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Polymorphism and desolvation of flupirtine maleate.

Crystallizates of the analgesic agent flupirtine maleate (Katadolon; ASTA Medica, Dresden, Germany) obtained from isopropanol are examined by X-ray diffractometry, polarization microscopy and thermoanalysis. Depending on the crystallizing conditions, the modifications A and B as well as an isopropanol solvate are observed. The inversion temperature A-->B of the enantiotropic modifications is 164 degrees C (differential scanning calorimetry (DSC) onset). During thermal desolvation, modification B is formed well below the inversion temperature. In concentrated isopropanol suspensions, the solvate and modification B are rapidly transformed into modification A. It is shown how phase-pure products consisting of modification A, which is better wettable with water and stable at room temperature, can be obtained.

Aminopyridines↗

Dynamic polarization of the microtubule cytoskeleton during CTL-mediated killing.

Efficient unidirectional killing by cytotoxic T lymphocytes (CTL) requires translocation of the microtubule organizing center (MTOC) to the target cell contact site. Here we utilize modulated polarization microscopy and computerized 3D reconstruction of tubulin and LFA-1 immunofluorescence images to investigate how this is accomplished. The results show that the MTOC is drawn vectorially to the contact site by a microtubule sliding mechanism. Once the MTOC arrives at the contact site, it oscillates laterally. Microtubules loop through and anchor to a ring-shaped zone (pSMAC) defined by the dense clustering of LFA-1 at the target contact site. Microtubules that run straight between the MTOC and pSMAC and then turn sharply may indicate the action of a microtubule motor such as dynein.

Animals↗

Light retardance by human oocyte spindle is positively related to pronuclear score after ICSI.

Disturbed spindle assembly increases risks of chromosome mal-segregation. Non-invasive polarization microscopy (PolScope) was employed in two centres to assess spindle integrity for the first time quantitatively in human oocytes from consenting patients undergoing intracytoplasmic sperm injection (ICSI) with respect to pronuclear (PN) score after fertilization. In one centre oocytes were selected before ICSI, in another selection was after ICSI according to PN score. In both centres, mean retardance of light by birefringent spindles in oocytes forming a pre-embryo with good PN score after ICSI was significantly higher compared with spindles in oocytes developing into a lower PN score pre-embryo with limited developmental potential (P < 0.001). Transfers involving oocytes with high retardance and at least one good PN score embryo resulted more frequently in a conception than transfers from oocytes with spindles of lower mean retardance and lower PN score embryos. There was a trend for an inverse relationship between age and magnitude of retardance in a small oocyte cohort. The study suggests that quantitative evaluation of mean retardance of light by the oocyte spindle predicts oocyte health, is related to PN score of the embryo and may be especially useful to assess oocyte quality in countries with legal restrictions to select after fertilization.

Adult↗

Visualization of flow-aligned type I collagen self-assembly in tunable pH gradients.

Collagen is a major component of the extracellular matrix that exhibits unique hierarchical organization at multiple length scales ranging from nano to macroscale. Despite numerous methods to create collagen-based biomaterials, the self-assembly process of collagen ex vivo is poorly understood. Here, we describe a system that uses a microfluidic method to investigate the dynamics of collagen self-assembly. A main inlet stream of semidilute soluble collagen-I is hydrodynamically focused by two side inlet streams, which gradually increases the pH in the main stream. This enables dynamic nonequilibrium investigation of the self-assembly process simultaneously at different positions and therefore different stages in the assembly process within the same system. The device is designed for in situ monitoring and characterization of collagen assembly using polarization microscopy and X-ray diffraction: the continuous extensional flow provides highly ordered phases of the macromolecules over a large distance in the outlet microchannel and allows for data collection without material damage. We further demonstrate that finite element method simulations provide a good description of our experimental results regarding the diffusive phenomena, flow profile, and pH distribution. Our approach has broad impact, since it provides a powerful means of controlling and investigating the dynamic self-assembly process of biomacromolecules.

Animals↗

[Calcium pyrophosphate deposits--a chameleon].

Calcium Pyrophosphate Dihydrate (CPPD) crystals deposit in articular fibro- or hyaline cartilage (chondrocalcinosis), joint capsules, synovium, periarticular ligaments and tendons resulting in an age dependent prevalence. These calcifications may be asymptomatic or may manifest as acute pseudogout arthritis, pseudorheumatoid arthritis, bursitis, tenosynovitis, tendinitis, polymyalgic syndrome or chronic pyrophosphate arthropathy. The diagnosis is based on the presence of intracellular CPPD crystals in synovial fluid detected by polarizing microscopy, the characteristic radiological changes and the typical clinical presentations. The therapy is symptom oriented or disease specific in case of an underlying metabolic disease such as hemochromatosis, hyperparathyroidism, hypophosphatasia, hypomagnesemia or hypothyroidism.

Calcium Pyrophosphate↗

Organic material and the optical properties of the dark zone in caries lesions of enamel.

Sections of uncavitated natural caries lesions of human enamel were extracted with various solvents and examined by polarizing microscopy. After lipid extraction, the dark zone enlarged by 19-162% and its birefringence increased, while after protein extraction it shrank by 37-92% and became less birefringent. It is concluded that occlusion of submicroscopic pores by organic material largely accounts for the optical properties of the dark zone. The results are not consistent with occlusion of pores by reprecipitation of mineral. On the basis of previous work, organic material in this location could influence demineralization and remineralization.

Calcium↗

Pathological mineralizations: calcifications and Si-bearing particles in soft tissues and their eventual relationship to different prostheses.

Polarizing microscopy (PM), scanning electron microscopy (SEM), x-ray dispersive analysis (EDAX), x-ray diffraction (XRD), and infrared spectrometry (IR) were used to study the following pathological mineralizations: calcifications and silicon(Si)-bearing mineralizations in cerebral tissue from an epileptic child; traces of Si-bearing particles in periprosthesic mammarian tissue, and calcifications in capsular mammarian tissue from a patient with a silicone gel mammarian implant, and 2 calcium-bearing compounds, a typical apatitic calcification, and a nonphosphorous-bearing calcification in arterial tissues. In this tissue we also found Si-bearing particles due to an artifact from glassware.

Aged↗

Early alterations in the collagen meshwork and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs.

OBJECTIVE: The purpose of this study was to evaluate matrix changes in knee cartilage prior to development of surface disruptions and to examine the ankle for evidence of osteoarthritis (OA)-like lesions. DESIGN: Guinea-pig ankles and knees were examined histologically or viewed with polarization microscopy to reveal changes in orientation of the collagen fibers. RESULTS: The medial femoral condyles were virtually free of histologic changes at 3 months of age. Changes were present by 6 months. Medial tibial plateau histologic changes were seen at 3 months which became more pronounced with age. Alterations in the collagen meshwork corresponding to areas of proteoglycan (PG) loss were noted in animals with an intact articular surface as early as 3 months. Histologic changes were noted in the ankles as early as 3 months of age which included surface disruptions, cell loss and loss of PG staining. Only knee joint composite histology scores were significantly elevated at 3 months while both knee and ankle scores were significantly elevated at 6 months. Knee and ankle joint scores were not different from each other at 6 months. CONCLUSIONS: Alterations in the orientation of the collagen network of the cartilage correlated with Safranin-O loss suggesting that alterations in collagen:PG interactions play a role in the early phases of the OA process and precede frank histologic changes in the articular surface. The results in this study report for the first time OA-like lesions occurring spontaneously in articular cartilage of the ankle in the guinea-pig.

Aging↗

Foreign material as a cause of choledocholithiasis.

BACKGROUND AND STUDY AIMS: There are sporadic reports concerning foreign material as a cause of choledocholithiasis. Gallstones obtained endoscopically from the biliary duct were analyzed to determine their structure and the presence of foreign material. The possible correlation between foreign material in gallstones and clinical data was investigated. PATIENTS AND METHODS: Gallstones were examined qualitatively using a crystallographic analysis by X-ray powder diffraction, and the foreign material which was found was subjected to morphological analysis using polarization microscopy. Patients' clinical data were obtained from clinical records, physical examination and documentation. RESULTS: Out of a group of 54 patients, foreign material was found in CBD stones of six individuals (11.1%). Four of these patients were from the group who had undergone a previous cholecystectomy (29/54, 13.8%), and surgical suturing material was found in these four. On the other hand, in the group who had not undergone the operation, foreign material was found in two patients out of 25 (8%); in one case it was cellulose and in the other case the fibre was not identified. CONCLUSIONS: Foreign material can account for lithiasis in the biliary duct. In contrast to present reports in the literature, we have proved that this is possible even in patients without previous surgical intervention in the pancreatobiliary region.

Adult↗

Congo red dichroism with dispersed amyloid fibrils, an extrinsic cotton effect.

The spectral absorption, optical rotatory dispresion, and circular dichroism associated with interaction of Congo red dye with partly purified suspensions of amyloid fibril fragments were examined. A set of phenomena consistent with a Cotton effect was found. A nearly identical set of phenomena was obtained with poly-L-lysine in its alpha-helical form. The dichroism seen when Congo red binds to amyloid substance in tissue sections can also be interpreted as a Cotton effect. This suggests that some special conformation, presumably in protein, is present in a major constituent of amyloid. This conformation is not present in gamma globulin, Bence-Jones protein, albumin, fibrinogen, or other proteins tested so far. These and other optical properties of amyloid substance can be used to compare amyloid deposits in different human cases and in different species. Extension of the use of polarization microscopy with other dyes that bind to other substances in tissue sections should permit more exquisite probing of the conformation of important macromolecules in situ in cells and tissues than has hitherto been possible.

Amyloid↗

A general native-state method for determination of proliferation capacity of human normal and tumor tissues in vitro.

An important need in cancer research and treatment is a physiological means in vitro by which to assess the proliferation capacity of human tumors and corresponding normal tissue for comparison. We have recently developed a native-state, three-dimensional, gel-supported primary culture system that allows every type of human cancer to grow in vitro at more than 90% frequency, with maintenance of tissue architecture, tumor-stromal interaction, and differentiated functions. Here we demonstrate that the native-state culture system allows proliferation indices to be determined for all solid cancer types explanted directly from surgery into long-term culture. Normal tissues also proliferate readily in this system. The degree of resolution of measurement of cell proliferation by histological autoradiography within the cultured tissues is greatly enhanced with the use of epi-illumination polarization microscopy. The histological status of the cultured tissues can be assessed simultaneously with the proliferation status. Carcinomas generally have areas of high epithelial proliferation with quiescent stromal cells. Sarcomas have high proliferation of cells of mesenchymal organ. Normal tissues can also proliferate at high rates. An image analysis system has been developed to automate proliferation determination. The high-resolution physiological means described here to measure the proliferation capacity of tissues will be important in further understanding of the deregulation of cell proliferation in cancer as well as in cancer prognosis and treatment.

Breast Neoplasms↗

Biomesogenic matrix systems.

The bifunctionally reactive nucleoside and distant nucleoside analogs adenosine (Ado), S-[(adenine-9-yl)methoxyethyl]-L-cysteine (Na-salt) (cysA) and 9-vinyladenine (vA) in aqueous solutions assemble on complementary polyuridylic acid templates to form complex lyomesophases. The systems are investigated by polarizing microscopy, differential scanning calorimetry (DSC) and 1H- and 31P-nmr spectroscopies, assisted by molecular modeling studies. The results indicate the importance of biomesogenic (pre)ordering in nucleic acid native and artificial matrix reactions.

Adenine↗

Self-assembly and lyomesophases formed by long-chain alkoxymethyl-nucleobase derivatives.

Amphiphilic complementary nucleobase derivatives, containing n-octadecyloxymethyl substituents at the N1 position of pyrimidine and N9 of purine, dissolved in chloroform form non-specific lyotropic mesophases, which were analyzed by optical polarizing microscopy. Molecular modeling studies visualize hypothetical horizontal and vertical nucleobase hydrogen-bonding and stacking arrangements, as well as aliphatic long-chain interstrand interaction.

Chloroform↗

Physical characterization of polymer electrolytes as novel iontophoretic drug delivery devices.

Polymer electrolytes are solid-like materials formed by dispersing a salt at the molecular level in a high molecular weight polymer such as poly(ethylene oxide) (PEO). They have been extensively studied for use in electrochemical applications such as batteries and display devices. This paper considers a novel application of polymer electrolytes as the basis of iontophoretic drug delivery systems. Polymer electrolyte films were cast from solutions of PEO and various drug salts using either water or an acetonitrile/ethanol mixture as the solvent. These films were characterized by variable-temperature polarizing microscopy (VTPM), differential scanning calorimetry (DSC), and alternating current (AC) impedance analysis. The films were around 100-micron thick and mechanically strong; the optical and thermal methods provided evidence that the polymer electrolytes had crystalline and amorphous phases, although some drugs may exist in films as nanodispersions. The amorphous phase is important as ions have greater mobility in this phase and therefore allow a current to be passed when the material is incorporated into a device such as one suitable for drug delivery by iontophoresis. The AC impedance analysis showed that the conductivity of the films varied between 10(-6) and 10(-3) S cm-1, depending on the salt, casting solvent, and temperature. Two drugs in particular were shown to be promising candidates in these systems: lidocaine hydrochloride and lithium chloride.

Drug Delivery Systems↗

Sarcomere size in developing muscles of a tarsonemid mite.

The embryo of a tarsonemid mite was found to be suitable for in vivo observations of muscle development by polarization microscopy. The four dorsal muscles of the metapodosoma each contain three sarcomeres, the anterior two of which can be seen clearly. These sarcomeres can be identified and followed during much of their development. Sarcomeres are about 2.5 micra long when first detected and increase in length until they are about 10 micra long. The change in length is associated with a slow, approximately constant rate of increase in the length of the A region, and an initially slow then much more rapid increase in the length of the I band. Preceding the period when the I band elongates rapidly there is an increase in the diameter of the muscle fibers and an increase in the retardation of the A band. A, I, Z, and H bands are visible during most of these changes. The change in A band length has been interpreted in terms of the growth of the A filaments which have been observed by electron microscopy in muscles of other animals. It is suggested that the exceptionally long sarcomeres in this mite result from the early fixing of the number of sarcomeres in a given muscle fiber.

Animals↗

The impact of chromosomes and centrosomes on spindle assembly as observed in living cells.

We analyzed the role that chromosomes, kinetochores, and centrosomes play in spindle assembly in living grasshopper spermatocytes by reconstructing spindles lacking certain components. We used video-enhanced, polarization microscopy to distinguish the effect of each component on spindle microtubule dynamics and we discovered that both chromosomes and centrosomes make potent and very different contributions to the organization of the spindle. Remarkably, the position of a single chromosome can markedly affect the distribution of microtubules within a spindle or even alter the fate of spindle assembly. In an experimentally constructed spindle having only one chromosome, moving the chromosome to one of the two poles induces a dramatic assembly of microtubules at the nearer pole and a concomitant disassembly at the farther pole. So long as a spindle carries a single chromosome it will persist normally. A spindle will also persist even when all chromosomes are detached and then removed from the cell. If, however, a single chromosome remains in the cell but is detached from the spindle and kept in the cytoplasm, the spindle disassembles. One might expect the effect of chromosomes on spindle assembly to relate to a property of a specific site on each chromosome, perhaps the kinetochore. We have ruled out that possibility by showing that it is the size of chromosomes rather than the number of kinetochores that matters. Although chromosomes affect spindle assembly, they cannot organize a spindle in the absence of centrosomes. In contrast, centrosomes can organize a functional bipolar spindle in the absence of chromosomes. If both centrosomes and chromosomes are removed from the cell, the spindle quickly disappears.

Animals↗

The mitotic apparatus. Structural changes after isolation.

The fibrous structure of the mitotic apparatus (MA) isolated from dividing sea urchin eggs undergoes no changes visible in phase contrast during extended storage, but the solubility of the MA rapidly decreases after isolation. Polarization microscopy shows that a decrease in the birefringence of the MA also occurs after isolation and is correlated with the loss of solubility. This loss of birefringence indicates that some structural change takes place during this period, and such a change was demonstrated by means of electron microscopy. The tubular filaments which form the spindle of the intracellular MA and of the freshly isolated MA were found to break down during storage to rows of dense granules, this loss of continuity presumably accounting for the loss of birefringence. The interrelations of the observed changes and the significance of these observations for investigations on the isolated MA are discussed.

Animals↗

Anomalous contraction of invertebrate striated muscle.

The phenomenon of A band shortening or contraction has been investigated in glycerinated myofibrils of Pecten irradians, Homarus americanus, Cambarus virilis, and Limulus polyphemus through the techniques of ultraviolet microbeam inactivation and polarization microscopy. With the former method, it has been shown that these muscles, even though exhibiting the shortening effect, contract in a manner consistent with only the sliding filament model. Intrinsic birefringence studies have indicated no significant changes in mass distribution or orientation within the shortened A bands. Except in the case of Limulus muscle, the shortening effect was seen only in contraction under tension. The magnitude of this anomalous phenomenon was dependent upon glycerination time and has been duplicated in rabbit psoas muscle through brief trypsin treatment. A band shortening could not be observed in glutaraldehyde-fixed muscle or in myofibrils glycerinated for only short periods. It has been concluded that the phenomenon of A band contraction is an artifact induced by the glycerination procedure, possibly through weakening of the sarcomere structure. However, the fact that the A band shortens under tension rather than lengthens poses an interesting paradox.

Adenosine Triphosphate↗