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PubMed1995

Robert Feulgen Prize Lecture 1995. New approaches to in situ detection of nucleic acids.

The present paper reviews recent results obtained by different molecular biology-based, immunocytological approaches to the localization and identification of nucleic acids in sections of biological material. Examples of sensitive, high-resolution detection methods for RNA, DNA or specialized DNA regions are presented. Special emphasis is placed on the potential values and limitations of these new methods.

Animals↗
PubMed1988

Prospects for NMR imaging in the study of biological morphogenesis.

Small objects can be visualised with a spatial resolution that approaches microscopic dimensions using the technique of high resolution nuclear magnetic resonance (NMR) imaging. Some important features of the method are described and the prospects for using the technique to study morphogenesis are discussed. It is concluded that NMR imaging, in conjunction with the related method of localised spectroscopy, is capable of producing novel structural information.

Animals↗
PubMed2002

First- and second-order multivariate calibration applied to biological samples: determination of anti-inflammatories in serum and urine.

First- and second-order multivariate calibration of fluorescence data have been compared as regards the determination of anti-inflammatories and metabolites in the biological fluids serum and urine. The simultaneous resolution of naproxen-salicylic acid mixtures in serum and naproxen-salicylic acid-salicyluric acid mixtures in urine was accomplished and employed for a discussion of the relative advantages of the applied chemometric tools. The analysis of second-order fluorescence excitation-emission matrices was performed using iteratively reweighted generalized rank annihilation method (IRGRAM), parallel factor analysis (PARAFAC), and self-weighted alternating trilinear decomposition (SWATLD). The results were compared with first-order fluorescence emission data analyzed with partial least-squares regression (PLS). In all cases, the performance of the methods was improved through the formation of inclusion complexes of the analytes with beta-cyclodextrin. The concentration ranges in which the analytes could be determined were as follows: naproxen, 0-250 ng mL(-1) in serum and 0-200 ng mL(-1) in urine; salicylic acid, 0-500 ng mL(-1) in serum and 0-300 ng mL(-1) in urine, and salicyluric acid, 0-300 ng mL(-1) in urine.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2005

Human gene therapy and imaging: cardiology.

This review discusses the basics of cardiovascular gene therapy, the results of recent human clinical trials, and the rapid progress in imaging techniques in cardiology. Improved understanding of the molecular and genetic basis of coronary heart disease has made gene therapy a potential new alternative for the treatment of cardiovascular diseases. Experimental studies have established the proof-of-principle that gene transfer to the cardiovascular system can achieve therapeutic effects. First human clinical trials provided initial evidence of feasibility and safety of cardiovascular gene therapy. However, phase II/III clinical trials have so far been rather disappointing and one of the major problems in cardiovascular gene therapy has been the inability to verify gene expression in the target tissue. New imaging techniques could significantly contribute to the development of better gene therapeutic approaches. Although the exact choice of imaging modality will depend on the biological question asked, further improvement in image resolution and detection sensitivity will be needed for all modalities as we move from imaging of organs and tissues to imaging of cells and genes.

Cardiology↗
PubMed1981

Glial-released proteins: II. Two-dimensional electrophoretic identification of proteins regulated by hydrocortisone.

The proteins released into the culture medium (CM) by confluent C6 glioma cell monolayers were analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE). These cells synthesize and release a complex set of proteins which can be resolved on the basis of molecular charge and size. Over 400 spots on fluorograms corresponding to radio-labeled glial-released protein (GRP) were detected and classified according to their positions in 2-D gels. The marked species charge heterogeneity of the 2-D pattern was used as a criterion to assign the majority of GRP components to "series'. Series are composed of families of related proteins or glycoproteins distributed in a line of evenly spaced members in the isoelectric focusing dimension. Long-term exposure of monolayers to 2 microM hydrocortisone influenced the accumulation in the culture medium of half of the classified GRP species. Five classes of GRP were identified based on their steroid-responsiveness as seen in the deviation of GRP radiolabel ratios from control and hormone-treated culture CM. Hormonal response, verified by reverse-label experiments, showed a number of GRP in CM are either consistently increased 1- to 7- fold (Classes I and II) or decreased 1- to 3-fold (Classes IV and V). The remaining GRP (Class III) included those proteins which were found to be uninfluenced or to change in a week or inconsistent manner. Some GRP series were coordinately induced while other series gave graded responses. These results represent the first high-resolution classification of GRP by physical and biological properties.

Clone Cells↗
PubMed1987

Preparative liquid chromatography of carbohydrates: mono- and di-saccharides, uronic acids, and related derivatives.

The general principles and practical aspects of preparative high-performance liquid chromatography (l.c.) of mono- and di-saccharides, sugars acids, lactones, and N-acetylated amino sugar derivatives are described. Milligram to gram quantities of these carbohydrates were isolated on semi-preparative (0.78 X 30 cm) or preparative (approximately 2.0 X 30 cm) columns packed with aminopropyl silica gel provided better resolution of individual mono- and di-saccharides, but columns of cation-exchange resin had higher capacity and were more durable and economical to use. Preparative, cation-exchange columns were operated at flow rates of less than 5 mL/min and pressures of approximately 1-2 MPa, allowing them to be used on unmodified analytical l.c. systems. Details are given for the efficient packing, use, and care of these columns, and on the effects of column selectivity, packing technique, and sample size on chromatographic resolution. Isolation of naturally occurring sugars from biological sources on a laboratory-packed column is described.

Carbohydrates↗
PubMed1993

Study of polychlorinated dibenzodioxins and furans from municipal waste incinerator emissions in The Netherlands: analytical methods and levels in the environment and human food chain.

An overview is given of the methods that have been used in the study of polychlorodibenzo-p-dioxins and polychlorodibenzofurans in agriculture and the human food chain in a national survey and monitoring programme, including sampling strategies, sampling in the field and clean-up and analysis in various biological and environmental samples by high-resolution gas chromatography-high resolution mass spectrometry. The quality of data was evaluated as a result of internal quality control protocols and participation in interlaboratory comparison studies. Statistical analysis techniques and modelling were applied in order to compare and relate congener profiles in various matrices and to evaluate levels found in field studies for their use for regulatory purposes.

Air Pollutants↗
PubMed1989

Visualization of alpha-helices in tobacco mosaic virus by cryo-electron microscopy.

We have used tobacco mosaic virus (TMV) as a test specimen, in order to develop techniques for the analysis of high-resolution structural detail in electron micrographs of biological assemblies with helical symmetry. It has previously been shown that internal details of protein structure can be visualized by processing electron micrographs of unstained specimens of extended two-dimensional crystalline arrays. However, the techniques should in principle be applicable to other periodic specimens, such as assemblies with helical symmetry. We show here that data to spacings better than 10 A can be retrieved from electron images of frozen hydrated TMV. The three-dimensional computed map agrees well with that derived from X-ray diffraction and shows the two pairs of alpha-helices forming the core of the coat subunit, the C alpha-helix and the viral RNA. The results demonstrate that it is possible to determine detailed internal structure in helical particles.

Freezing↗
PubMed1994

Identification and separation of five cephalosporins by micellar electrokinetic capillary chromatography.

The cephalosporins are a group of structurally related, broad spectrum beta-lactam antibiotics isolated from the mold Cephalosporium. Methods of analysis of cephalosporin antibiotics include microbiological, titrimetric and chromatographic assays. Chromatographic techniques, including high-performance liquid chromatography, have been extensively utilized for specific and sensitive assays of beta-lactam antibiotics in a variety of matrices, i.e. clinical and pharmaceutical. Several of the drawbacks of HPLC in the analysis of cephalosporins in food and biological samples include matrix interferences and low resolution due to column adsorption. Recently, the applicability of capillary electrophoresis to the resolution of beta-lactam antibiotics has been demonstrated in the literature. In this paper we employed sodium borate and an anionic surfactant, sodium dodecyl sulfate (SDS), in a separation technique called micellar electrokinetic capillary chromatography with UV detection, to resolve a mixture of five cephalosporins--cefuroxime, cephalexin, cephapirin, cefamandole nafate and cephalothin. The presence of SDS in the running buffer above the critical micelle concentration, creates a pseudostationary phase enabling high-efficiency chromatographic separations. The effect of the ion-pairing reagent, pentanesulfonic acid sodium salt, on the resolution of the cephalosporin mixture in conjunction with SDS was also examined.

Cephalosporins↗
PubMed1994

A new method to measure bilayer thickness: cryo-electron microscopy of frozen hydrated liposomes and image simulation.

A new method to measure the bilayer thickness of liposomes is described. Frozen hydrated liposomes composed of either phosphatidylcholine (PC) or PC containing 30 mol% cholesterol were observed in vitreous ice with a cryo-electron microscope, which enables high-resolution observation of frozen-hydrated unstained biological specimens. The examined PCs were di-(saturated acyl) PCs (di-C12:0 (DL), diC14:0 (DM), di-C16:0 (DP)), unsaturated acyl PCs (diC18:1 (DO), C16:0-C18:1 (PO)) and PC from a natural source (egg-yolk (EY) PC). Every liposome image displayed a pair of concentric circles indicating a different bilayer structure due to the acyl-chain species. The observation of the liposomes showed the following results; (1) liposomes including unsaturated acyl PC (DOPC, POPC and EYPC) displayed a smooth and regular image of the bilayer and sharp distributions of the bilayer thickness, while the images of those composed of di-(saturated acyl) PC (DLPC, DMPC and DPPC) showed irregular shapes and wide distributions of the bilayer thickness, (2) addition of 30 mol% cholesterol to the di-(saturated acyl) PCs dispelled the irregularity in the shape of the membrane and the distribution of the bilayer thickness, and (3) the mean bilayer thickness of the liposome composed of di-(saturated acyl) PCs increased corresponding to the carbon numbers of the acyl chains. The reliability of the observations and measurements was confirmed by coupling of the cryo-electron microscopy with image simulation using the MULTI SLICE computer program and the error estimation of +/- 0.63 A using images of crystalline chlorinated copper phthalocyanine.

Computer Simulation↗
PubMed2004

A dynamic phase microscopic study of optical characteristics of individual chloroplasts.

Dynamic phase microscopy (DPM) allows the monitoring of optical path difference (or phase height), h(x,y,t) approximately integraln(x,y,z,t)dz, an integral refractive index projection of the medium, n(x,y,z,t), in optically transparent biological specimens at high spatial and temporal resolutions. In this study, DPM was used for the analysis of fluctuations in the optical characteristics of individual bean chloroplasts in various metabolic states. A "phase image" of an individual chloroplast, which represents a three-dimensional plot of the "phase height", was obtained for the first time, and the frequency spectra of the fluctuations of h(x,y,t) were investigated. The fluctuation patterns, i.e., the intensity and the frequency spectra of phase height fluctuations in bean chloroplasts (Class B) were found to depend on their metabolic state. Under conditions of noncyclic (or pseudocyclic) electron transport, the fluctuations displayed characteristic frequencies in the range of 0.25-0.6 Hz and were space-time-correlated in the chloroplast domains with the cross sizes of approximately 2 microm. The fluctuation intensity decreased in the presence of uncouplers (nigericin and valinomycin, 20 microM). A stronger (in comparison with 20 microM valinomycin) effect of 20 microM nigericin suggests that the light-induced generation of the transmembrane pH difference (DeltapH) makes the main contribution to the increment of space-correlated fluctuations of h(x,y,t). Studies of chloroplasts incubated in media of various osmolarity (50-500 mM sucrose) have shown that structural changes in thylakoids are among other factors responsible for phase height fluctuations.

Chloroplasts↗
PubMed2005

Crystallographic studies on two bioisosteric analogues, N-acetyl-beta-D-glucopyranosylamine and N-trifluoroacetyl-beta-D-glucopyranosylamine, potent inhibitors of muscle glycogen phosphorylase.

Structure-based inhibitor design has led to the discovery of a number of potent inhibitors of glycogen phosphorylase b (GPb), N-acyl derivatives of beta-D-glucopyranosylamine, that bind at the catalytic site of the enzyme. The first good inhibitor in this class of compounds, N-acetyl-beta-D-glucopyranosylamine (NAG) (K(i) = 32 microM), has been previously characterized by biochemical, biological and crystallographic experiments at 2.3 angstroms resolution. Bioisosteric replacement of the acetyl group by trifluoroacetyl group resulted in an inhibitor, N-trifluoroacetyl-beta-D-glucopyranosylamine (NFAG), with a K(i) = 75 microM. To elucidate the structural basis of its reduced potency, we determined the ligand structure in complex with GPb at 1.8 angstroms resolution. To compare the binding mode of N-trifluoroacetyl derivative with that of the lead molecule, we also determined the structure of GPb-NAG complex at a higher resolution (1.9 angstroms). NFAG can be accommodated in the catalytic site of T-state GPb at approximately the same position as that of NAG and stabilize the T-state conformation of the 280 s loop by making several favourable contacts to Asn284 of this loop. The difference observed in the K(i) values of the two analogues can be interpreted in terms of subtle conformational changes of protein residues and shifts of water molecules in the vicinity of the catalytic site, variations in van der Waals interaction, and desolvation effects.

Crystallography↗
PubMed2005

Short order nanohole arrays in metals for highly sensitive probing of local indices of refraction as the basis for a highly multiplexed biosensor technology.

A small array of subwavelength apertures patterned in a gold film on glass was characterized for use as a biosensor. It is widely believed that such arrays allow the resonance of photons with surface plasmons in the metallic film. Surface plasmon methods (and other evanescent wave methods) are extremely well suited for the measure of real time biospecific interactions. An extremely high sensitivity of 88,000%/refractive index unit was measured on an array with theoretical active area of .09 microm2. The formation of a biological monolayer was monitored. Both sensitivity and resolution were determined through measurement. The measured resolution, for a sensor with an active area of less than 1.5 microm2, is 9.4 x 10(-8) refractive index units which leads to a calculated sensitivity of 3.45E6%/refractive index unit. These values far exceed theoretical and calculated values of other grating coupled surface plasmon resonance (SPR) detectors and prism based SPR detectors. Because the active sensing area can be quite small (.025 microm2) single molecule studies are possible as well as massive multiplexing on a single chip format.

Biosensing Techniques↗
PubMed2003

AFM characterization of tilt and intrinsic flexibility of Rhodobacter sphaeroides light harvesting complex 2 (LH2).

Atomic force microscopy (AFM) has developed into a powerful tool to investigate membrane protein surfaces in a close-to-native environment. Here we report on the surface topography of Rhodobacter sphaeroides light harvesting complex 2 (LH2) reconstituted into two-dimensional crystals. These photosynthetic trans-membrane proteins formed cylindrical oligomeric complexes, which inserted tilted into the lipid membrane. This peculiar packing of an integral membrane protein allowed us to determine oligomerization and tilt of the LH2 complexes, but also protrusion height and intrinsic flexibility of their individual subunits. Furthermore the surface contouring reliability and limits of the atomic force microscopy could be studied. The two-dimensional crystals examined had sizes of up to 5 microm and, as revealed by a 10 A cryo electron microscopy projection map, p22(1)2(1) crystal symmetry. The unit cell had dimensions of a = b = 150 A and gamma = 90 degrees, and housed four nonameric complexes, two pointing up and two pointing down. AFM topographs of these 2D crystals had a lateral resolution of 10 A. Further, the high vertical resolution of approximately 1 A, allowed the protrusion height of the cylindrical LH2 complexes over the membrane to be determined. This was maximally 13.1 A on one side and 3.8 A on the other. Interestingly, the protrusion height varied across the LH2 complexes, showing the complexes to be inserted with a 6.2 degree tilt with respect to the membrane plane. A detailed analysis of the individual subunits showed the intrinsic flexibility of the membrane protruding peptide stretches to be equal and independent of their protrusion height. Furthermore, our analysis of membrane proteins within this peculiar packing confirmed the high vertical resolution of the atomic force microscopy on biological samples, and led us to conclude that the image acquisition function was equally accurate for contouring protrusions with heights up to approximately 15 A.

Amino Acid Sequence↗
PubMed1996

Imaging developing neural morphology using optical coherence tomography.

Imaging technologies offer numerous possibilities to investigate the processes involved in neural development. The optical coherence tomography (OCT) technology is analogous to ultrasound backscatter microscopy except reflections of light are detected rather than sound. The OCT technology combines high-resolution in vivo imaging in a diode-based benchtop instrument capable of micron-scale resolution in transparent and non-transparent biological specimens. In this paper, we examine the potential of using OCT for the investigation of developing neural morphology. To demonstrate the capabilities of this technique in assessing neural development, we have chosen to image early normal and abnormal neural morphology in a common developmental biology model, Xenopus laevis. In vivo images clearly identify gross and subtle differences in neural structure and may offer an alternative to the costly and time-consuming process of repeated histological preparation for neural developmental studies. Because imaging can be performed rapidly and repeatedly, the morphological changes of single specimens can be followed throughout development. To illustrate the future potential of this technique, a state-of-the-art Cr4+:forsterite modelocked laser is used as a broad bandwidth light source to image individual cells in a developing specimen.

Animals↗
PubMed1998

Mass spectrometry of peptides in neuroscience.

This review focuses on the contributions of modern mass spectrometry to neuropeptide research. An introduction to newer mass spectrometric techniques is provided. Also, the use of mass spectrometry in combination with high-resolution separation techniques for neuropeptide identification in biological samples is illustrated. The amino acid sequence information that is important for the identification and analysis of known, novel, or chemically modified neuropeptides may be obtained using mass spectrometric techniques. Because mass spectrometry techniques can be used to reflect the dynamic properties associated with neuropeptide processing in biological systems, they may be used in the future to monitor peptide profiles within organisms in response to environmental challenges such as disease and stress.

Mass Spectrometry↗
PubMedDate not supplied

Declarative simulation of dynamicals systems: the 812 programming language and its application to the simulation of genetic networks.

A major part of biological processes can be modeled as dynamical systems (DS), that is, as a time-varying state. In this article, we advocate a declarative approach for prototyping the simulation of DS. We introduce the concepts of collection, stream and fabric. A fabric is a multi-dimensional object that represents the successive values of a structured set of variables. A declarative programming language, called 8 1/2 has been developed to support the concept of fabrics. Several examples of working 8 1/2 programs are given to illustrate the relevance of the fabric data structure for simulation applications and to show how recursive fabric definitions can be easily used to model various biological phenomena in a natural way (a resolution of PDE, a simulation in artificial life, the Turing diffusion-reaction process and various examples of genetic networks). In the conclusion, we recapitulate several lessons we have learned from the 8 1/2 project.

Programming Languages↗
PubMed1997

[Tuberculous pneumopathy in the course of cystic fibrosis].

BACKGROUND: Tuberculosis is rarely seen in patients with cystic fibrosis. CASE REPORT: A 14-year old female adolescent, regularly followed for a well-tolerated form of cystic fibrosis, developed an acute respiratory infection with consolidation of the left inferior lobe, and no response to the usual antibiotic treatment of cystic fibrosis. Mycobacterium tuberculosis was found in aspirate by fibroscopy, on Loewenstein medium. No familial or social infection contact were identified. Antituberculous chemotherapy with three drugs brought about a prompt improvement of sytemic signs, weight gain, resolution of pulmonary foci and sedation of biological findings referable to inflammation. CONCLUSION: This case report reminds us that tuberculosis may occur in cystic fibrosis patients. Loewenstein cultures should routinely be made when faced with an unexplainable worsening of the condition.

Adolescent↗