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PubMed2005

Cytochrome P450: nature's most versatile biological catalyst.

The author describes studies that led to the resolution and reconstitution of the cytochrome P450 enzyme system in microsomal membranes. The review indicates how purification and characterization of the cytochromes led to rigorous evidence for multiple isoforms of the oxygenases with distinct chemical and physical properties and different but somewhat overlapping substrate specificities. Present knowledge of the individual steps in the P450 and reductase reaction cycles is summarized, including evidence for the generation of multiple functional oxidants that may contribute to the exceptional diversity of the reactions catalyzed.

Animals↗
PubMed2005

Recombinant human activated protein C upregulates cyclooxygenase-2 expression in endothelial cells via binding to endothelial cell protein C receptor and activation of protease-activated receptor-1.

Prostacyclin (PGI(2)) has beneficial cytoprotective properties, is a potent inhibitor of platelet aggregation and has been reported to improve microcirculatory blood flow during sepsis. The formation of PGI(2) in response to proinflammatory cytokines is catalysed by the inducible cyclooxygenase (COX) isoform COX-2. Recombinant human activated protein C (rhAPC, drotrecogin alfa (activated)) was shown to have multiple biological activities in vitro and to promote resolution of organ dysfunction in septic patients. Whether rhAPC exerts its beneficial effects by modulating prostanoid generation is unknown up to now. It was therefore the aim of the study to examine the in vitro effect of rhAPC on COX-2-mRNA-expression and PGI(2) release from human umbilical vein endothelial cells (HUVEC). We found that rhAPC, at supra-therapeutical concentrations (500 ng/ml-20 microg/ml), upregulated the amount of COX-2-mRNA in HUVEC at t=3-9 h and caused a time- and dose-dependent release of 6-keto PGF(1 alpha), the stable hydrolysis product of prostacyclin. RhAPC further increased the stimulating effect of tumor necrosis factor-alpha (TNF-alpha) and thrombin on COX-2-mRNA-levels. Transcript levels of cyclooxygenase-1 (COX-1) and prostaglandin 12 synthase, however, were unaffected by the stimulation with rhAPC or thrombin. The upregulatory effect on COX2-mRNA levels was specific for rhAPC since the zymogen protein C in equimolar concentrations had no effect on COX-2-mRNA-levels or 6-keto PGF(1 alpha)-release. Western Blot analysis revealed an increase of COX-2-protein content in HUVEC after treatment with rhAPC. As shown by experiments using monoclonal antibodies against the thrombin receptor PAR-1 (mAb=ATAP2) and against the endothelial protein C receptor (EPCR; mAb=RCR-252), the effect of rhAPC on COX-2-mRNA upregulation was mediated by binding to the EPCR-receptor and signaling via PAR-1. These results demonstrate that induction of COX-2-expression is an important response of HUVEC to stimulation with rhAPC and may represent a new molecular mechanism, by which rhAPC promotes upregulation of prostanoid production in human endothelium.

Blood Coagulation Factors↗
PubMed2005

A specific role of integrin Mac-1 in accelerated macrophage efflux to the lymphatics.

In response to injury, monocytes migrate to the site of inflammation, where they differentiate into macrophages and participate in various biologic processes. However, their fate during the resolution of acute inflammation is not fully understood. Here, we show that inflammatory macrophages do not die locally by apoptosis; rather, they migrate across the peritoneal mesothelium to the lymphatics, through which they further migrate to the lymph nodes and to the blood circulation. Macrophage efflux is enhanced considerably on cell activation, and such accelerated macrophage migration is dependent specifically on integrin Mac-1, and can be blocked by addition of its antagonist. Thus, genetic inactivation of Mac-1 in mice inhibits the accelerated macrophage efflux from the inflammatory site to the lymphatics, but it does not compromise the accumulation of blood monocytes into the inflammatory site. Together, our study demonstrates that Mac-1 is involved specifically in the efflux of activated macrophages to the lymphatics, suggesting that Mac-1 may play an important role in the removal of local inflammatory macrophages and in their subsequent migration to the lymph nodes, a process that is critical to the development of the adaptive immunity.

Animals↗
PubMed2004

Thin is better!: ultrathin cryosection immunocytochemistry.

In immunofluorescence microscopy (IFM), the repression of out of focus fluorescence signal is crucial in order to obtain high-resolution images. One option to acquire high vertical resolution (z-axis resolution) is to produce optical sections with a confocal microscope. The z-axis resolution usually obtained with confocal microscopy of biological samples is about 500 nm. Another option is to produce very thin sections with a cryo-ultramicrotome (physical sections). The ultrathin cryosections we employ are about 100 nm in thickness: thus all of the fluorescence must come from within this 100 nm thickness. The use of ultrathin cryosections permits the acquisition of extremely high-quality images and minimizes the possibility for false localization in IFM (Fig. 1). Ultrathin cryosections can be applied to immunoelectron microscopy (IEM) as well as IFM (Fig. 2). We show new methods of ultrathin cryosection immunocytochemistry(1-3). Human full-term placentas were fixed with 4% paraformaldehyde, solidified with 10% gelatin, infiltrated with 2.3 M sucrose, and then frozen in liquid nitrogen. Ultrathin cryosections were cut with a cryo-ultramicrotome and then transferred to glass cover slips for IFM or to nickel grids for IEM. Cryosections were incubated with mouse anti-p230, a trans-Golgi network marker, and subsequently incubated with Alexa 488-labeled goat anti-mouse IgG or with goat anti-mouse 5-nm colloidal gold particles. For visualization and preservation of ultrastructure of cryosections at the electron microscopic level, the sections on grids were postfixed with ferrocyanide-reduced osmium and then stained with uranyl acetate and lead citrate in polyvinyl alcohol(1). Ultrathin cryosection immunocytochemistry should be an important technique for functional genomics research, especially for the analysis of the in situ expression of target molecules(2,3).

Cryoultramicrotomy↗
PubMed2025

The challenge of chromatin model comparison and validation: A project from the first international 4D Nucleome Hackathon.

The computational modeling of chromatin structure is highly complex due to the hierarchical organization of chromatin, which reflects its diverse biophysical principles, as well as inherent dynamism, which underlies its complexity. Chromatin structure modeling can be based on diverse approaches and assumptions, making it essential to determine how different methods influence the modeling outcomes. We conducted a project at the NIH-funded 4D Nucleome Hackathon on March 18-21, 2024, at The University of Washington in Seattle, USA. The hackathon provided an amazing opportunity to gather an international, multi-institutional and unbiased group of experts to discuss, understand and undertake the challenges of chromatin model comparison and validation. Here we give an overview of the current state of the 3D chromatin field and discuss our efforts to run and validate the models. We used distance matrices to represent chromatin models and we calculated Spearman correlation coefficients to estimate differences between models, as well as between models and experimental data. In addition, we discuss challenges in chromatin structure modeling that include: 1) different aspects of chromatin biophysics and scales complicate model comparisons, 2) large diversity of experimental data (e.g., population-based, single-cell, protein-specific) that differ in mathematical properties, heatmap smoothness, noise and resolutions complicates model validation, 3) expertise in biology, bioinformatics, and physics is necessary to conduct comprehensive research on chromatin structure, 4) bioinformatic software, which is often developed in academic settings, is characterized by insufficient support and documentation. We also emphasize the importance of establishing guidelines for software development and standardization.

Chromatin↗
PubMed2002

Effect of dexamethasone on the metabonomics profile associated with phosphodiesterase inhibitor-induced vascular lesions in rats.

Metabonomics is the evaluation of the multiparametric metabolic response of biological systems to pathophysiological stimuli. High-resolution nuclear magnetic resonance (NMR) spectroscopy of biofluids coupled with pattern recognition-based chemometric analysis is an emerging approach to the study of metabonomics and may be used for the prediction of toxicity in vivo and for identification of surrogate markers of toxicity. Previously, we established that metabonomic analysis of urine samples has significant potential for identification of phosphodiesterase type 4 (PDE-4) inhibitor-induced vascular lesions in rats. It was not clear, however, whether the observed changes in metabonomics profile were related mechanistically to the pathogenesis of these vascular lesions or whether these changes were reflective primarily of the ensuing inflammatory response. In the present study, dexamethasone was used to suppress inflammation associated with vascular lesions induced in rats by the PDE-4 inhibitor CI-1018 and urine samples were evaluated for resultant changes in metabonomic profile. Female Wistar rats were given CI-1018 by gavage at 750 mg/kg with or without concurrent intraperitoneal administration of dexamethasone at 1 mg/kg for 4 days. Dexamethasone induced a characteristic lymphoid depletion and lymphocytolysis but no evidence of vascular lesions. Rats dosed with CI-1018 had mild vascular changes in liver and/or marked vascular lesions in mesentery characterized by medial necrosis, hemorrhage, and/or edema accompanied by perivascular mixed inflammatory cell infiltrates. Inflammatory infiltrates associated with these lesions were eliminated in rats given dexamethasone, yet minimal medial smooth muscle necrosis and degeneration still occurred, suggestive of etiologic changes rather than effects secondary to the inflammatory response. Principle component analysis of urine NMR spectra produced a clear pattern separation within 48 to 72 h between CI-1018-treated rats with vascular lesions and vehicle controls or rats given dexamethasone alone. There was no pattern separation, however, between rats given CI-1018 alone and rats given CI-1018 and dexamethasone concurrently, suggesting that CI-1018-induced urine spectral changes were associated with the vascular lesions, yet were independent of the inflammatory response. These findings provide new insight into the mechanism(s) of PDE inhibitor-induced vasculitis and support the potential use of metabonomics for developing reliable noninvasive methods for detecting vascular changes in rats.

Animals↗
PubMed2004

New laboratory technique measures projected dynamic area of prosthetic heart valves.

BACKGROUND AND AIM OF THE STUDY: Fluid dynamic forces, valve design factors and gravity interactively determine the complex motion of prosthetic heart valve occluders. Although motion has been investigated, in vitro, using high-speed image recording, the technique has significant cost and limitations on resolution. METHODS: The kinematics of mechanical and biological valve occluders in the aortic and mitral positions were assessed by measuring projected dynamic valve area (PDVA). Valves were tested in a pulse duplicator system simulating normal cardiac conditions. To quantify PDVA, light passage through back-illuminated valves was measured by a calibrated photosensor with high-frequency response up to 150 kHz. Ten consecutive cycles were sampled using a PC data acquisition system. The system was calibrated under static conditions using reference areas. RESULTS: Several characteristics can be obtained from PDVA measurement including: maximum and minimum PDVA; rate of change of valve opening and closing PDVA; occluder rebound; and oscillatory open occluder behavior. Biological valves open more rapidly, close more gently, and exhibit no occluder rebound. They are also unaffected by gravity, and vary little in behavior from cycle to cycle compared with mechanical valves. CONCLUSION: A new method for measuring PDVA has been developed. Distinct differences in performance between valves were identified. It is hypothesized that, aside from patient factors and differences in materials, mechanical valves that mimic the PDVA behavior of biological valves, will lead to reduction of thrombogenicity, cavitation and high-intensity transient signals (HITS), and also reduce sound level and regurgitation.

Coronary Circulation↗
PubMed2001

[Androgen receptor isoforms expression and its novel cDNA cloning in human prostate].

OBJECTIVE: To investigate the androgen receptor isoforms expression and its novel eDNA cloning in prostate tissue. METHODS: The tissue from a case of benign prostatic hyperplasia (BPH) was analyzed by high resolution isoelectric focusing (IEF) technique and molecular biological technique of cDNA cloning was used to determine its AR isoforms expression and genes of AR isoforms. RESULTS: From the prostatic specimen, three types of AR isoforms were detected with pI values of 6.5, 6.0 and 5.3 respectively. A novel AR subtype cDNA cloning, named as PHAR-1 and including 1 913 nucleotides was isolated from the same BPH tissue. The GenBank accession number for the nucleotide sequence of this AR cDNA was BankIT372205 AF324243. The comparison between PHAR-1 and AR nucleotide sequence registered in GenBank showed that 961 nucleotides in three part of nucleotide sequence were the same, while the other 952 nucleotides were different. CONCLUSION: These three AR isoforms may originate from the same gene. The results may be important for the study of BPH, or prostatic growth. It is also valuable in the field of pharmacotherapy for treatment of BPH and prostatic cancer.

DNA, Complementary↗
PubMed1980

Application of cryosectioning to x-ray microanalysis of biological tissue.

Cryosectioning biological tissue is a critical and difficult step when applied to x-ray microanalysis of diffusible elements. Conventional metal machining theory can explain and predict results obtained by cryosectioning more consistantly than a freeze-thaw model. Temperature of a critical zone where shearing occurs is probably the most important factor for cryosectioning. This is presumed to be a result of thermally induced changes in tissue brittleness. Two other factors which may change effective cutting temperature are section thickness and cutting speed. Tissue brittleness also depends upon the extent of ice crystal damage from initial freezing which is largely determined by heat removal rate (freezing rate), water content, size, and composition. Antiroll plates are effective devices for counteracting bending forces which occur as the section is sheared from the block. Cryosectioning does not appear to affect elemental distribution or tissue morphology in either bounded or unbounded systems. Thus cryosectioning appears to be a predictable and controllable step which is suitable for x-ray microanalysis of diffusible elements in biological tissue. Deterioration of spatial and analytical resolution does not appear to occur through the process of cryosectioning; rather these limits are set by initial freezing.

Electron Probe Microanalysis↗
PubMed1994

[The value of bone marrow scintigraphy and magnetic resonance tomography in diagnostic imaging of bone marrow].

This article provides an overview of the possibilities and indications for magnetic resonance imaging (MRI) and bone marrow scintigraphy in imaging the bone marrow. After a brief review of the anatomical and physiologic structures of bone marrow, the principles of both methods are described. The radiopharmaceuticals of choice for bone marrow scintigraphy are the newly developed 99mTc-labelled anti-granulocyte monoclonal antibodies. They allow a high-quality, whole-body visualization of haematopetically active bone marrow by specifically targeting granulopoetic bone marrow cells and mature granulocytes. MRI enables us to visualize the bone marrow with high contrast and spatial resolution by assessing different properties of the biological tissue--mainly fat, water, and mineral content. Both methods provide a non-invasive and sensitive approach for imaging the bone marrow in different benign and malignant disorders.

Antibodies, Monoclonal↗
PubMedDate not supplied

Intact-protein based sample preparation strategies for proteome analysis in combination with mass spectrometry.

The complexity of tissue and cell proteomes and the vast dynamic range of protein abundance present a formidable challenge for analysis that no one analytical technique can overcome. As a result, there is a need to integrate technologies to achieve the high-resolution and high-sensitivity analysis of complex biological samples. The combined technologies of separation science and biological mass spectrometry (Bio-MS) are the current workhorse in proteomics, and are continuing to evolve to meet the needs for high sensitivity and high throughput. They are relied upon for protein quantification, identification, and analysis of post-translational modifications (PTMs). The standard technique of two dimensional poly-acrylamide gel electrophoresis (2D PAGE) offers relatively limited resolution and sensitivity for the simultaneous analysis of all cellular proteins, with only the most highly abundant proteins detectable in whole cell or tissue-derived samples. Hence, many alternative strategies are being explored. Numerous sample preparation procedures are currently available to reduce sample complexity and to increase the detectability of low-abundance proteins. Maintaining proteins intact during sample preparation has important advantages compared with strategies that digest proteins at an early step. These strategies include the ability to quantitate and recover proteins, and the assessment of PTMs. A review of current intact protein-based strategies for protein sample preparation prior to mass spectrometry (MS) is presented in the context of biomedically driven applications.

Animals↗
PubMed2006

Recombinant G protein-coupled receptors from expression to renaturation: a challenge towards structure.

G protein-coupled receptors (GPCRS) represent a class of integral membrane proteins involved in many biological processes and pathologies. Fifty percent of all modern drugs and almost 25% of the top 200 bestselling drugs are estimated to target GPCRs. Despite these crucial biological implications, very little is known, at atomic resolution, about the detailed molecular mechanisms by which these membrane proteins are able to recognize their extra-cellular stimuli and transmit the associated messages. Obviously, our understanding of GPCR functioning would be greatly facilitated by the availability of high-resolution three-dimensional (3D) structural data. However, expression, solubilization and purification of these membrane proteins are not easy to achieve, and at present, only one 3D structure has been determined, that of bovine rhodopsin. This review presents and compares the different successful strategies which have been applied to solubilize and purify recombinant GPCRs in the perspective of structural biology experiments.

Animals↗
PubMed1993

Regional cerebral blood flow in affective disorder.

The results of two-dimensional cerebral blood flow (CBF) studies in depression and the results of measurements of regional CBF (rCBF) using single photon emission computerized tomography (SPECT) are briefly reviewed. A technical outline of both types of method is described. The majority of SPECT studies point to a decreased CBF in untreated patients with a tendency towards normalization, but other studies suggest that, following treatment, there is an increase above normal either from a decreased or an abnormally high initial level. One study has pointed to unipolar patients having lower flow in the right temporal and parietal lobes relative to normals, whereas non-endogenous patients did not differ significantly from normal controls. Bipolar depressives have been found to have higher flow values than normals in the parietal and temporal lobes in the left hemispheres. This relevant aspect or a possible relationship between type of depression and hemispheric asymmetry needs further study for full classification. Thus, most investigations designed to determine which brain structures might be involved in regulating affect have not been very successful, which may be a consequence of the biological heterogeneity of these disorders or of the limited spatial resolution of the techniques used for investigation. Even if some of these technical difficulties can be overcome with measurements of receptor kinetics supplemented, other biological variables that have proved valuable in psychiatry (such as rapid eye movement latency and biogenic amines) should be studied to find more precise biological markers for depressive illness.

Cerebrovascular Circulation↗
PubMed2003

Nanodissection and high-resolution imaging of the Rhodopseudomonas viridis photosynthetic core complex in native membranes by AFM. Atomic force microscopy.

In photosynthesis, highly organized multiprotein assemblies convert sunlight into biochemical energy with high efficiency. A challenge in structural biology is to analyze such supramolecular complexes in native membranes. Atomic force microscopy (AFM) with high lateral resolution, high signal-to-noise ratio, and the possibility to nanodissect biological samples is a unique tool to investigate multiprotein complexes at molecular resolution in situ. Here we present high-resolution AFM of the photosynthetic core complex in native Rhodopseudomonas viridis membranes. Topographs at 10-A lateral and approximately 1-A vertical resolution reveal a single reaction center (RC) surrounded by a closed ellipsoid of 16 light-harvesting (LH1) subunits. Nanodissection of the tetraheme cytochrome (4Hcyt) subunit from the RC allows demonstration that the L and M subunits exhibit an asymmetric topography intimately associated to the LH1 subunits located at the short ellipsis axis. This architecture implies a distance distribution between the antenna and the RC compared with a centered location of the RC within a circular LH1, which may influence the energy transfer within the core complex. The LH1 subunits rearrange into a circle after removal of the RC from the core complex.

Image Processing, Computer-Assisted↗
PubMed2000

Real-time high-resolution optical sectioning suggests biphasic cytokinetic mechanism in Dictyostelium discoideum.

Despite its biological significance, much of the mechanism of cytokinesis is not yet resolved. The problems include: (1) signaling mechanism determining the position of the cleavage furrow, (2) molecular and mechanistic nature of the contractile ring, and (3) the origin of forces responsible for cleavage. Using high-resolution imaging technique, the present study analyzes morphometric changes of cytokinesis in wild type (NC4) Dictyostelium discoideum amoeba. A sample was prepared by the agar-overlay method, creating 3-mm-thick, nearly two-dimensional cells; and high-resolution image was acquired at 16.7 milliseconds' temporal, 234 nm x, y-, and 100 nm z-axis resolutions. Under this condition, the formation of cleavage furrow initiates at mitotic telophase, and daughter cells separate 18-22 minutes after the furrow initiation. We found that the compression of cells and the room temperature need to be carefully controlled for cytokinesis to proceed in an orderly manner. The results demonstrate that the pole-to-pole distance increases by 83% during the initial 5 minutes of cytokinesis, while the distance of equator only decreases by 56%. In contrast, during the subsequent 5 minutes, the pole-to-pole distance only increases by 17%, while the equator distance decreases as much as by 44%. This study indicates that cytokinesis consists of at least two different phases, each of which results from a different mechanism.

Actins↗
PubMed2006

A comparison of liquid nitrogen and liquid helium as cryogens for electron cryotomography.

The principal resolution limitation in electron cryomicroscopy of frozen-hydrated biological samples is radiation damage. It has long been hoped that cooling such samples to just a few kelvins with liquid helium would slow this damage and allow statistically better-defined images to be recorded. A new "G2 Polara" microscope from FEI Company was used to image various biological samples cooled by either liquid nitrogen or liquid helium to approximately 82 or approximately 12 K, respectively, and the results were compared with particular interest in the doses (10-200 e-/A2) and resolutions (3-8 nm) typical for electron cryotomography. Simple dose series revealed a gradual loss of contrast at approximately 12K through the first several tens of e-/A2, after which small bubbles appeared. Single particle reconstructions from each image in a dose series showed no difference in the preservation of medium-resolution (3-5 nm) structural detail at the two temperatures. Tomographic reconstructions produced with total doses between 10 and 350 e-/A2 showed better results at approximately 82 K than approximately 12 K for every dose tested. Thus disappointingly, cooling with liquid helium is actually disadvantageous for cryotomography.

Cryoelectron Microscopy↗
PubMed2004

A general framework for analyzing the genetic architecture of developmental characteristics.

The genetic architecture of growth traits plays a central role in shaping the growth, development, and evolution of organisms. While a limited number of models have been devised to estimate genetic effects on complex phenotypes, no model has been available to examine how gene actions and interactions alter the ontogenetic development of an organism and transform the altered ontogeny into descendants. In this article, we present a novel statistical model for mapping quantitative trait loci (QTL) determining the developmental process of complex traits. Our model is constructed within the traditional maximum-likelihood framework implemented with the EM algorithm. We employ biologically meaningful growth curve equations to model time-specific expected genetic values and the AR(1) model to structure the residual variance-covariance matrix among different time points. Because of a reduced number of parameters being estimated and the incorporation of biological principles, the new model displays increased statistical power to detect QTL exerting an effect on the shape of ontogenetic growth and development. The model allows for the tests of a number of biological hypotheses regarding the role of epistasis in determining biological growth, form, and shape and for the resolution of developmental problems at the interface with evolution. Using our newly developed model, we have successfully detected significant additive x additive epistatic effects on stem height growth trajectories in a forest tree.

Algorithms↗
PubMed1999

An external ion microbeam for studies of biological samples.

A new external ion-beam system was developed and combined with a light ion-microbeam system in JAERI Takasaki. The system is designed for micro-particle-induced X-ray emission analysis of biological samples in air environment with 1 micron spatial resolution. One of the most serious problems in keeping such a high spatial resolution is multiple scattering in a beam exit window. Thin Kapton film (7.5 microns thick) was adopted as the exit window as well as a sample backing foil to minimize the distance between the film and samples. The lifetime of the foil under ion irradiation and spatial resolution of the external microbeam were investigated. The results shows that the film can endure sufficient long-time irradiation to take elemental maps and the resolution can be kept nearly 1 micron.

Biological Assay↗