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At least 631 records · Page 35Linked to original sources
PubMed2005

Automated microscope system for determining factors that predict neuronal fate.

Unraveling cause-and-effect relationships in the nervous system is challenging because some biological processes begin stochastically, take a significant amount of time to unfold, and affect small neuronal subpopulations that can be difficult to isolate and measure. Single-cell approaches are slow, subject to user bias, and sometimes too laborious to achieve sample sizes large enough to detect important effects. Here, we describe an automated imaging and analysis system that enables us to follow the fates of individual cells and intracellular proteins over time. Observations can be quantified in a high-throughput manner with minimal user bias. We have adapted survival analysis methods to determine whether and how factors measured during longitudinal analysis predict a particular biological outcome. The ability to monitor complex processes at single-cell resolution quickly, quantitatively, and over long intervals should have wide applications for biology.

Animals↗
PubMed2005

Heptameric (L12)6/L10 rather than canonical pentameric complexes are found by tandem MS of intact ribosomes from thermophilic bacteria.

Ribosomes are universal translators of the genetic code into protein and represent macromolecular structures that are asymmetric, often heterogeneous, and contain dynamic regions. These properties pose considerable challenges for modern-day structural biology. Despite these obstacles, high-resolution x-ray structures of the 30S and 50S subunits have revealed the RNA architecture and its interactions with proteins for ribosomes from Thermus thermophilus, Deinococcus radiodurans, and Haloarcula marismortui. Some regions, however, remain inaccessible to these high-resolution approaches because of their high conformational dynamics and potential heterogeneity, specifically the so-called L7/L12 stalk complex. This region plays a vital role in protein synthesis by interacting with GTPase factors in translation. Here, we apply tandem MS, an approach widely applied to peptide sequencing for proteomic applications but not previously applied to MDa complexes. Isolation and activation of ions assigned to intact 30S and 50S subunits releases proteins S6 and L12, respectively. Importantly, this process reveals, exclusively while attached to ribosomes, a phosphorylation of L12, the protein located in multiple copies at the tip of the stalk complex. Moreover, through tandem MS we discovered a stoichiometry for the stalk protuberance on Thermus thermophilus and other thermophiles and contrast this assembly with the analogous one on ribosomes from mesophiles. Together with evidence for a potential interaction with the degradosome, these results show that important findings on ribosome structure, interactions, and modifications can be discovered by tandem MS, even on well studied ribosomes from Thermus thermophilus.

Amino Acid Sequence↗
PubMed1998

Single-particle selection and alignment with heavy atom cluster-antibody conjugates.

A method is proposed for selecting and aligning images of single biological particles to obtain high-resolution structural information by cryoelectron microscopy. The particles will be labeled with multiple heavy atom clusters to permit the precise determination of particle locations and relative orientations even when imaged close to focus with a low electron dose, conditions optimal for recording high-resolution detail. Heavy atom clusters should also allow selection of images free from many kinds of defects, including specimen movement and particle inhomogeneity. Heavy atom clusters may be introduced in a general way by the construction of "adaptor" molecules based on single-chain Fv antibody fragments, consisting of a constant framework region engineered for optimal cluster binding and a variable antigen binding region selected for a specific target. The success of the method depends on the mobility of the heavy atom cluster on the particle, on the precision to which clusters can be located in an image, and on the sufficiency of cluster projections alone to orient and select particles for averaging. The necessary computational algorithms were developed and implemented in simulations that address the feasibility of the method.

Antibodies↗
PubMed2000

Optical coherence tomography: an introduction to the technique and its use.

This report describes the new optical imaging technique of optical coherence tomography (OCT). OCT is capable of high-resolution, micrometer-scale, cross-sectional imaging of biological tissue. The OCT for ophthalmic application uses 843-nm, near-infrared light, which produces a longitudinal resolution of 10 to 20 microm and a penetration depth of a few millimeters. The scans are displayed in a false color representation scale on which warm colors represent areas of high optical reflectivity and cool colors represent areas of minimal or no reflectivity. A cross-sectional view similar to a histology section is obtained. The cornea, iris, and lens may be visualized as well as the retina and optic nerve. OCT has been used to investigate several ocular diseases. These include macular disease, genetic retinal disease, retinal detachment and retinoschisis, choroidal tumors, optic nerve disorders, and glaucoma.

Diagnostic Techniques, Ophthalmological↗
PubMed2005

Eddy variability east of Madagascar.

A strong but variable western boundary current flows south along the east coast of Madagascar, and at the island's southern end it interacts with eddies propagating zonally from the east. These two routes of variability are compared using altimetric sea-surface-height data and dynamic height from a high-resolution numerical model. The effects on biological productivity are also discussed.

Journal Article↗
PubMed2002

Lipid-derived mediators in endogenous anti-inflammation and resolution: lipoxins and aspirin-triggered 15-epi-lipoxins.

It is well appreciated that lipid-derived mediators play key roles in inflammation and many other physiologic responses where multicellular processes are involved. Among them, lipoxins (LX) and aspirin-triggered LX (ATL) evoke actions of interest in a range of physiologic and pathophysiologic processes, and these two series have emerged as founding members of the first class of lipid/chemical mediators "switched on" in the resolution phase of an inflammatory reaction. These unique compounds possess a trihydroxytetraene structure and are both structurally and functionally distinct among the many groups of lipid-derived bioactive mediators. LXA4 and 15-epi-LXA4 (a member of the ATL series) display leukocyte-selective actions that enable them to serve as endogenous "stop signals" in multicellular events in that they modulate adherence, transmigration, and chemotaxis. Both LXA4 and 15-epi-LXA elicit these responses via a G protein-coupled receptor (GPCR), termed ALXR, identified in human and murine tissues. Among eicosanoids, ALXR is stereoselective for LXA4 (5S,6R,15S-trihydroxy-7,9,13- trans-11-cis-eicosatetraenoic acid). Its aspirin-triggered 15 R epimer (15-epi-LXA4) and their bioactive stable analogs act in the subnanomolar to nanomolar range in human cellular systems and murine models of acute inflammation and reperfusion. ALXR also has the ability to interact with a wide panel of small peptides that give different signaling responses in vitro than LXA4 or its analogs, suggesting that ALXR is capable of serving as a multirecognition receptor in immune responses. Characterization of ALXR and development of metabolically stable LX and ATL analogs that are mimetics rapidly advanced our appreciation of the mechanism of LX actions and the potential utility of these counter-regulatory biocircuits in the quest to control local inflammatory events. In this on-line update, LX and ATL biosynthesis and the LXA4 specific receptor, termed ALXR, are reviewed with a focus on their roles in inflammation and resolution with respect to pharmacology, molecular biology, and signal transduction in several cell types and animal models investigated thus far.

Animals↗
PubMed2005

Production and X-ray crystallographic analysis of fully deuterated cytochrome P450cam.

Neutron protein crystallography allows H-atom positions to be located in biological structures at the relatively modest resolution of 1.5-2.0 A. A difficulty of this technique arises from the incoherent scattering from hydrogen, which considerably reduces the signal-to-noise ratio of the data. This can be overcome by preparing fully deuterated samples. Efficient protocols for routine and low-cost production of in vivo deuterium-enriched proteins have been developed. Here, the overexpression and crystallization of highly (>99%) deuterium-enriched cytochrome P450cam for neutron analysis is reported. Cytochrome P450cam from Pseudomonas putida catalyses the hydroxylation of camphor from haem-bound molecular O(2) via a mechanism that is thought to involve a proton-shuttle pathway to the active site. Since H atoms cannot be visualized in available X-ray structures, neutron diffraction is being used to determine the protonation states and water structure at the active site of the enzyme. Analysis of both hydrogenated and perdeuterated P450cam showed no significant changes between the X-ray structures determined at 1.4 and 1.7 A, respectively. This work demonstrates that the fully deuterated protein is highly isomorphous with the native (hydrogenated) protein and is appropriate for neutron protein crystallographic analysis.

Camphor 5-Monooxygenase↗
PubMed2006

MUMBO: a protein-design approach to crystallographic model building and refinement.

In recent years, significant progress has been achieved in automation of the crystal structure-determination process. However, the final part of this process, namely the refinement of the atomic model, is still tedious for biological macromolecules because, especially at lower resolution, it requires extensive manual intervention. Here, it is shown that computer algorithms widely used in protein-design approaches can substantially simplify this process, helping to identify the correct orientation of the side chains during refinement. This approach was implemented into the computer program MUMBO. As in many protein-design programs, side-chain rotamer diversity is generated using rotamer libraries. The selection of the best combination of side-chain orientations is based on either the dead-end elimination (DEE) theorem or a Metropolis Monte Carlo approach and on a detailed atomic scoring function that describes the molecular interactions between the rotamers. We show that this scoring function can be easily extended and complemented through the introduction of an X-ray pseudo-energy calculated from the electron density present at the position of the rotamer. This extension is fully compatible with present protein-design algorithms and it is shown for a number of test cases that using this approach model refinement is simplified and convergence occurs faster.

Algorithms↗
PubMed2006

Automatic recovery of the optic nervehead geometry in optical coherence tomography.

Optical coherence tomography (OCT) uses retroreflected light to provide micrometer-resolution, cross-sectional scans of biological tissues. OCT's first application was in ophthalmic imaging where it has proven particularly useful in diagnosing, monitoring, and studying glaucoma. Diagnosing glaucoma is difficult and it often goes undetected until significant damage to the subject's visual field has occurred. As glaucoma progresses, neural tissue dies, the nerve fiber layer thins, and the cup-to-disk ratio increases. Unfortunately, most current measurement techniques are subjective and inherently unreliable, making it difficult to monitor small changes in the nervehead geometry. To our knowledge, this paper presents the first published results on optic nervehead segmentation and geometric characterization from OCT data. We develop complete, autonomous algorithms based on a parabolic model of cup geometry and an extension of the Markov model introduced by Koozekanani, et al. to segment the retinal-nervehead surface, identify the choroid-nervehead boundary, and identify the extent of the optic cup. We present thorough experimental results from both normal and pathological eyes, and compare our results against those of an experienced, expert ophthalmologist, reporting a correlation coefficient for cup diameter above 0.8 and above 0.9 for the disk diameter.

Algorithms↗
PubMed2001

Detection of tumorigenesis in rat bladders with optical coherence tomography.

Optical coherence tomography (OCT) is a novel technique that enables noninvasive cross-sectional imaging of biological tissues. Because of its high resolution (approximately 10 microm), superior dynamic range (140 dB in our case) and up to 2-3 mm penetration depth, OCT is potentially useful for noninvasive screening of superficial lesions. Bladder cancer arises within the transitional epithelium. Despite the ability to visualize the epithelium via cystoscopy, it is often difficult to detect early epithelial cancers and to determine their penetration to the underlying layers. To investigate the potential of OCT to enhance imaging of bladder cancers and other epithelial lesions, we applied OCT to normal and diseased bladder epithelium, and correlated the results with histological findings. OCT images of porcine bladder (a close homolog of human bladder) confirm the ability of this method to image human tissues. To determine whether OCT can track the course of bladder cancer, a standard rat model of bladder cancer in which Fisher rats are exposed to methyl-nitroso-urea (MNU), was followed both with OCT and histological studies. Our results show that the micro morphology of porcine bladder such as the urothelium, submucosa and muscles is identified by OCT and well correlated with the histological evaluations. OCT detected edema, inflammatory infiltrates, and submucosal blood congestion as well as the abnormal growth of urothelium (e.g., papillary hyperplasia and carcinomas). By contrast, surface imaging, which resembles cystoscopy, provided far less sensitivity and resolution than OCT. This is the first OCT study of any tumor documented in a systematic fashion, and the results suggest the potential of OCT for the noninvasive diagnosis of both bladder inflammatory lesions and early urothelial abnormalities, which conventional cystoscopy often misses, by imaging characterization of the increases in urothelial thickening and backscattering. However, because of the depth limitation, OCT may have limited applications in staging the invasion of higher-state urothelial cancers, especially for papillary carcinomas.

Animals↗
PubMed1972

Membrane-associated replication complex in arbovirus infection.

Cytoplasmic extracts of chicken embryo fibroblast cells infected with Semliki Forest virus were subjected to isopycnic centrifugation in discontinuous sucrose gradients. Seven distinct bands were usually formed. The four upper bands contained predominantly smooth membranes and the lowest band was enriched in rough endoplasmic reticulum. One fraction (fraction 5), banding at a density of 1.16 g/cm(3), was found to be heavily enriched in pulse-labeled ribonucleic acid (RNA), viral RNA polymerase, and viral RNA forms associated with RNA replication. Thus, fraction 5 evidently contained a membrane-associated viral replication complex of a type previously defined in picornavirus infections. Fraction 5 was also consistently enriched with unique membranous structures previously observed in intact cells as type 1 cytopathic vacuoles (CPV-1). When the CPV-1 in fraction 5 were isolated from cells briefly incubated with (3)H-uridine and (3)H-adenosine prior to cell disruption, a large proportion was found to be labeled by high-resolution autoradiography. Thus, ultrastructural, biochemical, and biological evidence were all consistent with the interpretation that the CPV-1 membranes represent a significant element of the viral replication complex.

Adenosine↗
PubMed1985

Hepatic copper distribution in primary biliary cirrhosis shown by the scanning proton microprobe.

A number of conditions are associated with abnormalities of trace metal handling by the liver. We report the application of the Oxford scanning proton microprobe to the analysis of hepatic copper in one such condition, primary biliary cirrhosis. The scanning proton microprobe analyses conventional tissue sections (5-10 micron thickness) and produces simultaneous elemental distribution maps of biologically relevant elements with a spatial resolution of 1 micron and a detection limit better than 1 ppm. We have confirmed the localisation of excess copper to periportal areas and suggest that such accumulation is confined to a proportion of periportal hepatocytes. We have also shown a close spatial correlation between regions of copper accumulation and areas of high sulphur concentration. The copper to sulphur ratio in these areas is consistent with their identity as aggregates of copper loaded metallothionein, and the scanning proton microprobe was further able to show that the aggregates contain less than 30 ppm zinc.

Computers↗
PubMed1995

Biological atomic force microscopy: from microns to nanometers and beyond.

In recent years, atomic force microscopy (AFM) has become the most rapidly developing imaging method. We summarize its recent applications in structural biology, with emphasis on high-resolution imaging, and illustrate these applications with images of cell membranes, DNA, and soluble and membrane proteins. With present technology, and at room temperature, nanometer resolution of DNA and soluble proteins is achievable, although the resolution attained on cell surfaces is more limited (10-50 nm). We suggest that high-resolution imaging of cell surfaces and very high (sub-nanometer) resolution molecular imaging requires an approach that increases specimen rigidity. Operation at cryogenic temperatures, the most promising approach, is discussed, and a cryo-AFM designed for operating at ambient pressure and liquid nitrogen temperature is described. The results include high-resolution images and evidence of increased molecular rigidity at low temperatures.

Animals↗
PubMed2002

The Feline Genome Project.

The compilation of a dense gene map and eventually a whole genome sequence (WGS) of the domestic cat holds considerable value for human genome annotation, for veterinary medicine, and for insight into the evolution of genome organization among mammals. Human association and veterinary studies of the cat, its domestic breeds, and its charismatic wild relatives of the family Felidae have rendered the species a powerful model for human hereditary diseases, for infectious disease agents, for adaptive evolutionary divergence, for conservation genetics, and for forensic applications. Here we review the advantages, rationale, and present strategy of a feline genome project, and we describe the disease models, comparative genomics, and biological applications posed by the full resolution of the cat's genome.

Animals↗
PubMed1999

Probing Nanometer Structures with Atomic Force Microscopy.

Atomic force microscopy (AFM) can generate high-resolution images of the surface of biological specimens and can also probe the interactions between and within single macromolecules. Thus isolated heterogeneous biological structures can be studied in submolecular detail with AFM.

Journal Article↗
PubMedDate not supplied

Cholinergic and purinergic neurohumoral signalling in the inner ear: a molecular physiological analysis.

The ability to identify the expression of the protein subunits which assemble to form ionotropic receptors for acetylcholine and extracellular adenosine 5'-triphosphate (ATP) in individual cells of the inner ear provides examples of the high resolution and exquisite sensitivity which molecular biology brings to the study of hearing and balance. The data from these studies provide both fine detail with respect to the classification of the elements involved and an overview of the sites of potential interaction of both extracellular and intracellular signalling pathways. The high sensitivity necessitates a molecular physiological approach when using these techniques so that these data on the site and extent of expression can be balanced against functional significance. With the demonstration of expression of the alpha 9 subunit of the nicotinic acetylcholine receptor in cochlear outer hair cells, molecular biology has provided an explanation for the unusual cholinergic receptor pharmacology of the olivocochlear efferent innervation which has confounded investigators for decades. In addition, a role for extracellular ATP as a signalling molecule regulating electrochemical gradients and neurotransmission within the inner ear is supported by the extent of P2 receptor expression in this tissue, data which beg for intense functional study.

Adenosine Triphosphate↗
PubMed2005

Structural characterization of complex bacterial glycolipids by Fourier transform mass spectrometry.

Bacterial glycolipids are complex amphiphilic molecules which are on the one hand of utmost importance for the organization and function of bacterial membranes, and which on the other hand play a major role in the activation of cells of the innate and adaptive immune system of the host. Already small alterations of their chemical structure may influence the biological activity tremendously. Due to their intrinsic biological heterogeneity [number and type of fatty acids, saccharide structures, and substitution with e.g. phosphate (P), 2-aminoethyl- (pyro)phosphate groups (P-Etn) or 4-amino-4-deoxyarabinose (Ara4N)], separation of the different components are a prerequisite for unequivocal chemical and NMR structural analyses. In this contribution the structural information which can be obtained from heterogeneous samples of glycolipids by Fourier transform (FT) ion cyclotron resonance mass spectrometric methods is described. By means of recently analysed complex biological samples the possibilities of high resolution electrospray ionization FT-MS are demonstrated. Capillary skimmer dissociation, as well as tandem mass spectrometry MS/MS analysis utilizing collision-induced dissociation and infrared multiphoton dissociation, are compared and their advantages to provide structural information of diagnostic importance are discussed.

Carbohydrate Sequence↗
PubMed2003

Common-path interferometer for frequency-domain optical coherence tomography.

A Michelson-type spectral interferometer that uses a common beam path for the reference and the sample arms is described. This optical arrangement is more compact and stable than the more commonly used dual-arm interferometer and is well suited for frequency-domain optical coherence tomography of biological samples. With a 16-bit CCD camera, the instrument has sufficient dynamic range and resolution for imaging to depths of 2 mm in scattering biological materials. Images obtained with this spectral interferometer are presented, including cross-sectional images in a Xenopus laevis tadpole.

Abdomen↗