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PubMed2006

High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9.

High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by approximately 100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contig-based physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize.

Chromosome Mapping↗
PubMed2002

Candidate sites of action for microdosimetry associated with exposure to extremely-low-frequency magnetic fields, electric fields and contact currents.

Recent advances enable one to apply numerical techniques to anatomically-correct human models to compute current densities and electric fields in tissue due to exposure to electric fields, magnetic fields, or contact currents. These methods have proved to be informative in estimating exceedance of basic restrictions prescribed by exposure guideline organizations. To date, the analyses have been conducted with a resolution on the order of millimeters. However, these techniques have future roles to play at higher levels of resolution at those sites in target tissues suspected of transducing local electric fields into biological responses. Two specific cases in which high resolution "microdosimetry" would yield value involve (a) residential settings and childhood leukemia and (b) worker exposure and cardiovascular disease. Recent research suggests that residential contact currents on the order of microamperes can produce biologically significant dose (expressed as the local electric field) to the bone marrow of a child. Microdosimetry would focus on pluripotent progenitor cells resident in the marrow compartment, as well as anatomic features that distinguish a child's from an adult's marrow. Laboratory and epidemiologic research has suggested that magnetic field exposure may affect heart rate variability, a measure reflective of autonomic nervous system control of cardiac activity. Given the physical attributes of the central nervous system and the sites that could serve as substrates for field interactions, future microdosimetry addressing heart rate variability effects may be well-advised to focus on the electrically excitable dendritic arborizations of neurons. In both cases, microdosimetry will help shed light on primary interactions in tissue.

Adult↗
PubMed1998

High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity.

A human tumor necrosis factor-alpha (TNF-alpha) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-A resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the N-terminal seven amino acids that is disordered in the structure of wild-type TNF-alpha. Compared with wild-type TNF-alpha, it exhibits 11- and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect and in vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-alpha. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29-36 into a rigid segment as a consequence of induced formation of intra- and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe56 projects into a hydrophobic cavity, while Ile52 is exposed to the bulk solvent. Ile52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-alpha. It is also expected that the 1.8-A resolution structure will serve as an accurate model for explaining the structure-function relationship of wild-type TNF-alpha and many TNF-alpha mutants reported previously and for the design of new TNF-alpha mutants.

Animals↗
PubMed2005

Sample preparation procedures for biological atomic force microscopy.

Since the late 1980s, atomic force microscopy (AFM) has been increasingly used in biological sciences and it is now established as a versatile tool to address the structure, properties and functions of biological specimens. AFM is unique in that it provides three-dimensional images of biological structures, including biomolecules, lipid films, 2D protein crystals and cells, under physiological conditions and with unprecedented resolution. A crucial prerequisite for successful, reliable biological AFM is that the samples need to be well attached to a solid substrate using appropriate, nondestructive methods. In this review, we discuss common techniques for immobilizing biological specimens for AFM studies.

Animals↗
PubMed2004

Definition of pertinent parameters for the evaluation of articular cartilage repair tissue with high-resolution magnetic resonance imaging.

To evaluate articular cartilage repair tissue after biological cartilage repair, we propose a new technique of non-invasive, high-resolution magnetic resonance imaging (MRI) and define a new classification system. For the definition of pertinent variables the repair tissue of 45 patients treated with three different techniques for cartilage repair (microfracture, autologous osteochondral transplantation, and autologous chondrocyte transplantation) was analyzed 6 and 12 months after the procedure. High-resolution imaging was obtained with a surface phased array coil placed over the knee compartment of interest and adapted sequences were used on a 1 T MRI scanner. The analysis of the repair tissue included the definition and rating of nine pertinent variables: the degree of filling of the defect, the integration to the border zone, the description of the surface and structure, the signal intensity, the status of the subchondral lamina and subchondral bone, the appearance of adhesions and the presence of synovitis. High-resolution MRI, using a surface phased array coil and specific sequences, can be used on every standard 1 or 1.5 T MRI scanner according to the in-house standard protocols for knee imaging in patients who have had cartilage repair procedures without substantially prolonging the total imaging time. The new classification and grading system allows a subtle description and suitable assessment of the articular cartilage repair tissue.

Adult↗
PubMed2001

Genetic information, genomic technologies, and the future of drug discovery.

The completion of the first draft of the human genome has provided an unprecedented opportunity to understand the genetic and molecular basis of disease. Parallel developments of new biological technologies, such as transcript profiling, allow scientists to examine almost any biological system in high molecular resolution. Contemporary drug discovery research is now focusing on the identification and validation of pharmaceutical targets in the molecular pathways/systems embedded in this information. Novel therapeutic interventions are being developed and evaluated as a result of this research which will be the basis of innovative pharmaceuticals of the future.

Animals↗
PubMed1987

The molecular genetics of HLA-related disorders.

The HLA region on the short arm of chromosome 6 contains a set of highly polymorphic loci responsible for regulating the immune response. Particular haplotypes, defined serologically, have been associated with a risk of developing certain autoimmune diseases such as insulin-dependent (juvenile-onset) diabetes mellitus, multiple sclerosis and rheumatoid arthritis. Recent developments in molecular biology have permitted an improved resolution of the locus and of the sequential arrangement of the susceptibility determinants on these haplotypes. Restriction fragment length polymorphisms have allowed subdivisions of serological haplotypes to be made. These correlate with disease susceptibility in some cases. Amplification of specific HLA class II alleles and nucleic acid sequencing have resulted in the identification of the structural determinants in the HLA that underlie some of these diseases.

Arthritis, Rheumatoid↗
PubMed2002

Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery.

The function of the human nucleotide excision repair (NER) apparatus is to remove bulky adducts from damaged DNA. In an effort to gain insights into the molecular mechanisms involved in the recognition and excision of bulky lesions, we investigated a series of site specifically modified oligonucleotides containing single, well-defined polycyclic aromatic hydrocarbon (PAH) diol epoxide-adenine adducts. Covalent adducts derived from the bay region PAH, benzo[a]pyrene, are removed by human NER enzymes in vitro. In contrast, the stereochemically analogous N(6)-dA adducts derived from the topologically different fjord region PAH, benzo[c]phenanthrene, are resistant to repair. The evasion of DNA repair may play a role in the observed higher tumorigenicity of the fjord region PAH diol epoxides. We are elucidating the structural and thermodynamic features of these adducts that may underlie their marked distinction in biologic function, employing high-resolution nuclear magnetic resonance studies, measurements of thermal stabilities of the PAH diol epoxide-modified oligonucleotide duplexes, and molecular dynamics simulations with free energy calculations. Our combined findings suggest that differences in the thermodynamic properties and thermal stabilities are associated with differences in distortions to the DNA induced by the lesions. These structural effects correlate with the differential NER susceptibilities and stem from the intrinsically distinct shapes of the fjord and bay region PAH diol epoxide-N(6)-adenine adducts.

Base Sequence↗
PubMed2005

Laser-guided direct writing for three-dimensional tissue engineering.

One of the principal limitations to the size of an engineered tissue is oxygen and nutrient transport. Lacking a vascular bed, cells embedded in an engineered tissue will consume all available oxygen within hours while out branching blood vessels will take days to vascularize the implanted tissue. One possible solution is to directly write vascular structures within the engineered tissue prior to implantation, reconstructing the tissue according to its native architecture. The cell patterning technique, laser-guided direct writing (LGDW), can pattern multiple cells types with micrometer resolution on arbitrary surfaces, including biological gels. Here we show that LGDW can pattern human umbilical vein endothelial cells (HUVEC) in two- and three-dimensions with micrometer accuracy. By patterning HUVEC on Matrigel, we can direct their self-assembly into vascular structures along the desired pattern. Finally, co-culturing the vascular structures with hepatocytes resulted in an aggregated tubular structure similar in organization to a hepatic sinusoid. This capability can facilitate studies of tissue architecture at the single cell level, and of heterotypic interactions underlying processes such as liver and pancreas morphogenesis, differentiation, and angiogenesis.

Animals↗
PubMed1989

Determination of pipecolic acid in serum or plasma by solid-phase extraction and isotope dilution mass spectrometry.

The determination of pipecolic acid in serum or plasma by positive chemical ionization gas chromatography/mass spectrometry is assessed. This quantitative method involves stable isotope dilution and cation-exchange solid-phase extraction. Several derivatives of pipecolic acid and its octadeuterated analogue were investigated for their mass spectrometric characteristics. The heptafluorobutyric methyl ester derivatives afford optimal resolution on gas chromatography of biological extracts. Moreover, the derivatizing reagent (methanolic HCl) allows a combined elution and derivatization. Selected ion monitoring is performed on the [M + H]+ ions of both analyte and internal standard, at m/z 340 and 348, respectively. Serum or plasma samples from healthy subjects and patients suspected of peroxisomal diseases have been examined.

Child↗
PubMed2005

A vision-based, 3D reconstruction technique for scanning electron microscopy: direct comparison with atomic force microscopy.

High-resolution, detailed 3D reconstructions of biological specimens obtained from scanning electron microscopy stereo-micrographs and proprietary software were compared with Tapping-Mode AFM datasets of the same fields. The reconstruction software implements several original solutions including a neural adaptive point-matching technique, the ability to build an irregular triangulated mesh rather than a regular orthogonal grid, and the ability to re-map one of the original images exactly onto the reconstructed surface. The technique was applied to human nerve tissue to obtain 1,424 x 968-pixel, texture-mapped datasets, which were subsequently compared against 512 x 512-pixel AFM datasets from the same viewfields. Accounting for the inherent differences of the two techniques, direct comparison revealed an excellent visual match. The correspondence was also quantified by calculating the cross-correlation coefficient between corresponding altimetric profiles in SEM and AFM data, which consistently exceeded a figure of 0.9, with a rate of point mismatch in the order of 0.01%. Research is still underway to improve the robustness of the technique when applied to arbitrary images

Humans↗
PubMed1993

MR microscopy of the rat lung using projection reconstruction.

Projection reconstruction has been implemented with self-refocused selection pulses on a small bore, 2.0 T MR microscope, to allow imaging of lung parenchyma. Scan synchronous ventilation and cardiac gating have been integrated with the sequence to minimize motion artifacts. A systematic survey of the pulse sequence parameters has been undertaken in conjunction with the biological gating parameters to optimize resolution and signal-to-noise (SNR). The resulting projection images with effective echo time of < 300 microseconds allow definition of lung parenchyma with an SNR improvement of approximately 15 x over a more conventional 2DFT short echo gradient sequence.

Animals↗
PubMed2005

Microscopic magnetic resonance elastography (microMRE).

Magnetic resonance elastography (MRE) was extended to the microscopic scale to image low-frequency acoustic shear waves (typically less than 1 kHz) in soft gels and soft biological tissues with high spatial resolution (34 micromx34 micromx500 microm). Microscopic MRE (microMRE) was applied to agarose gel phantoms, frog oocytes, and tissue-engineered adipogenic and osteogenic constructs. Analysis of the low-amplitude shear wave pattern in the samples allowed the material stiffness and viscous loss properties (complex shear stiffness) to be identified with high spatial resolution. microMRE experiments were conducted at 11.74 T in a 56-mm vertical bore magnet with a 10 mm diameterx75 mm length cylindrical space available for the elastography imaging system. The acoustic signals were generated at 550-585 Hz using a piezoelectric transducer and high capacitive loading amplifier. Shear wave motion was applied in synchrony with the MR pulse sequence. The field of view (FOV) ranged from 4 to 14 mm for a typical slice thickness of 0.5 mm. Increasing the agarose gel concentration resulted in an increase in shear elasticity and shear viscosity. Shear wave motion propagated through the frog oocyte nucleus, enabling the measurement of its shear stiffness, and in vitro shear wave images displayed contrast between adipogenic and osteogenic tissue-engineered constructs. Further development of microMRE should enable its use in characterizing stiffer materials (e.g., polymers, composites, articular cartilage) and assessing with high resolution the mechanical properties of developing tissues.

Adipose Tissue↗
PubMed2003

Nano-high-performance liquid chromatography in combination with nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysis.

Fourier transform ion-cyclotron resonance (FTICR) mass spectrometry offers several advantages for the analysis of biological samples, including excellent mass resolution, ultra-high mass measurement accuracy, high sensitivity, and wide mass range. We report the application of a nano-HPLC system coupled to an FTICR mass spectrometer equipped with nanoelectrospray source (nano-HPLC/nano-ESI-FTICRMS) for proteome analysis. Protein identification in proteomics is usually conducted by accurately determining peptide masses resulting from enzymatic protein digests and comparing them with theoretically digested protein sequences from databases. A tryptic in-solution digest of bovine serum albumin was used to optimize experimental conditions and data processing. Spots from Coomassie Blue and silver-stained two-dimensional (2D) gels of human thyroid tissue were excised, in-gel digested with trypsin, and subsequently analyzed by nano-HPLC/nano-ESI-FTICRMS. Additionally, we analyzed 1D-gel bands of membrane preparations of COS-6 cells from African green monkey kidney as an example of more complex protein mixtures. Nano-HPLC was performed using 1-mm reverse-phase C-18 columns for pre-concentration of the samples and reverse-phase C-18 capillary columns for separation, applying water/acetonitrile gradient elution conditions at flow rates of 200 nL/min. Mass measurement accuracies smaller than 3 ppm were routinely obtained. Different methods for processing the raw data were compared in order to identify a maximum number of peptides with the highest possible degree of automation. Parallel identification of proteins from complex mixtures down to low-femtomole levels makes nano-HPLC/nano-ESI-FTICRMS an attractive approach for proteome analysis.

Animals↗
PubMedDate not supplied

Application of confocal laser scanning microscopy to analysis of H2O2-induced DNA damage in human cells.

Confocal laser scanning microscopy (CLSM) offers improved depth discrimination and spatial resolution to the analysis of biologic samples. We demonstrate in this paper that such technology is valuable in examining DNA single-strand breaks in human cells. The single-cell-gel (SCG) assay is a new technique for measuring DNA strand breaks in individual cells. Cells embedded in low-melting-point agarose are treated with varying concentrations of hydrogen peroxide to induce DNA strand breaks. Following cell lysis and alkaline electrophoresis, which enables single-stranded break detection, analysis of the resulting "comets" provides an accurate method of comparing changes in DNA migration patterns, which have been shown to reflect the DNA damage levels. A statistically significant difference (p < 0.01) in single-stranded DNA damage levels was detected in cells exposed to hydrogen peroxide concentrations as low as 10 nm for 2 min. LSM analysis of the SCG technique allows rapid, sensitive, and reproducible quantitation of single-stranded breaks of cellular DNA.

Cell Line↗
PubMed1997

High-Resolution Atomic Force Microscopy of Native Valonia Cellulose I Microcrystals

We have studied the surface of native Valonia cellulose I microcrystals under propanol and waterby atomic force microscopy (AFM). Ultra-high-resolution images of the surface are presented, as well as lower resolution morphological observations of whole crystals. The pitch of 0.52 nm along the molecule due to the asymmetrical glucose unit and the intermolecular spacing of approximately 0.6 nm are clearly resolved in both imaging environments. The relationship between the crystalline bulk and the surface are discussed, with particular attention being paid to previous crystallographic studies. We also show that the glucose units along the cellulose chains are not topographically equivalent due to the twofold screw symmetry and accordingly present strong evidence of triclinic character by direct surface imaging, rather than by taking average measurements in reciprocal space. The crystallographic distinction between monoclinic and triclinic structure is a displacement of the cellulose chains by a quarter of the c axis period, resulting in either a stagger or a diagonal shifting, respectively, of the cellobiose unit along the chain axis by 0.26 nm. This structural identification (in real space) represents, as far as we are aware, the highest resolution AFM imaging of a biological specimen to date. This study opens up the future possibility of identifying the localized triclinic or monoclinic nature of the Valonia cellulose surface with AFM.

Journal Article↗
PubMed1998

Pilus biogenesis via the chaperone/usher pathway: an integration of structure and function.

The molecular basis of how pathogenic bacteria cause disease has been studied by blending a well-developed genetic system with X-ray crystallography, protein chemistry, high resolution electron microscopy, and cell biology. Microbial attachment to host tissues is one of the key events in the early stages of most bacterial infections. Attachment is typically mediated by adhesins that are assembled into hair-like fibers called pili on bacterial surfaces. This article focuses on the structure-function correlates of P pili, which are produced by most pyelonephritic strains of Escherichia coli. P pili are assembled via a chaperone/usher pathway. Similar pathways are responsible for the assembly of over 30 adhesive organelles in various Gram-negative pathogens. P pilus biogenesis has been used as a model system to elucidate common themes in bacterial pathogenesis, namely, the protein folding, secretion, and assembly of virulence factors. The structural basis for pilus biogenesis is discussed as well as the function and consequences of microbial attachment.

Amino Acid Sequence↗
PubMed2001

Time-resolved hydroxyl-radical footprinting of RNA using Fe(II)-EDTA.

Chemical footprinting methods have been used extensively to probe the structures of biologically important RNAs at nucleotide resolution. One of these methods, hydroxyl-radical footprinting, has recently been employed to study the kinetics of RNA folding. Hydroxyl radicals can be generated by a number of different methods, including Fe(II)-EDTA complexes, synchrotron radiation, and peroxynitrous acid disproportionation. The latter two methods have been used for kinetic studies of RNA folding. We have taken advantage of rapid hydroxyl-radical generation by Fe(II)-EDTA-hydrogen peroxide solutions to develop a benchtop method to study folding kinetics of RNA complexes. This technique can be performed using commercially available chemicals, and can be used to accurately define RNA folding rate constants slower than 6 min(-1). Here we report the method and an example of time-resolved footprinting on the hairpin ribozyme, a small endoribonuclease and RNA ligase.

DNA Footprinting↗