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PubMed1999

Real time imaging of rotating molecular machines.

Observation of true rotation has been relatively rare in living systems, but there may be many molecular machines that rotate. Molecular rotations accompanying function can be imaged in real time under an optical microscope by attaching to the protein machine either a small tag such as a single fluorophore or a tag that is huge compared with the size of the protein. As an example of the former approach, axial rotation of an actin filament sliding over myosin has been measured quantitatively by attaching a fluorophore rigidly to the filament and imaging the orientation of the fluorophore continuously by polarization microscopy. As a huge tag in the latter approach, an actin filament turned out to be quite useful. Using this tag, the enzyme F1-ATPase has been shown to be a rotary stepper motor made of a single molecule. Further, the efficiency of this ATP-fueled motor has been shown to reach almost 100%. The two examples above demonstrate that one can now image conformational changes, which necessarily involve reorientation, in a single protein molecule during function. Single-molecule physiology is no longer a dream.

Actins↗

PubMed2000

Foreign bodies in sarcoidosis.

Sarcoidosis is a multisystem disease of unknown etiology. The demonstration of polarizable foreign bodies in cutaneous granulomas is generally thought to exclude a diagnosis of sarcoidosis. Nevertheless. some investigators have reported systemic sarcoidosis with cutaneous manifestations in which polarizable particles were associated with granuloma formation in the skin. We searched the biopsy specimens of granulomatous lesions from 50 patients with cutaneous sarcoidosis using polarization microscopy to estimate the frequency of polarizable foreign bodies in cutaneous lesions of sarcoidosis. Using electron probe microanalysis, we sought to determine what elements compose these foreign bodies. Polarizable foreign bodies were found in the granulomatous skin lesions of 12 of 50 patients with cutaneous sarcoidosis. All 12 patients also had at least one other granulomatous systemic lesion, and 4 had biopsy specimens of a systemic lesion available for review. Polarizable foreign bodies were found in two cases. The elements identified were calcium, phosphorus, silicon, and aluminum. Polarizable foreign bodies were found in cutaneous sarcoidosis far more often than expected. Foreign bodies were also found in granulomatous systemic lesions. The foreign body may serve as an inciting stimulus for granuloma formation in selected cases of sarcoidosis.

Adult↗

PubMed1985

Pseudogout presenting with low synovial fluid glucose: identification of crystals by gram stain.

A man developed acute monoarticular ankle arthritis caused by calcium pyrophosphate dihydrate (CPPD) crystals. The clinical syndrome resembled that of a pyogenic arthritis. Synovial fluid analysis revealed a glucose concentration of 13 mg/dL and 99,000 white blood cells/mm3. Only one other report of an extremely low synovial fluid glucose associated with pseudogout could be found. The diagnosis of pseudogout was initially suggested when rhomboidal forms were seen during synovial fluid Gram stain examination. Synovial fluid examination with polarized microscopy was initially negative, but revealed numerous CPPD crystals when repeated on the third hospital day. This case serves to illustrate how pseudogout can mimic pyogenic arthritis in both clinical presentation and low synovial fluid glucose concentration. The examination of Gram-stained synovial fluid can reveal the rhomboidal forms of CPPD crystals. The appearance of these crystals is documented in this report.

Arthritis, Infectious↗

PubMed1999

Endochondral vs. intramembranous demineralized bone matrices as implants for osseous defects.

This study focuses on the difference in regenerative capacity between endochondral and intramembranous demineralized bone matrices (DBMs) when implanted into bony defects. It also focuses on the possible influence of the type of skeletal recipient site (orthotopic or heterotopic). Of 34 Wistar rats, 10 served as a source of DBM, and 24 were divided into two groups of 12 animals. In group A identical defects were produced in the parietal bones, whereas in group B the defects were produced in each radius. The right defects were implanted with endochondral DBM and the left defects were implanted with intramembranous DBM. Descriptive and/or histomorphometric analyses were performed by means of light and polarized microscopy, and radiography (group B). Right and left data were compared to disclose differences in bone-healing capacity. The quantitative results demonstrated that endochondral DBM displays a greater regenerative capacity than intramembranous DBM when implanted heterotopically. The different clinical performances of endochondral and intramembranous bone grafts might be explained on the basis of the mechanical rather than the osteoinductive principle. The qualitative results suggest that the type of bone deposition induced by the DBMs is not related to the type of implanted DBM. Recipient site characteristics and/or environmental factors seem decisive in the occurrence of either types of ossification.

Animals↗

PubMed1991

Amyloidosis.

Amyloidosis is a disease involving the fibrillar deposition of proteins in a manner that uniformly leads to the presence of green birefringence on polarization microscopy after staining the involved tissues with Congo red. In the year summarized, a wide range of new information has accumulated about this disease. In this article, attention has been paid to several newly described proteins now known to precipitate into amyloid deposits, including the proteins transthyretin, apolipoprotein A-1, cystatin C, gelsolin, amyloid beta protein, beta 2-microglobulin, scrapie protein, and islet amyloid polypeptide. The number of these amyloid-related proteins has resulted in the need for a revised nomenclature and classification scheme. The results of a recent international symposium addressing this issue are summarized in table form. The varied clinical manifestations of amyloidosis are described according to organ system, with unusual or unique areas of involvement noted. Finally, the treatment of amyloidosis and its prognosis are addressed, and new areas of possible intervention suggested.

Amyloid↗

PubMed2001

Tophaceous gout of the spine mimicking epidural infection: case report and review of the literature.

OBJECTIVE AND IMPORTANCE: Tophaceous gout uncommonly affects the axial skeleton. The clinical presentations of gout of the spine range from back pain to quadriplegia. Gout that presents as back pain and fever may be difficult to distinguish from spinal infection. We present a case of a patient with tophaceous gout of the lumbar spine who was initially diagnosed with and treated for an epidural infection. The clinical and diagnostic features of tophaceous gout of the spine are reviewed. CLINICAL PRESENTATION: A 70-year-old man presented with a 2-day history of fever and back pain. A physical examination revealed that he had flank tenderness and evidence of polyarthritis affecting the elbows, knees, and right first metatarsophalangeal joint. A magnetic resonance imaging scan of the patient's lumbar spine showed an extensive area of abnormal gadolinium enhancement of the paramedian posterior soft tissues from L3 to S1 with an area of focal enhancement extending into the right L4-L5 facet joint. INTERVENTION: A laminectomy was performed at L4-L5, and a chalky white material in the facet joint was found eroding into the adjacent pars intra-articularis. Light and polarizing microscopy confirmed the presence of gouty tophus. No evidence of infection was found. CONCLUSION: Gouty arthritis of the spine is rare. Thirty-seven previous cases have been reported. When the clinical presentation includes acute back pain and fever, differentiation of spinal gout from spinal infection may be difficult. The clinical suspicion of spinal gout may lead to the correct diagnosis by a less invasive approach than exploration and laminectomy.

Aged↗

PubMed1998

Human wound contraction: collagen organization, fibroblasts, and myofibroblasts.

The closure of ungrafted sacrococcygeal pilonidal sinus excisional wounds was studied in 15 patients. Wound punch biopsies were taken on a regular basis, and histologic sections were made. To document changes, computer-assisted morphometric image analysis was employed. Initial average wound depth was 37.8 +/- 4.6 mm, and complete closure (0 wound depth) was reached by 68 days. Wound contraction contributed 88 percent to wound closure, whereas the deposition of scar only contributed 12 percent. Maximum cells density within granulation tissue was reached by day 18. Myofibroblasts, identified by alpha-smooth muscle actin immunostaining, first appeared on day 11. Unlike those observed in laboratory animals, myofibroblasts were a minor cell population of granulation tissue, never exceeding 10 percent of the cells. The pattern of collagen fiber organization was documented by polarized light microscopy of Sirius red-stained sections. Early granulation tissue collagen fibers demonstrated a fine greenish birefringence, whereas more mature granulation tissue collagen fibers were thicker, displaying orange-yellowish birefringence. Myofibroblasts were associated exclusively with thicker collagen fibers, whereas fibroblasts were associated with both fine and thick collagen fibers. It is proposed that human wound contraction involves a volume change whereby normal dermal and adipose tissues are pulled into the defect by forces generated within fibroblasts.

Actins↗

PubMed1999

Analysis of breast implant capsular tissue for crystalline silica and other refractile phases.

This study questions previous reports of the presence of micrometer-sized areas of crystalline silica in pathologic tissue sections that are based exclusively on polarized-light microscopy. By using optical principles, it can be argued that it is impossible to identify unambiguously or to detect the birefringence of crystalline silica in 5-microm-thin sections. To clarify whether silicone, amorphous silica, or crystalline silica occurs in micrometer-sized moieties in standard 5-microm-thick tissue sections, one needs to apply a structural means of analysis in addition to optical microscopy. This study recommends the use of the laser Raman spectroscopic technique, which is very well suited to clarify this highly controversial issue in future pathologic studies.

Breast↗

PubMed2001

Renal microtophi in a patient with lupus nephritis and tophaceous gout.

This case describes a 58-year-old female with systemic lupus erythematosus (SLE) and coexistent chronic tophaceous gout. A renal biopsy showed concurrent lupus nephritis and renal medullary microtophi, confirmed by electron and polarizing microscopy, respectively. Whereas clinical SLE and gout have already been shown to be rarely associated, this case further illustrates the presentation of these two diseases in a single renal specimen. In this patient the gout began shortly after menopause without known risk factors and before any overt renal disease or signs of SLE. The tophaceous gout antedating the SLE, as well as the apparently benign course of illness, suggest that the pathologic effects of SLE and gout on the kidneys are based on independent mechanisms and may not necessarily aggravate each other. Treatment of the gout with allopurinol may have contributed to improved renal function.

Journal Article↗

PubMed2003

The origin of chondrocytes in the nucleus pulposus and histologic findings associated with the transition of a notochordal nucleus pulposus to a fibrocartilaginous nucleus pulposus in intact rabbit intervertebral discs.

STUDY DESIGN: Intact rabbit lumbar intervertebral discs were examined histologically. OBJECTIVES: To demonstrate the origin of chondrocytes in the nucleus pulposus, and to document histologic findings associated with the transition of a notochordal to a fibrocartilaginous nucleus pulposus. SUMMARY OF BACKGROUND DATA: A human nucleus pulposus undergoes a chronological transition from a notochordal to a fibrocartilaginous nucleus pulposus. However, the origin of chondrocytes forming fibrocartilage in the nucleus pulposus and the mechanisms of transition remain unknown. METHODS: Hematoxylin-eosin- and safranin O-stained slides obtained from 125 intact rabbit intervertebral discs were observed with light and polarized light microscopy. RESULTS: Of the 125 intervertebral discs examined, 58 had a notochordal nucleus pulposus. The remaining intervertebral discs had a nucleus pulposus with fibrocartilage lamellas or fibrocartilage fibers. All forms of fibrocartilage lamellas and fibers found in the nucleus pulposus were formed by chondrocytes that had originated and migrated from the cartilage endplate. The origin of chondrocytes proceeded in a centripetal direction from the periphery toward the center of the cartilage endplate. The newly formed fibrocartilage lamellas and fibers, therefore, initially involved replacement of the peripheral regions of the nucleus pulposus, followed by replacement of the central region. This centripetal sequential replacement mechanism decreased the size of the notochordal tissue while increasing the lamellar structure of the intervertebral disc. CONCLUSIONS: Chondrocytes in the intact rabbit nucleus pulposus originated and migrated from the cartilage endplate. The chondrocytes changed notochordal nucleus pulposus into fibrocartilaginous nucleus pulposus by depositing fibrocartilage lamellas and fibers in a centripetal direction.

Animals↗

PubMed2005

Protective effects of a selective neutrophil elastase inhibitor (sivelestat) on lipopolysaccharide-induced acute dysfunction of the pulmonary microcirculation.

OBJECTIVE: The purpose of this study was to evaluate the effect of a neutrophil elastase inhibitor, sivelestat, on lipopolysaccharide-induced acute lung injury through analysis of hemodynamic changes in the pulmonary microcirculation. DESIGN: Randomized animal study. SETTING: Medical school laboratory. SUBJECTS: Twenty-seven Wistar rats (15 rats for microspectroscopic observations, 12 rats for measurements of neutrophil elastase activity and wet-to-dry ratio). INTERVENTIONS: Thoracosternotomy was performed on male Wistar rats under continuous anesthesia and mechanical ventilation. Rats were divided into three groups (n = 5 each groups) on the basis of the reagent used: lipopolysaccharide group (100 microg/kg lipopolysaccharide intravenously), sivelestat group (10 mg/kg sivelestat; 100 microg/kg lipopolysaccharide intravenously), and control group (saline only, intravenously). MEASUREMENTS AND MAIN RESULTS: We measured morphologic changes and hemodynamic variables, including tissue blood flow, erythrocyte velocity, erythrocyte count, thickness of interalveolar septa, and leukocyte adhesion in the pulmonary microcirculation, with a video-rate (33 msec/frame) dual-spot microspectroscopy system (DSMSS) and a laser-Doppler flowmeter. Blood-free wet-to-dry ratio and neutrophil elastase activity in bronchoalveolar lavage fluid, serum, and supernatant of lung homogenate were measured in another set of experiments (n = 4 for each group). Sixty minutes after lipopolysaccharide administration, severe thickening of the interalveolar septa was observed in the lipopolysaccharide but not the sivelestat group. In the lipopolysaccharide group, DSMSS measurements of erythrocyte velocity and hemoglobin oxygenation in single capillaries were decreased significantly (vs. control p < .05, vs. sivelestat p < .01), whereas tissue blood flow and erythrocyte velocity measurements from laser-Doppler flowmeter were increased significantly (vs. control p < .05, vs. sivelestat p < .01). The number of adherent leukocytes was increased significantly in the lipopolysaccharide group at 30, 45, and 60 mins after lipopolysaccharide administration (vs. control p < .01, vs. sivelestat p < .05). The number of adherent leukocytes did not increase in the sivelestat group. The wet-to-dry ratio was significantly higher in the lipopolysaccharide group than in control (p < .05) and sivelestat (p < .05) groups. Neutrophil elastase activities in the bronchoalveolar lavage fluid, serum, and lung tissue were all significantly lower in the sivelestat group than in the lipopolysaccharide group (p < .05). CONCLUSIONS: Lipopolysaccharide induces leukocyte adhesion in the pulmonary microcirculation, resulting in decreased tissue hemoglobin oxygen and alveolar and interstitial edema. The selective neutrophil elastase inhibitor sivelestat reduces neutrophil elastase activity and attenuates acute changes in the pulmonary microcirculation.

Analysis of Variance↗

PubMed2006

Orthogonal (transverse) arrangements of actin in endothelia and fibroblasts.

Though actin filaments running across the cell (transverse actin) have been occasionally reported for epithelial cells in groups and for cells growing on fibres, there has been no report heretofore of transverse actin in cells grown on planar substrata. This paper describes evidence in support of this possibility derived from actin staining, polarization microscopy and force measurements. The paper introduces two new methods for detecting the orientation and activity of contractile elements in cells. The orthogonal actin is most obvious in cells grown on groove ridge structures, but can be detected in cells grown on flat surfaces.

Actins↗

PubMed2000

Realignment of a smectic-A phase with applied electric field

The realignment of a smectic-A phase contained in a conventional display device (with a mesophase layer approximately 10 &mgr;m thick) in response to an applied electric field has been studied using polarizing microscopy and x-ray diffraction. The initial state of the mesophase was a highly aligned focal conic texture. The applied field created a striated texture with parallel (but not equally spaced) disclination lines which appeared to be created in pairs. The lines seem to grow from disclination "eyes" in the original texture, linking them first in pairs, and then in long chains, as the field is increased. We suggest that the center of each pair of striations corresponds to a disclination wall and that the texture consists of a parallel array of smectic layers arranged in concentric flattened half-cylinders. Previous electro-optic studies of cells of this type have described the striated, field-on texture but appear to have overlooked the role of the defects in the original texture as growth points for the striations. However, there are structural similarities with two related studies where the field-induced disturbance of the mesophase starts with the production of toroidal focal conics-similar to those we propose.

Journal Article↗

PubMed2005

Highly ordered monodomain ionic self-assembled liquid-crystalline materials.

Liquid-crystalline properties of the ionic self assembled complex benzenehexacarboxylic- (didodecyltrimethylammonium)6 [BHC- (C12D)6] were investigated by polarizing microscopy, differential scanning calorimetry (DSC), x-ray analysis, null ellipsometry, UV and IR spectroscopy. The complex exhibits a bilayer smectic Sm- A2 liquid-crystalline phase and aligns spontaneously. Alignment properties do not depend on the hydrophobic or hydrophilic treatment of the surfaces. The aligned complex possesses a negative (delta n=-0.02) homeotropically oriented optical axis, with layers aligned parallel to the surface. X-ray analysis of the aligned sample revealed a lamellar structure with a d spacing of 3.15 nm, consisting of sublayers of thicknesses d1 = 1.41 and d2 = 1.74 nm . This was confirmed by simple geometrical calculations and detailed temperature-dependent investigations, revealing that the first layer contains the BHC molecules and oppositely charged groups of the surfactants, and the second the alkyl tails of the surfactant. Changes in the order parameters (as calculated from the IR investigations) are correlated with the phase transitions as found by DSC. The properties of the complex are strongly influenced by the ionic interactions within the complex. The presence of these groups slows down the dynamics within the material sufficiently to allow for crystallization of the complex from an aligned LC phase into a single crystal domain, as well as restricting the transition to the isotropic phase.

Journal Article↗

PubMed2005

Electric-field-induced deformation dynamics of a single nematic disclination.

Disclinations in nematic liquid crystals usually adopt a straight shape in order to minimize their elastic energy. Once created in the course of a nonequilibrium process such as a temperature quench from the isotropic to the nematic phase, the topologically stable disclinations of half-integer strength either annihilate each other in pairs of opposite strength or form topologically unstable disclinations of integer strength. In this article, we demonstrate that the annihilation process can be inhibited and the defects can be deformed by an applied electric field. We study the disclination lines in the deep uniaxial nematic phase, located at the boundary between two different types of walls, the so-called pi wall (a planar soliton stabilized by the surface anchoring) and the Brochard-Léger (BL) wall stabilized by the applied electric field. By changing the electric voltage, one can control the energy of director deformations associated with the two walls and thus control the deformation and dynamics of the disclination line. At small voltages, the disclinations are straight lines connecting the opposite plates of the cell, located at the two ends of the pi walls. The pi walls tend to shrink. When the voltage increases above E(F), the Fréedericksz threshold, the BL walls appear and connect pairs of disclinations along a path complementary to the pi wall. At E>2 E(F), the BL walls store sufficient energy to prevent shrinking of the pi walls. Reconstruction of the three-dimensional director configuration using a fluorescent confocal polarizing microscopy demonstrates that the disclinations are strongly bent in the region between the pi and the BL walls. The distortions and the related dynamics are associated with the transformation of the BL wall into two surface disclination lines; we characterize it experimentally as a function of the applied electric field, the cell thickness, and the sample temperature. A simple model captures the essential details of the experimental data.

Journal Article↗

PubMed2005

Electric-field-induced nematic-cholesteric transition and three-dimensional director structures in homeotropic cells.

We study the phase diagram of director structures in cholesteric liquid crystals of negative dielectric anisotropy in homeotropic cells of thickness d which is smaller than the cholesteric pitch p. The basic control parameters are the frustration ratio d/p and the applied voltage U. Upon increasing U, the direct transition from completely unwound homeotropic structure to the translationally invariant configuration (TIC) with uniform in-plane twist is observed at small d/p < or = 0.5. Cholesteric fingers that can be either isolated or arranged periodically occur at 0.5 < or = d/p<1 and at the intermediate U between the homeotropic unwound and TIC structures. The phase boundaries are also shifted by (1) rubbing of homeotropic substrates that produces small deviations from the vertical alignment; (2) particles that become nucleation centers for cholesteric fingers; (3) voltage driving schemes. A novel reentrant behavior of TIC is observed in the rubbed cells with frustration ratios 0.6 < or = d/p < or = 0.75, which disappears with adding nucleation sites or using modulated voltages. In addition, fluorescence confocal polarizing microscopy (FCPM) allows us to directly and unambiguously determine the three-dimensional director structures. For the cells with strictly vertical alignment, FCPM confirms the director models of the vertical cross sections of four types of fingers previously either obtained by computer simulations or proposed using symmetry considerations. For rubbed homeotropic substrates, only two types of fingers are observed, which tend to align along the rubbing direction. Finally, the new means of control are of importance for potential applications of the cholesteric structures, such as switchable gratings based on periodically arranged fingers and eyewear with tunable transparency based on TIC.

Journal Article↗

PubMed2006

Three-dimensional imaging of dielectric patterns in electrohydrodynamic convection of a nematic liquid crystal.

The transition from surface to bulk normal dielectric rolls in a nematic liquid crystal is imaged by fluorescence confocal polarizing microscopy. The three-dimensional director structure and the liquid flow are scanned in both the layer plane and the transverse plane. Two systems of small-scale convective flow are formed, one at each electrode. Strong anchoring makes director oscillations difficult and charges accumulate by the Carr-Helfrich mechanism. The middle region is a structureless convection where the director oscillates with the frequency of the applied voltage. The small-scale flow eventually fills the cell from one electrode to the other as one system of thin and elongated rolls. The true dielectric mode is not a director pattern, rather a surface flow instability.

Journal Article↗

PubMed2000

Coupling between flow and structure for a lamellar surfactant phase

The flow-structure relation of lamellar phases is studied using rheometry and cross-polarized microscopy under flow. The equilibrium phases show different defects. Low salinities lead to very viscous, "onion" phases, whereas at high salinity, a low viscosity plane lamellar phase is found. Under shear, the latter shows a sudden transition to a viscoelastic gel, with a texture and viscosity very similar to that of the onions. Gelation occurs after a certain delay time, increasing rapidly with salinity, by the nucleation of onions. This allows one to relate the delay time to the defect energy.

Journal Article↗