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PubMed2005

Evaluation of the transdermal permeation behavior of Proterguride from drug in adhesive matrix patches through hairless mouse skin.

The transdermal in vitro permeation behavior of the highly potent dopamine agonist Proterguride was investigated using hairless mouse skin as a model membrane. Drug in adhesive matrix formulations based on different types of pressure-sensitive adhesives (Eudragit E 100 and Gelva7883 as acrylates, Oppanol B 15 SFN as polyisobutylene, and BioPSA 7-4202 as silicone) with a drug load of 3% by weight were manufactured. All patches were examined for drug crystallization by polarized microscopy immediately after the manufacturing process and after storage for 30 days in sealed aluminium laminate bags at ambient temperature and at 40 degrees C, respectively. Furthermore, the influence of the drug load in acrylate-based formulations onto the steady-state flux of Proterguride was examined. The Eudragit E 100 system delivered a significantly higher steady-state flux than the systems based on Oppanol B 15 SFN and also a somewhat higher steady-state flux than the Gelva-based patch. An addition of 10% by weight of the crystallization inhibitor povidone 25 did not significantly influence the steady-state flux of Proterguride from acrylate matrices. The lipophilic silicone and polyisobutylene adhesives facilitated drug crystallization within the short storage periods at both conditions, probably due to the absence of povidone 25, which was incompatible with these polymers. Varying the drug load in acrylate-based formulations led to a linear increase of the steady-state flux until the steady-state flux of Proterguride leveled off and the patches tended to drug crystallization. It was found that Gelva-based patches show good physical stability, good skin adhesion, and moderate flux values and, thus, can be evaluated as a basis for a suitable formulation for the transdermal administration of Proterguride.

Acrylates↗

PubMed2002

Multinuclear NMR investigation of the NaDNA/ethidium bromide anisotropic system.

A combined use of (31)P, (23)Na, (2)H and (17)O NMR spectroscopies and polarized light microscopy has been employed to investigate the effect of the ethidium bromide (EB) binding on the liquid crystalline phase of concentrated double stranded DNA solutions. The optical textures and the (31)P and (23)Na NMR spectra of the DNA anisotropic solutions show that the intercalation of EB induces significant modifications either in the arrangements of the DNA rods and the surrounding ionic atmosphere. On the contrary, no indication of significant changes of the orientational order of the water molecules around DNA emerges from the water (2)H and (17)O NMR spectra.

Animals↗

PubMed2003

Asbestos release during removal of resilient floor covering materials by recommended work practices of the resilient floor covering institute.

The release of asbestos during maintenance and removal of resilient floor covering is of concern to health professionals and many regulators. This study assesses the asbestos levels observed during removal of resilient floor covering products using the "Recommended Work Practices" (1995) of the Resilient Floor Covering Institute or other methods requiring containment (Controls). The 1995 "work practices" require wet removal or dry heat removal but do not require the use of respirators. Wet removals of sheet vinyl/separated backing, 12" x 12" vinyl asbestos tile/mastic, and 9" x 9" asphalt tiles/mastic were conducted and the air was sampled during each procedure. Settled dust samples were collected at the sites of RFCI square tile removal and pieces of each type of tile were broken in a mini-enclosure to evaluate asbestos emissions. Analyses of the air samples collected during the removals showed that the RFCI methods did not produce asbestos counts significantly different from the Control methods requiring containment. Only a small number (0.7%) of fibers and structures, counted and measured by Analytical Transmission Electron Microscopy, would have been counted using the rules for Phase Contrast Microscopy in the 7400 method specified by Occupational Safety and Health Administration regulations. This indicates workers in similar situations without respirators are likely to have unknown exposure levels. A high percentage of these fibers and structures are 5 micrometers or less in length, smaller than 0.5 micrometer in diameter, and are easily inhaled. The RFCI air sample and settled dust data may cause regulators to consider requiring respiratory protection, cleanup procedures, and methods to control asbestos migration. Other areas that might be addressed are clearance levels and their measurement, removal area size, bulk sample analysis by transmission electron microscopy if polarized light microscopy reports less than 1 percent asbestos, better worker exposure evaluation, and supervisor/worker training in accordance with the Model Accreditation Plan.

Air Pollutants, Occupational↗

PubMed2004

A tribute to Shinya Inoue and innovation in light microscopy.

The 2003 International Prize for Biology was awarded to Shinya Inoue for his pioneering work in visualizing dynamic processes within living cells using the light microscope. He and his scientific descendants are now pushing light microscopy even further by developing new techniques such as imaging single molecules, visualizing processes in living animals, and correlating results from light and electron microscopy.

Animals↗

PubMed1967

Polarized light observations on striated muscle contraction in a mite.

Contraction of individual sarcomeres within the living mite Tarsonemus sp. was observed by polarized light microscopy. In unflattened animals the usual range of contraction was such that the minimum sarcomere length approximated the length of the A region, and the maximum sarcomere length was about twice the length of the A region. The central sarcomeres of the dorsal metapodosomal muscles were observed in detail. The A band length increased slightly with increasing sarcomere length since the regression of I region length on sarcomere length had an average slope of 0.91. When the A band length in a sarcomere which was shortening was compared with the length when the same sarcomere lengthened, no significant difference was seen. The A band of each sarcomere seemed to act as a not too rigid limit to further shortening; this agreed with the reversible shortening of a muscle in which the A band had been experimentally shortened. An H region was visible at long sarcomere lengths and was not visible at short sarcomere lengths, even when the muscle was actively shortening. The rate of change of H region length with sarcomere length suggested that I filament length may increase as sarcomere length increases. Despite this effect and the small increase in A length with sarcomere length, the results are considered to be consistent with a model in which shortening occurs by the relative movement of A and I filaments, with little or no change in length of either set of filaments. Sarcomere shortening was clearly associated with an increase in the retardation of the A region.

Animals↗

PubMed1975

Pressure-induced depolymerization of spindle microtubules. I. Changes in birefringence and spindle length.

Changes in birefringence retardation (BR) and length of Chaetopterus meiotic metaphase-arrested spindles produced by increased hydrostatic pressure were observed with polarized-light microscopy using a newly developed optical pressure chamber. Increased pressure produced rapid, reversible decreases in spindle BR and length. Pressures of 3,500 psi or higher at 22 degrees C caused complete disappearance of spindle BR within 3 min. Up to 6,000 psi, the rates of both BR decay and spindle shortening increased progressively with increasing pressure. At 6,000 psi or above, the BR decreased rapidly but there was no evidence of spindle shortening. The general observations are consistent with results of earlier classical experiments on effects of pressure on mitosis, and with experiments that used colchicine or low temperature as microtubule-depolymerizing agents. The kinetics of spindle depolymerization and repolymerization showed two phases: an initial phase of rapid decreases or increase in half-spindle microtubule BR; and a second phase of nearly constant BR during which most of the spindle shortening or growth occurs. BR is assumed to be directly related to the number of microtubules in a spindle cross section. It is hypothesized that microtubules in the spindle have different stabilities depending on the attachment of nonattachment of their ends. This hypothesis is used to explain the two phases of spindle depolymerization and repolymerization as well as several other observations.

Animals↗

PubMed1979

Micromanipulation studies of chromosome movement. I. Chromosome-spindle attachment and the mechanical properties of chromosomal spindle fibers.

We have used micromanipulation to study the attachment of chromosomes to the spindle and the mechanical properties of the chromosomal spindle fibers. Individual chromosomes can be displaced about the periphery of the spindle, in the plane of the metaphase plate, without altering the structure of the spindle or the positions of the nonmanipulated chromosomes. From mid-prometaphase through the onset of anaphase, chromosomes resist displacement toward either spindle pole, or beyond the spindle periphery. In anaphase a chromosome can be displaced either toward its spindle pole or laterally, beyond the periphery of the spindle; however, the chromosome resists displacement away from the spindle pole. When an anaphase half-bivalent is displaced toward its spindle pole, it stops migrating until the nonmanipulated half-bivalents reach a similar distance from the pole. The manipulated half-bivalent then resumes its poleward migration at the normal anaphase rate. No evidence was found for mechanical attachments between separating half-bivalents in anaphase. Our observations demonstrate that chromosomes are individually anchored to the spindle by fibers which connect the kinetochores of the chromosomes to the spindle poles. These fibers are flexible, much less extensible than the chromosomes, and are to pivot about their attachment points. While the fibers are able to support a tensile force sufficient to stretch a chromosome, they buckle when subjected to a compressive force. Preliminary evidence suggests that the mechanical attachment fibers detected with micromanipulation correspond to the birefringent chromosomal spindle fibers observed with polarization microscopy.

Animals↗

PubMed1982

Flagellated ectosymbiotic bacteria propel a eucaryotic cell.

A devescovinid flagellate from termites exhibits rapid gliding movements only when in close contact with other cells or with a substrate. Locomotion is powered not by the cell's own flagella nor by its remarkable rotary axostyle, but by the flagella of thousands of rod bacteria which live on its surface. That the ectosymbiotic bacteria actually propel the protozoan was shown by the following: (a) the bacteria, which lie in specialized pockets of the host membrane, bear typical procaryotic flagella on their exposed surface; (b) gliding continues when the devescovinid's own flagella and rotary axostyle are inactivated; (c) agents which inhibit bacterial flagellar motility, but not the protozoan's motile systems, stop gliding movements; (d) isolated vesicles derived from the surface of the devescovinid rotate at speeds dependent on the number of rod bacteria still attached; (e) individual rod bacteria can move independently over the surface of compressed cells; and (f) wave propagation by the flagellar bundles of the ectosymbiotic bacteria is visualized directly by video-enhanced polarization microscopy. Proximity to solid boundaries may be required to align the flagellar bundles of adjacent bacteria in the same direction, and/or to increase their propulsive efficiency (wall effect). This motility-linked symbiosis resembles the association of locomotory spirochetes with the Australian termite flagellate Mixotricha (Cleveland, L. R., and A. V. Grimstone, 1964, Proc. R. Soc. Lond. B Biol. Sci., 159:668-686), except that in our case propulsion is provided by bacterial flagella themselves. Since bacterial flagella rotate, an additional novelty of this system is that the surface bearing the procaryotic rotary motors is turned by the eucaryotic rotary motor within.

Animals↗

PubMed2006

Comparative study using 685-nm and 830-nm lasers in the tissue repair of tenotomized tendons in the mouse.

OBJECTIVE: The objective of this study was to evaluate the effects of 685- and 830-nm laser irradiations, at different fluences on the healing process of Achilles tendon (Tendon calcaneo) of mice after tenotomy. BACKGROUND DATA: Some authors have shown that low-level laser therapy (LLLT) is able to accelerate the healing process of tendinuos tissue after an injury, increasing fibroblast cell proliferation and collagen synthesis. However, the mechanism by which LLLT acts on healing process is not fully understood. METHODS: Forty-eight male mice were divided into six experimental groups: group A, tenomized animals, treated with 685 nm laser, at the dosage of 3 J/cm(2); group B, tenomized animals, treated with 685-nm laser, at the dosage of 10 J/cm(2); group C, tenomized animals, treated with 830-nm laser, at dosage of 3 J/cm(2); group D, tenomized animals, treated with 830-nm laser, at the dosage of 10 J/cm(2); group E, injured control (placebo treatment); and group F, non-injured standard control. Animals were killed on day 13 post-tenotomy, and their tendons were surgically removed for a quantitative analysis using polarization microscopy, with the purpose of measuring collagen fibers organization through the birefringence (optical retardation [OR]). RESULTS: All treated groups showed higher values of OR when compared to injured control group. The best organization and aggregation of the collagen bundles were shown by the animals of group A (685 nm, 3 J/cm(2)), followed by the animals of group C and B, and finally, the animals of group D. CONCLUSION: All wavelengths and fluences used in this study were efficient at accelerating the healing process of Achilles tendon post-tenotomy, particularly after the 685-nm laser irradiation, at 3 J/cm(2). It suggests the existence of wavelength tissue specificity and dose dependency. Further studies are required to investigate the physiological mechanisms responsible for the effects of laser on tendinuos repair.

Achilles Tendon↗

PubMed2005

Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles.

Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopus extract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.

Adenylyl Imidodiphosphate↗

PubMed1988

Contamination of perfused donor kidneys by starch from surgical gloves.

Starch induces a variety of inflammatory reactions in humans and may contaminate surgical procedures from surgical gloves. Using polarized microscopy and electron microscopy, the authors observed starch particles in renal perfusate and in glomeruli of perfused donor kidneys. Group 1 consisted of 10 unusable kidneys handled with standard concern of glove sterility. Eight other kidneys (Group 2) were perfused with particular attention toward avoidance of starch contamination. The gloves were rinsed five times, and the cuffs were not dipped into the perfusate. Two kidneys (Group 3), deemed unsuitable for transplantation, were perfused for 24 hours with perfusate swished with unwashed sterile gloves. Group 4 consisted of five transplant biopsies performed within one week after transplantation. Perfusates alone were also circulated through the Waters Perfusion Machine continuously for 24, 72, and 188 hours with and without the use of gloves. The number of birefringent crosses were counted in each of 25 glomeruli per specimen. Group 1 displayed a mean of 1.8 birefringent crosses per glomerulus; Group 2, 0; Group 3, 5.3; and Group 4, 4.4. Groups 1 and 3 also exhibited birefringent crosses in peripheral renal vessels. Perfusate alone, handled without gloves, showed no birefringent crosses; by contrast, perfusate handled with gloves showed numerous birefringent crosses. The authors conclude that starch from surgical gloves can enter the perfusate and lodge in glomeruli and other sites of donor kidneys. Rinsing gloves five times and avoiding contact of the perfusate with the glove cuff effectively eliminates the contamination.

Birefringence↗

PubMed1993

Histiocytic lymphadenitis associated with a large joint prosthesis.

Distinctive microscopic findings in enlarged axillary lymph nodes removed during modified radical mastectomy in a 76-year-old woman with a previous ipsilateral total shoulder joint replacement are described. The sinuses of the lymph nodes were distended by numerous large macrophages that had abundant granular eosinophilic cytoplasm. Periodic acid-Schiff staining was positive, and polarization microscopy revealed birefringent particles of polyethylene. These findings were diagnostic of the reactive lymphadenopathy that is seen uncommonly in lymph nodes draining large joint prostheses.

Aged↗

PubMed2005

Physical compatibility of neonatal total parenteral nutrient admixtures containing organic calcium and inorganic phosphate salts.

PURPOSE: The compatibility of calcium and phosphate salts in total parenteral nutrient (TPN) admixtures at the highest concentrations recommended for preterm and term infants was studied. METHODS: Particulate matter from eight different macronutrient combinations was measured and counted (range, 1.8-50 mum) by a laser-based, single-particle optical sensing technique. Measurements were performed at four intervals after compounding the formulations under aseptic conditions (within 1 hour of preparation and at 6, 24, and 30 hours) at 23-27 degrees C. The number of particles measuring >or=5, >or=10, and >or=25 microm per milliliter of TPN admixture was recorded. Detailed visual inspections were also performed at these intervals, and pH was measured at the beginning (time 0) and end of the study (30 hours). Precipitated material was characterized by polarized microscopy and infrared spectroscopy. RESULTS: The TPN admixture with the lowest concentration of amino acids (0.5%), as well as the highest pH, resulted in significant growth of particulate matter over time. At 30 hours, the particle growth was accompanied by visible evidence of precipitation, which was confirmed to be dibasic calcium phosphate. Neither significant particle growth nor precipitation was noted in the remaining seven formulations, which had amino acid concentrations of 1-4%. CONCLUSION: Commonly used organic calcium and inorganic phosphate salts in cysteine-added, lipid-free TPN formulations at the highest recommended amounts for neonates were compatible when the amino acid concentration was between 1% and 4% and the dextrose concentration was 5% or 10%. The salts remained compatible for up to 30 hours at a room temperature of up to 27 degrees C. Precipitation of dibasic calcium phosphate occurred with lower amino acid concentrations and higher pH values.

Amino Acids↗

PubMed2002

Structure and development of stomata on the primary root of Ceratonia siliqua L.

Stomata of various sizes are produced on the primary root of Ceratonia siliqua L. Most are generated during embryogenesis, prior to seed desiccation. They can be detected on the dry embryo in a wide zone just above the root tip. Initially, large stomata are formed. These have the ability to induce divisions of their neighbouring cells, creating particular cell patterns around them. Later, small perigenous stomata are generated. As the root grows following seed germination, the stomatal zone overlaps with that of the root hairs. Although root stomata of C. siliqua undergo a structural differentiation that seems almost identical to that of the elliptical stomata formed on leaves, they are unable to move and remain permanently open. Polarizing microscopy of fully differentiated stomata and young stomata at the stage of stomatal pore formation revealed deposition of radial cellulose microfibril systems on their periclinal walls. However, these systems were less developed than those on leaf stomata, a feature that might be responsible for their inactivity. Besides, plastids of the root guard cells (GCs) do not differentiate into chloroplasts but function solely as amyloplasts. Root stomata have a short life span. During rapid and intense root growth, GCs cannot keep pace with the elongation of their neighbouring rhizodermal cells. They therefore split in their mid-region, transversely to the stoma axis. The two parts of the transversely torn stoma are dragged apart and a large opening is formed on the root surface, just above the substomatal cavity. The root stomata, together with these openings, may facilitate increased gaseous exchange during respiration and/or an increased transfer of some nutrients and water in the rapidly growing primary root.

Cell Wall↗

PubMed2002

Unique condensation patterns of triplex DNA: physical aspects and physiological implications.

Triple-stranded DNA structures can be formed in living cells, either by native DNA sequences or following the application of antigene strategies, in which triplex-forming oligonucleotides are targeted to the nucleus. Recent studies imply that triplex motifs may play a role in DNA transcription, recombination and condensation processes in vivo. Here we show that very short triple-stranded DNA motifs, but not double-stranded segments of a comparable length, self-assemble into highly condensed and ordered structures. The condensation process, studied by circular dichroism and polarized-light microscopy, occurs under conditions that mimic cellular environments in terms of ionic strength, ionic composition and crowding. We argue that the unique tendency of triplex DNA structures to self-assemble, a priori unexpected in light of the very short length and the large charge density of these motifs, reflects the presence of strong attractive interactions that result from enhanced ion correlations. The results provide, as such, a direct experimental link between charge density, attractive interactions between like-charge polymers and DNA packaging. Moreover, the observations strongly support the notion that triple-stranded DNA motifs may be involved in the regulation of chromosome organization in living cells.

Chromosomes↗

PubMed1990

Synovial fluid calcium pyrophosphate dihydrate crystals and alizarin red positivity: analysis of 3000 samples.

Three thousand synovial fluids (1312 patients: chronic pyrophosphate arthropathy (CPA), 41%; osteoarthritis (OA), 12%; rheumatoid arthritis (RA), 16%) were examined for crystals, including calcium pyrophosphate dihydrate (CPPD), by polarized microscopy (score 0-3); calcific particles, by alizarin red positivity (ARP; 0-3); and total cell count. For 1150 fluids, local joint inflammation was assessed as 'active' or 'inactive' using a summated score of six clinical variables. CPPD and ARP scores did not correlate, but each showed positive correlation with age (P less than 0.01, P less than 0.02 respectively). Pseudogout had the highest mean CPPD score (P less than 0.001); intermittent CPPD positivity (range 8-100%) was seen in serially aspirated CPA joints, and there was no difference in CPPD positivity or score between active and inactive CPA. ARP was most frequent in OA subsets (72% of CPA, 46% of OA, 31% of RA; P less than 0.001). ARP was more frequent in active than inactive OA (P less than 0.05) but showed no association with inflammation in CPA or RA. Cell counts were higher in RA and pseudogout compared to OA and CPA, and in active compared to inactive RA. No correlation was found between ARP or CPPD scores and cell count. Cholesterol crystals were uncommon (0.2%) and showed no disease or joint predilection. In arthritic joints, CPPD and calcific particles particularly associate with the OA process and ageing. CPPD may contribute to acute and other calcific particles to chronic inflammation in OA.

Adolescent↗

PubMed2001

The utilization of synovial fluid analysis in the UK.

OBJECTIVE: To ascertain what use is being made of synovial fluid analysis in the UK, who is carrying out polarized light microscopy (PLM), and what confidence clinicians have in the results. Subjects and methods. A postal survey was developed, piloted, adjusted and then posted to 535 people, 90% of whom were senior rheumatologists and 10% orthopaedic surgeons, whose names had been obtained from professional lists. RESULTS: Three hundred and eleven replies (59%) were obtained after 1 month. Analysis of the replies showed that only microbiological tests and PLM are used regularly, that these are used mainly for the diagnosis of acute arthritis, and that the majority of respondents would like data from these assays to be available within 24 h. The majority of the respondents (95%) had access to PLM and 80% said that clinicians should be able to use it. However, PLM is currently being undertaken by a variety of people: non-specialist technicians (34% of respondents), specialist technicians (35%) and clinicians (31%). Respondents were confident in microbiological assays but not in cell counts or PLM, unless they were undertaking it themselves. CONCLUSIONS: There is an urgent need for guidelines, standardization and education about the use of synovial fluid assays in the UK.

Crystallization↗

PubMed2007

2-methoxyestradiol induces spindle aberrations, chromosome congression failure, and nondisjunction in mouse oocytes.

2-Methoxyestradiol (2-ME) is a metabolite of 17beta-estradiol and a natural component of follicular fluid. Local concentrations of 2-ME may be increased by exposure to environmental pollutants that activate the expression of enzymes in the metabolic pathway from 17beta-estradiol to 2-ME. It has been suspected that this may have adverse effects on spindle formation in maturing oocytes, which would affect embryo quality. To study the dose-response patterns, we exposed denuded mouse oocytes to 2-ME during in vitro maturation. Meiotic progression, spindle morphology, centrosome integrity, and chromosome congression were examined by immunofluorescence and noninvasive polarizing microscopy (PolScope). Chromosomal constituents were assessed after spreading and C-banding. 2-ME sustained MAD2L1 expression at the centromeres and increased the number of meiosis I-blocked oocytes in a dose-dependent manner. 2-ME also caused dramatic dose-dependent increases in the hyperploidy of metaphase II oocytes. Some of these meiosis II oocytes contained anaphase I-like chromosomes, which suggests that high concentrations of the catecholestradiol interfere with the physical separation of chromosomes. Noninvasive PolScope analysis and tubulin immunofluorescence revealed that perturbations in spindle organization, which resulted in severe disturbances of the chromosome alignment at the spindle equator (congression failure), were caused by 2-ME at meiosis I and II. Pericentrin-positive centrosomes failed to align at the spindle poles, and multipolar spindles and prominent arrays of cytoplasmic microtubule asters were induced in 2-ME-exposed metaphase II oocytes. In conclusion, a micromolar level of 2-ME is aneugenic for mammalian oocytes. Therefore, exposure to 2-ME and conditions that increase the intrinsic local concentration of 2-ME in the ovary may affect fertility and increase risks for chromosomal aberrations in the oocyte and embryo.

2-Methoxyestradiol↗