections derived from intact plant structures such as roots or florets. The method is based on the combination of vibratome sectioning with confocal microscopy. The protocol provides an excellent tool to investigate chromosome organization in plant nuclei in all cell types and has been used on tissues of both monocot and dicot plant species. The visualization of 3D well-preserved tissues means that cell types can be confidently identified. For example, meiocytes can be clearly identified at all stages of meiosis and can be imaged in the context of their surrounding maternal tissue. FISH can be used to localize centromeres, telomeres, repetitive regions as well as unique regions, and total genomic DNAs can be used as probes to visualize chromosomes or chromosome segments. The method can be adapted to RNA FISH and can be combined with immunofluorescence labeling. Once the desired plant material is sectioned, which depends on the number of samples, the protocol that we present here can be carried out within 3 d.     

Modification of commerical and custom-built slab gel electrophoresis units for two-dimensional polyacrylamide gel electrophoresis

Many commercial and custom-built slab gel electrophoresis units can be modified to function as two-dimensional polyacrylamide gel electrophoresis units with the insertion of Plexiglas adapters. These adapters can be made for about $50 a pair and can be used for either temporary or permanent modification of the slab gel units. The physical dimensions of the adapters can be varied to permit great flexibility in the diameter of cylinder gels and the thickness of slab gels that can be run together. For example, proteins from 6-mm cylinder gels can be easily separated on 1-mm slab gels, which can then be dried for autoradiography.     

The genetical analysis of covariance structure.

The analysis of covariance structures (J?reskog, 1973) is adapted to the simultaneous maximum likelihood estimation of genetical and environmental factor loadings and specific variances. The goodness of fit is tested by chi square and standard errors of parameter estimates can be obtained. Any linear model used in univariate genetical analyses can be extended to the multivariate case. Most biological hypotheses about the relationships between variables can be specified by a variety of factor models. Individual parameters can be given fixed values or set to zero and hypotheses concerning the congruence of genetical and environmental correlations can be tested. The method is illustrated with published twin data on cognitive abilities.     

Stratification of solids,nitrogen and phosphorus in swine manure in deep pits under slatted floors

Manure slurries stored in pits under slatted floors of both finishing and nursery barns were sampled at four different depths to study stratifications of total solids (TS) and nutrients (nitrogen and phosphorus), and to determine the relationship between the stratification of TS and nutrients. The results obtained can be used to improve the management and handling of swine manure in the under-slat storage pits. A management scheme that can be adopted for both the finishing and the nursery barns' pits is the layer-by-layer harvesting of the manure. The thinner manure, which is lower in nutrients, can be spread on land near the production units in larger volumes or it may pumped to land remote from the production units without causing many clogging problems. The thicker manure, higher in nutrients, can be transported to land further away and spread in smaller volumes. The TS content of each stratum can be used to accurately estimate the nitrogen and phosphorus levels in the respective strata so that application rates can easily be adjusted accordingly during the time of land application.     

Visual analysis of trabecular bone structure

Acquiring image data of bone biopsies by a micro-CT scanner is today a common technique. The amount of data to be assessed is huge. The task to assess quantitative measures requires a concise visualization. We present visualization techniques that can be used interactively on state-of-the-art PCs and demonstrate how the frontier can be pushed further. A skeletonization process is applied to the image of the bone to create the central surface. After triangulation this surface can be renderd at interactive frame rates. When the surface is additionally colored by local measures (mean grey value of image data, local thickness) the overall structure and details can be recognized at the same time. This can facilitate the exploration of the biopsy and can help finding special features.     

Use of bioluminescence in nucleic acid hybridization reactions

Luminescence reactions can be used to detect specific nucleic acid sequences hybridized with a nucleic probe. Different labels such as cytidine sulphone, fluorescein, and biotin can be incorporated into DNA or oligonucleotide molecules and detected by antibody or avidin conjugates coupled to glucose-6P dehydrogenase. On supports such as nitrocellulose filters, sensitivity is not greatly increased using luminescence, but detection is rapid and easy to perform using polaroid film. Moreover, hybridization can be performed with different labelled probes on the same sample. In solution, luminescence can be used to monitor sandwich reactions. The method is less sensitive than detection on filters but can easily be automated. The performance of these assays can be increased considerably by enzymatic amplification of the target catalysed by a thermostable polymerase.     

The use of controlled-release glass for the controlled delivery of bioactive materials

CRG can provide a useful supplement to other methods of controlled delivery. Some advantages of this approach are: The constituents of the CRG can be limited to biologically safe ionic species such as Na+, Ca2+ and PO4(3-). The release system is completely soluble in water and no residue remains. There is no evidence of any mechanism for biodegradation of the CRG and so one possible cause of premature or accelerated release is eliminated. The absolute release rate can be selected anywhere in a spectrum covering several orders of magnitude and the rate-controlling process has zero-order kinetics. The release rate can be selected to be pH-sensitive or pH-independent. Complex temporal release-rate patterns can be obtained readily by the choice of geometrical shape or composition profile of the device. The controlled release of organics or other heat-sensitive materials which cannot be incorporated in the glass can be realized by the use of composite structures in which the CRG is the rate-controlling constituent. A number of different functions can be performed by a single CRG-based device. For example, CRG can be used as a biomedical resorbable material in surgery and the CRG structural component can release an AM as it dissolves. Similarly the CRG of the sinter-composite used for organic AM release can itself release any selected inorganic adjuvant. CRG boluses containing either copper or cobalt and weighing approx. either 70 g or 15 g, have been administered to both cattle and sheep. More than 90% of the boluses remained either in the reticulum of trace element remained after dosing.(ABSTRACT TRUNCATED AT 250 WORDS)     

A shapeable foot-pressure measuring device

Measurement of foot pressure distribution for clinical purposes should ideally be made inside the shoe with a shaped insole and raised heel. Pressure measuring platforms cannot do this and transducers inserted inside the shoe can be obtrusive and inaccurate. The main clinical benefit of foot pressure measurement is the assistance it gives to shoe insole design. A shapeable foot pressure measuring device is being developed. The device can be accurately shaped to the contours of the foot and can indicate the pressure distribution over the entire surface of the foot. By adjusting the shape locally, high pressure areas can be relieved and the resulting shape can be used to vacuum-form a shoe insole. The device also has potential for the manufacture of special seating.     

Nucleic acid quantitation by continuous flow fluorometry

A system for rapid and sensitive fluorometric measurement of nucleic acids is described. Samples can be analyzed virtually as fast as they can be injected into the analyzer (three to five per minute). 5-microliter samples ranging in concentration from 0.05 to 40 micrograms/ml can be measured accurately. The sample can easily be recovered.     

The Gene Construction Kit: a new computer program for manipulating and presenting DNA constructs

The Gene Construction Kit is a new tool for manipulating and displaying DNA sequence information. Constructs can be displayed either graphically or as formatted sequence. Segments of DNA can be cut out with restriction enzymes and pasted into other sites. The program keeps track of staggered ends and notifies the user of incompatibilities and offers a choice of ligation options. Each segment of a construct can have its own defined thickness, pattern, direction and color. The sequence listing can be displayed in any font and style in user defined grouping. Nucleotide positions can be displayed as can restriction sites and protein sequences. The DNA can be displayed as either single- or double-stranded. Restriction sites can be readily marked. Alternative views of the DNA can be maintained and the history of the construct automatically stored. Gel electrophoresis patterns can be generated and can be used in cloning project design. Extensive comments can be stored with the construct and can be searched rapidly for key words. High quality illustrations showing multiple editable constructs with added graphics and text information can be generated for slides, posters or publication.     

龙葵果实综合利用的研究

龙葵是一种分布很广的野生植物。夏、秋之季果实累累,成熟果皮呈紫色,可作为提取天然食用色素的原料.果肉酸甜,可做成果酱.种籽中含油脂丰富,以不饱和酸酯为主,可提取特殊保健食用油.一物多用很有开发价值.     

A note on the occasional instability of detrending in correspondence analysis

When the two first eigenvalues of correspondence analysis are close to each other, their order can be reversed due to random variation in the data. The first axis can actually be in any direction in the plane defined by the two axes. However, the configuration of the points in the plane can remain unchanged but their projections onto any line in the plane can be very variable. The ordering in the first axis is preserved in detrending. The second axis is detrended with respect to the first one and therefore very variable configurations result when the orientation of the first axis in the plane is changed. This can lead to a situation where the detrended solutions are very unstable under random variation and therefore they can be only casually interpretable.     

Marine yeasts as biocontrol agents and producers of bio-products

As some species of marine yeasts can colonize intestine of marine animals, they can be used as probiotics. It has been reported that β-glucans from marine yeast cells can be utilized as immuno-stimulants in marine animals. Some siderophores or killer toxins produced by marine yeasts have ability to inhibit growth of pathogenic bacteria or kill pathogenic yeasts in marine animals. The virulent factors from marine pathogens can be genetically displayed on marine yeast cells, and the yeast cells displaying the virulent factors can stimulate marine animals to produce specific antibody against the pathogens. Some marine yeast cells are rich in proteins and essential amino acids and can be used in nutrition for marine animals. The marine yeast cells rich in lipid can be used for biodiesel production. Recently, it has been reported that some strains of Yarrowia lipolytica isolated from marine environments can produce nanoparticles. Because many marine yeasts can remove organic pollutants and heavy metals, they can be applied to remediation of marine environments. It has been shown that the enzymes produced by some marine yeasts have many unique properties and many potential applications.     

Hybridization-based karyotyping of mouse chromosomes: hybridization-bands.

We have developed a method, which we have named hybridization-banding, to identify simultaneously all chromosomes in a mouse metaphase spread. The method uses a combination of hybridization probes labeled with a single fluor to yield a simple, unique, readily identifiable hybridization pattern on each chromosome. The method is superior to Giemsa- or fluorescence-based banding methods for chromosome identification because the hybridization patterns are simpler and easier to identify, and unique patterns can be designed at will for each chromosome. Analysis can be performed with a standard fluorescence microscope, and images can be recorded on film with an ordinary 35-mm camera, making the method useful to many investigators. The method can also be applied to any species for which chromosomes and probes can be prepared.     

Enhancement of tomogram interpretability using the locked self-rotation function

The interpretation of cryo-electron tomograms of macromolecular complexes can be difficult because of the large amount of noise and because of the missing wedge effect. Here it is shown how the presence of rotational symmetry in a sample can be utilized to enhance the quality of a tomographic analysis. The orientation of symmetry axes in a sub-tomogram can be determined using a locked self-rotation function. Given this knowledge, the sub-tomogram density can then be averaged to improve its interpretability. Sub-tomograms of the icosahedral bacteriophage phiX174 are used to demonstrate the procedure.     

Protein φ and ψ dihedral restraints determined from multidimensional hypersurface correlations of backbone chemical shifts and their use in the determination of protein tertiary structures

The chemical shifts of the backbone atoms of proteins can be used to obtainrestraints that can be incorporated into structure determination methods. Eachchemical shift can be used to define a restraint and these restraints can besimultaneously used to define the local, secondary structure features. Theglobal fold can be determined by a combined use of the chemical shift basedrestraints along with the long-range information present in the NOEs ofpartially deuterated proteins or the amide–amide NOEs but not from suchlimited NOE data sets alone. This approach has been demonstrated to be capableof determining the overall folding pattern of four proteins. This suggeststhat solution-state NMR methods can be extended to the structure determinationof larger proteins by using the information present in the chemical shifts ofthe backbone atoms along with the data that can be obtained on a small numberof labeled forms.     

Shave excision of superficial solar skin lesions

Shave excision is a useful technique for the treatment of superficial solar lesions. The most common of these is the solar keratosis, which can be a red or gray scaling lesion. This can at times be difficult to differentiate from the superficial basal cell carcinoma or squamous cell carcinoma or their more infiltrating types. Shave excision gives the advantage of a histopathologic report. Lesions found to be infiltrating or in danger of recurrence can then be further excised. The cosmetic appearance of the healed shave excision site is generally quite good, although it can be paler than the surrounding skin. If this is likely to be a problem, the shave graft can be applied, as with deeper shave excisions. A thin shave graft also can be used to repigment pale scarred areas. A series of 1313 shave excised lesions is analyzed.     

Ultrafiltration membranes for chemical bonding of urease

Asymmetric ultrafiltration membranes suitable for covalent bonding of urease can be prepared from membranes based upon polyamide or polysulfone. Because the original membrane polymers are not chemically reactive, they have to be converted in such a way that known reactions for enzyme fixation can be used such as the diazo, the acyl-acid, the carbodiimide, and the methylbromide reaction. The enzyme was fixed within the porous substructure of the membrane. The amount of enzyme immobilized at the membrane dense skin was found to be negligible. The kinetics can be described according to the Michaelis-Menten model. Compared to the native urease, the activity of the membrane-bonded enzyme was very low. The reasons can be sought in the transmembrane transport and in the fixation procedure as well.     

Modeling cell concentration in complex media

A model that continuously predicts the concentration of microorganisms in complex medium fermentations is suggested. The model uses carbon dioxide evolution as its primary input and assumes that respiration activity can be differentiated into growth-related and maintenance-related functions. This model can be programmed on computer-coupled vessels and used to standardize on a physiological fermentation inoculum transfer time. The cell concentration estimate can also be used to calculate specific growth rate and can be combined with additional monitored information to calculate other important fermentation parameters such as specific oxygen uptake.     

Monitoring biotransformations in polyamide fibres

The enzymatic hydrolysis of polyamide fibres yields amino and carboxylic groups. These groups can be found in solution treatments as polyamide monomers and soluble oligomers. The amino groups can also be found at the surface of the fibres as end group chains. In this paper we report two methods to quantify the formation of these groups as a result of the enzymatic action. Soluble amino groups can be quantified with 2,4,6-trinitrobenzenesulfonic acid (TNBS), which yields a coloured complex which can be determined spectrophotometrically. The amino groups on the fibre surface can be quantified by reaction with a wool reactive dye and determination of colour intensities after a dyeing procedure below the glass transition temperature of polyamide.