Phase Contrast Light Microscopy Research Articles

Reconstruction of a century of airborne asbestos concentrations.

Airborne asbestos concentrations have been reconstructed for the entire 20th century for the first time through a combination of paleolimnological methods, particle-separation techniques, and analytical transmission electron microscopy. Pb concentrations and respirable aerosol mass concentrations in air and sediments yielded collection efficiencies of approximately 3000 m3 of air per gram of lake sediment. Airborne concentrations of chrysotile, the most common type of asbestos, reconstructed from control lake sediments echoed chrysotile's usage during the 20th century, with the highest concentrations mid-century (approximately 0.1 fibers/cm3) and then decreasing in the last quarter century. Reconstructed airborne concentrations of anthophyllite asbestos, a byproduct of local talc mining and milling, increased from <0.004 to 0.022 fibers/cm3 from 1846 to 1967. These anthophyllite concentrations during the approximately 100-year period of talc mining correlated well (r2 = 0.80, p < 0.01) with annual production of local talc and were much higher (p = 0.004) than concurrent concentrations in a control lake located upwind of the mines and mills. All of the chrysotile and more than 70% of the anthophyllite asbestos fibers were too narrow to be detected by phase-contrast light microscopy, the method used to measure airborne fiber concentrations before approximately 1980.

Aligned biodegradable nanofibrous structure: a potential scaffold for blood vessel engineering

A unique biodegradable nanofibrous structure, aligned poly( l-lactid- co- ε-caprolactone) [P(LLA-CL)] (75:25) copolymer nanofibrous scaffold was produced by electrospinning. The diameter of the generated fibers was around 500 nm with an aligned topography which mimics the circumferential orientation of cells and fibrils found in the medial layer of a native artery. A favorable interaction between this scaffold with human coronary artery smooth muscle cells (SMCs) was demonstrated via MTS assay, phase contrast light microscopy, scanning electron microscopy, immunohistology assay and laser scanning confocal microscopy separately. Tissue culture polystyrene and plane solvent-cast P(LLA-CL) film were used as controls. The results showed that, the SMCs attached and migrated along the axis of the aligned nanofibers and expressed a spindle-like contractile phenotype; the distribution and organization of smooth muscle cytoskeleton proteins inside SMCs were parallel to the direction of the nanofibers; the adhesion and proliferation rate of SMCs on the aligned nanofibrous scaffold was significantly improved than on the plane polymer films. The above results strongly suggest that this synthetic aligned matrix combines with the advantages of synthetic biodegradable polymers, nanometer-scale dimension mimicking the natural ECM and a defined architecture replicating the in vivo-like vascular structure, may represent an ideal tissue engineering scaffold, especially for blood vessel engineering.

Cellular toxicity induced by different pH levels on the R3230AC rat mammary tumour cell line. An in vitro model for investigation of the tumour destructive properties of electrochemical treatment of tumours

Introduction: The aim of this study was to evaluate the cellular toxicity of different pH levels on the R3230AC mammary tumour cell line (clone-D) in vitro and to determine in what way the pH affects the tumour cells. The results could be used to interpret the cell damaging effects seen in electrochemical treatment of tumours (EChT), where pH alteration in tissue is the major event. Methods: Tumour cells were treated with pH 3.5, 5, 7, 9, 10 and 11 for 10, 20 or 30 min, respectively, followed by studies with the viability assay 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl tetrazolium bromide (methyltetrazolium (MTT)), morphological observation in phase contrast microscope (PCM) and light microscope, nucleotide analogue incorporation (BrdU; 5-Brdmo-2′-deoxyuridine), Caspase-3 activity measurement and detection of DNA fragmentation by an agarose gel electrophoresis. Results: In the viability assay, it was found that different pH levels had cytotoxic effects; these effects were dependent on the pH value and on the time of exposure at a given pH. Morphologically, cells in pH 3.5 and 5 had shrunk, were rounded and had condensed chromatin, whereas prominent cell swelling and nuclear expansion were seen in the pH 9- and 10-treated cells. Gross cytolysis was found in pH 11. A BrdU incorporation assay indicated that proliferation rate is inhibited markedly both with decreasing and increasing pH. Significant Caspase-3 activity was found in pH 3.5 and 5 groups. Caspase-3 levels for the alkaline exposure were equal or below the normal control. DNA ladder formation, a characteristic of apoptosis, was only visualised in the treatment of pH 3.5 for 30 min. Conclusions: pH changes inhibit cell proliferation and decrease cell viability. The pathway of killing tumour cell in low pH probably has at least two directions: apoptosis and cell necrosis, whereas high pH results in only cell necrosis. The study suggests that low pH environment can induce apoptosis in unphysiological condition comparable with tissue pH at EChT. In addition, it seems that R3230AC mammary tumour cells are more tolerant to high pH than to acidic changes. This supports the theory that anodic EChT should be more efficient than cathodic.

Basic studies on gene therapy of human malignant melanoma by use of the human interferon β gene entrapped in cationic multilamellar liposomes.: 1. Morphology and growth rate of six melanoma cell lines used in transfection experiments with the human interferon β gene

Six cell lines of human malignant melanoma: A375, A375.2, G361, HMV-1, MM8.1 and WM115 were seeded at densities of 1x10(4) cells/ml, 2 x 10(4) cells/ml or 3 x 10(4) cells/ml of RPMI medium supplemented with 10% fetal calf serum and antibiotics in a humidified atmosphere of 5% CO2 at 37 degrees C. A375 cells were also grown in Dulbecco's minimum Eagle's medium (DMEM medium). The morphology was studied by phase contrast light microscopy. At 4 days after seeding the colonies of A375 cells and HMV-1 cells were oval-shaped, the cells were polyhedrical and were making contact with each other regularly. The remaining cells were scattered, more elongated, and made contact randomly. G361 cells and MM8.1 cells tended to form superposed layers before 100% confluency was achieved. There were great differences in the growth rate and doubling time of melanoma cells. The doubling time in day 1 was short (around 6-12 h) in the case of A375, G361 and HMV-1 cells, longer (around 18 h) in the case of MM8.1 cells and very long (ranging between 26 and 89 h) for A375.2 and WM115 cells. There were also differences in the doubling time of cells as a function of the cell density at seeding. On the other hand, except for MM8.1 cells, there were differences between the doubling time in day 2 compared to day 1.

Pseudoasbestos bodies and fibers in bronchoalveolar lavage of refractory ceramic fiber users.

Refractory ceramic fibers (RCF) are widely used to replace asbestos in applications requiring high heat resistance. Ferruginous bodies mimicking asbestos bodies (ABs) have been detected in the lungs of RCF production workers. This raises the question about their presence in other occupational groups and whether "typical ABs" still reflect past asbestos exposures in all settings. An AB counting by phase-contrast light microscopy and a screening test by analytical electron microscopy were systematically performed on all bronchoalveolar lavage fluids (BALF) submitted to our laboratory in 1992 through 1997 (n = 1,800). When RCF were detected in electron microscopy, the structures considered as "typical ABs" were marked under light microscopy and prepared for further chemical and structural analysis. Pseudo-ABs on RCF were detected in samples from nine subjects (0.5%). All of them had worked either as foundry workers, steel workers, or welders. In these subjects, alumino-silicate fibers compatible with RCF accounted for 42% of the core fibers analyzed, other nonasbestos fibers for 28%, and asbestos fibers for 30%. ABs thus remain a valid marker of asbestos retention but attention must be paid to a possible occurrence of pseudo-asbestos bodies on RCF and other nonasbestos fibers in end-users of refractory fibers.

Evaluation of ultra-thin poly(epsilon-caprolactone) films for tissue-engineered skin.

Various natural and synthetic polymeric materials have been used as scaffold matrices for tissue-engineered skin. However, the commercially available skin replacement products pose problems of poor mechanical properties and immunological rejection. We have thus developed a film of 5 microm thickness, via biaxial stretching of poly(epsilon-caprolactone) (PCL), as a potential matrix for living skin replacements. The aim of this study was to evaluate the feasibility of using biaxially stretched PCL films as matrices for culturing human dermal fibroblasts. For this purpose, we cultured human dermal fibroblasts for 7 days on the films. Glass cover slips and polyurethane (PU) sheets were used as controls. The data from phase contrast light, confocal laser, and scanning electron microscopy suggested that biaxially stretched PCL films support the attachment and proliferation of human dermal fibroblasts. Thymidine-labeling results showed quantitatively that cell proliferation on the PCL films was superior to that on the PU samples. These results indicated that biaxially stretched PCL films supported the growth of human dermal fibroblasts and might have potential to be applied in tissue engineering a dermal equivalent or skin graft.

Microstructure of acid–induced skim milk–locust bean gum–xanthan gels

Abstract The microstructure of acid skim milk gels (14% w/w milk protein low heat powder) with or without addition of locust bean gum (LBG), xanthan gum (XG) and LBG/XG blends was determined by transmission electron microscopy (TEM), phase-contrast light microscopy (PCLM) and scanning electron microscopy (SEM). Three polysaccharide concentrations (0.001%, 0.02% and 0.1%, w/w) were used for binary mixtures. In the case of ternary mixtures, three LBG/XG weight ratios were used (4/16, 11/9 and 16/4) at 0.02% total polysaccharide concentration. Control acid skim milk gels were structured by a homogeneous network of casein particles (0.1–0.7 μm in diameter) and clusters immobilizing whey in small pores (1–5 μm in diameter). Filamentous structures and small aggregates were observed at the surface of casein particles. Low concentration of LBG or XG (0.001% w/w) did not affect markedly the microstructure of acid skim milk gels. Conversely, LBG or XG at 0.02 or 0.1% concentration and LBG/XG blends at the three ratios selected had a great influence on the gel microstructure. Although the size and surface structure of the casein particles were not modified by the presence of polysaccharides, the primary casein network appeared very compact with a decrease of pore size and a large increase in the porosity of the network at the supramolecular level (sponge-like morphology). The effect is stronger for gels containing LBG and XG used at higher concentration and less apparent for gels containing LBG/XG blends. Skim milk/XG gels were highly organized into fibrous structures whereas skim milk/LBG gels were more heterogeneous. These structures were discussed in the light of volume-exclusion effects (demixing) and specific interactions between casein micelles and polysaccharides. At the three weight ratios, skim milk/LBG/XG gels displayed both jagged “coral-like”, “veil-like” and filamentous structures. These structures could originate from a secondary network constituted by the known LBG/XG synergistic interactions.

The effects of cell passages on the cell morphology and the outcome of herpes simplex virus type 1 infection

Because cell cultures are essential in biological research which involves the analysis of virus morphogenesis, this study focused on examining the significance of cell passages. Human embryonic lung fibroblasts (MRC-5) at passage (P) 27 were seeded twice a week to P 32, P 40, and P 48, when just at confluence and then infected with herpes simplex virus type 1 (HSV-1). The structure of the non-virus-infected (MOCK) and HSV-1 infected cells, the amount of cellular infectious virus particles and the capability to express HSV-1 glycoproteins C (gC-1) and D (gD-1) were investigated by phase-contrast and immunofluorescence light microscopy, immunogold cryosection EM, plaque assays, immunoblots, and total protein assays. Modified cell structure including fragmentation of tubulin fibers were visible in MOCK from P 38 onwards. The quantity of vimentin remained unchanged while actin accumulated and β-tubulin decreased in HSV-1 infected late P cells compared to early P cultures. Cells of high P counts contained significantly fewer infectious virus particles, very likely of lower virulence, and their expression of gC-1 and gD-1 were concordantly reduced. These observations indicate that the number of cell P must be considered in order to reproduce results of cell biology and viral morphogenesis. The MRC-5 cells ought not to be passaged more than ten times beyond P 27 in the laboratory.

Localisation of fungal hyphae in wood using immunofluorescence labelling and confocal laser scanning microscopy

This study explored the feasibility of using immunofluorescence labelling in conjunction with confocal laser scanning microscopy (CLSM) for detection of common fungal colonisers of unseasoned radiata pine in New Zealand. Wood sections infected with Ophiostoma piceae were treated with monoclonal antibody IF3 (1), and then Oregon green 514 goat anti-mouse IgG, a fluorescent secondary antibody. Additional wood sections infected with other Ophiostoma spp., Sphaeropsis sapinea, Leptographium procerum, Trichoderma sp. and Phlebiopsis gigantea were treated similarly to determine whether the antibody was specific to O. piceae or was recognising other fungal species. Sections were examined using phase contrast and fluorescence light microscopy prior to CLSM. Immunolabelled fungal hyphae showed relatively weak fluorescence compared to the strong autofluorescence of wood cell walls and extractives. Labelled hyphae of O. piceae were detected in wood using CLSM but not with ordinary fluorescence microscopy. This is because CLSM has stronger illumination power and superior imaging ability compared with ordinary fluorescence microscopy. The monoclonal antibody did not cross-react with the other Ophiostoma species. However, non-specific antibody binding was observed with L. procerum and Trichoderma species. Furthermore, cell walls of L. procerum showed strong autofluorescence with optical properties similar to wood extractives when examined prior to incubation with the monoclonal and secondary antibody, therefore cross-reactivity tests were inconclusive for Leptographium and Trichoderma species. The current investigation demonstrated that CLSM provides possibilities for future investigations on in situ interactions of common radiata pine fungal colonisers, with one another and with wood.

Effects of hydrostatic pressure on cultured bovine trabecular meshwork cells.

In order to explore the effects of pressure on trabecular meshwork cells, bovine trabecular meshwork cells were cultured in vitro and subjected to different levels of hydrostatic pressure. The cellular morphology, ultrastructure and phagocytosis were studied with inverted phase-contrast microscopy, light microscopy and transmission electron microscopy, etc. It was found that the cells subjected to 2.0 kPa or 2.67 kPa for 48 h had no remarkable difference as compared with the controls in terms of parameters observed. Those under 4.0 kPa for 24 h showed slight changes in structure and a mild decrease in phagocytic function. The damage appeared more severe if the pressure was higher or lasted longer. From the above we conclude that trabecular meshwork cells can only bear pressure below a certain level. They may be destroyed structurally or impaired functionally by pressure over this level.

Effects of temperature and heat activation on germination of individual spores of Bacillus subtilis.

Phase intensity changes of individual germinating spores of Bacillus subtilis were determined by phase-contrast light microscopy and image analysis. Two germination phases were investigated. The length of the time period before a change in phase brightness was evident and the duration of the phase intensity change until a constant greylevel was maintained. The incubation temperature (37 and 20 degrees C) and heat activation (10 min at 65 degrees C) had a distinct effect on both phases. At 37 degrees C, spores of B. subtilis 604 started to show a decrease in brightness in L-alanine buffer after 3-39 min and needed 10-39 min to complete the phase change. At 20 degrees C, lag times of 10-100 min were observed and the spores needed 30-100 min to reach a constant greylevel. Heat activation and subsequently exposure to L-alanine buffer at 20 degrees C reduced the lag phase to 6-90 min and the phase change was finished after 30-60 min. Our results indicate enzymatic involvement before and during the phase intensity change of germinating spores.

Determination of machinablity of PM materials by drilling tests : J.R.Spirko et al. (General Carbide Corp, Johnstown, USA.) Int. J. Powder Metall., vol 34, no 3, 1998, 37–46

We recently found that polyclonal antibodies to laminin, a basement membrane-related glycoprotein, inhibited murine lung morphogenesis when added to organ cultures of mouse embryonic lung. Using a series of monoclonal antilaminin antibodies with previously characterized subunit specificity (termed AL-1, AL-2, AL-3, AL-4, and AL-5), the deposition and functional involvement of different laminin domains in the developing lung were investigated. By immunohistochemistry the antibodies' reactivity was largely localized to the basement membrane, but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were cultured for 3 days in the presence of 50–100 μg/ml of each antibody or in the presence of the same concentration of immunoglobulins G and M, laminin-neutralized antibody, or medium alone. Cultures were monitored by phase-contrast microscopy, light microscopy, and immunofluorescence. Although all antibodies penetrated the tissues in culture, only two of them inhibited branching activity. These two antibodies were AL-1, which binds on or near the cross region of laminin, and AL-5, which binds to the lateral short arms at the globular end regions of the B chain of laminin. Inhibition of branching with these two antibodies was dose-dependent and statistically significant for the two concentrations used. AL-2, AL-3, AL-4, laminin-neutralized antibodies and control immunoglobulins did not alter lung morphogenesis. The two domains of laminin that promote lung branching morphogenesis have been reported by others to promote the attachment of a variety of cells and/or bind heparin. These domains of laminin may promote branching morphogenesis by facilitating cell attachment and, consequently, cell proliferation.

Assessment of environmental asbestos exposure in Turkey by bronchoalveolar lavage.

Environmental or domestic exposure to asbestos fibers originating from local soil is responsible for a high incidence of diseases in large rural areas of Turkey. Bronchoalveolar lavage fluids (BALF) were obtained for 65 Turkish subjects originating from these areas and for 42 Turkish controls. Asbestos bodies (ABs) and uncovered fibers (UFs) were quantified by phase contrast light microscopy. Total fiber burden was determined by transmission electron microscopy. The main asbestos types disclosed were tremolite and to a lesser extent chrysotile. AB and fiber concentrations were higher in environmentally exposed subjects (geometric mean [geometric standard deviation]: 5.20 [6.22] AB/ml, 444 [11.6] tremolite fibers/ml) than in control subjects (0.22 [1.45] AB/ml, 12.0 [15.4] tremolite fibers/ml) (p < 0.001). In subjects environmentally exposed in Turkey, AB burdens on tremolite were in the same range as those on commercial amphiboles in subjects occupationally exposed in Belgium. In Turkish subjects, values above either 1 AB/ml, 3 uncovered fiber/ml in light microscopy, or 300 fibers/ml in electron microscopy indicated usually an abnormal alveolar retention reflecting a significant cumulative exposure from environmental or domestic origin. These observations are probably valid for other areas in the world where diseases associated with environmental exposure to soil- derived asbestos fibers occur and for immigrants originating from these areas.

Comparison of the Effects of Potential Parenteral Vehicles for Poorly Water Soluble Anticancer Drugs (Organic Cosolvents and Cyclodextrin Solutions) on Cultured Endothelial Cells (HUV-EC)

□ The effect of dilution of parenteral vehicles (organic cosolvent and 0.1M cyclodextrin solutions) on cultured endothelial cells (HUV-EC) were compared in vitro. Cell morphology was observed by phase contrast light microscopy and cell viability by measuring 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction or intracellular lactate dehydrogenase (LDH) activity and total protein. Disruption of the HUV-EC monolayer was observed at dilutions of 1 in 20 for the melphalan and PEP cosolvents, 1 in 100 for an investigational drug cosolvent, and 1 in 10 for 0.1M dimethyl-β-cyclodextrin. In comparison, 0.1M SBE7M- and HP-β-cyclodextrin caused only minor disruption at a 1 in 5 dilution. MTT reduction, intracellular LDH, and total protein were decreased following exposure to 1 in 10 dilution of the melphalan cosolvent. For other test solutions, intracellular LDH activity and total protein were measured, and reductions were observed following exposure to 1 in 10, 20, and 50 dilutions of the investigational drug cosolvent and 1 in 5 dilution of DM-β-cyclodextrin (0.1M). At a dilution of 1 in 10, no delayed toxicity was observed for cosolvents or cyclodextrin solutions. Hence, 0.1M SBE7M- or HP-β-cyclodextrin formulations may be less damaging to the venous endothelium at the site of injection than organic cosolvent formulations.

Effect of Shear Flow on the Morphology and Phase Behavior of a Near-Critical SAN/PMMA Blend

An ongoing debate concerns whether experimental observations of flow-induced miscibility of polymer blends actually result from a true shift of the critical temperature. To elucidate the origin of the phenomenon more clearly, we have investigated the structural changes of phase domains of a number of polymer blends under the influence of flow. The most recent study used a near-critical blend of poly(styrene-co-acrylonitrile)/poly(methyl methacrylate), which exhibits LCST type phase behavior. Rheo-SALS (small angle light scattering) was used to probe the time-dependent structure evolution during shear flow. Quenched samples were examined with TEM and phase contrast light microscopy, and the Fourier transforms of digitized micrographs were compared with two-dimensional light scattering measurements of the same samples. The blends were also subjected to pressure-driven flows and drag flows at very high stresses, and their morphology was similarly studied. The structure evolution due to flow could be explained by hydrodynamic effects consistent with droplet breakup theory.

Transcrystallization in syndiotactic polypropylene induced by high-modulus carbon fibers

It was first shown that transcrystallization can be induced in syndiotactic polypropylene (sPP) when a carbon fiber (CF) of high-modulus (HM) is embedded in the melt of sPP crystallizing under quiescent conditions. High-tenacity carbon fiber (HTCF), on the other hand, did not cause transcrystalline growth. Coating of HMCF by silicon carbide (SiC) stopped the transcrystallization of sPP. The difference in the morphology of the transcrystalline layer between isotactic PP (iPP) and sPP was revealed by phase contrast light (PCLM), scanning electron (SEM) and atomic force microscopy (AFM) taken from the etched surface of single fiber microcomposite specimens.

Using confocal laser scanning microscopy, scanning electron microscopy and phase contrast light microscopy to examine marine biofilms

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 16:199-204 (1998) - doi:10.3354/ame016199 Using confocal laser scanning microscopy, scanning electron microscopy and phase contrast light microscopy to examine marine biofilms T. A. Norton1, R. C. Thompson1,*,**, J. Pope2, C. J. Veltkamp3, B. Banks4, C. V. Howard2, S. J. Hawkins1,** 1Port Erin Marine Laboratory, School of Biological Sciences, University of Liverpool, Port Erin, Isle of Man IM9 6JA, United Kingdom 2Department of Foetal and Infant Pathology, and 3School of Biological Sciences, University of Liverpool L69 3BX, United Kingdom 4School of Biological Sciences, University of Southampton, Southampton SO16 3BX, United Kingdom *Addressee for correspondence. E-mail: rct@soton.ac.uk **Present address: Division of Biodiversity & Ecology, School of Biological Sciences, Bassett Crescent East, University of Southampton, Southampton SO16 7PX, United Kingdom ABSTRACT: Confocal laser scanning microscopy (CLSM) was used to examine living organisms within marine biofilms growing on microscope slides and on natural opaque and uneven substrata such as rocks and shells. The results were compared to images of identical fields observed under transmitted light and phase contrast microscopy and scanning electron microscopy. Confocal microscopy proved superior in several respects: (1) It gave clear images of organisms even if they were overlaid with a thick layer of mucus, other organisms and debris. (2) It did not require the biofilm to be disrupted or dried and allowed samples to be re-examined at intervals. (3) It could also distinguish living (fluorescing) organisms from dead cells or inorganic matter. (4) Examining optical 'slices' of samples allowed the 3 dimensional structure of the biofilm to be visualised. However, species identification, particularly of diatoms, was much easier using scanning electron microscopy. Methods were developed for overlaying a fixed grid on samples so that specific sites or individual organisms could be accurately relocated for re-examination. Appropriate staining methods were also evaluated. Confocal microscopy will prove to be an invaluable aid for examining the structure and growth of living biofilms in studies of shore ecology and marine fouling. KEY WORDS: Confocal microscopy · SEM · Self-registration · Biofilms · Microalgae · Cyanobacteria · Biofouling Full text in pdf format PreviousExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 16, No. 2. Publication date: November 27, 1998 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 1998 Inter-Research.

Calcium and the anionic polypeptide fraction (APF) have opposing effects on cholesterol crystallization in model bile

Background/Aims: Cholesterol gallstones contain both calcium and biliary proteins, but their respective roles in gallstones pathogenesis are unknown. We have studied the effects of calcium and a major biliary protein, anionic polypeptide fraction, on the process of cholesterol crystallization in bile. Methods: Anionic polypeptide fraction was purified from human bile. Model bile composed of cholesterol, egg yolk lecithin and sodium taurocholate was prepared in a lipid concentration (18 mM, 37 mM, and 120 mM, respectively) simulating lithogenic human gallbladder bile. The crystallization process was observed by phase contrast light microscopy, and sequential separation of precipitable cholesterol structures by sucrose density gradient ultracentrifugation. Results: Addition of calcium, or anionic polypeptide fraction alone, or both together did not influence the crystal observation time of bile (the time which elapsed from initiation of supersaturation to the first appearance of crystals). However, the rate and quantity of cholesterol precipitation and crystal formation were affected by both. Calcium increased in a dose-dependent manner the cholesterol monohydrate crystal mass before apparent equilibrium was reached. This effect was inhibited by anionic polypeptide fraction, which increased the amount of cholesterol within precipitable phospholipid vesicles, and decreased the rate of crystal formation. Fluorescence-labeled anionic polypeptide fraction revealed that anionic polypeptide fraction (with and without calcium) was primarily associated with vesicle aggregates. Conclusions: Our data demonstrate that calcium and anionic polypeptide fraction have opposing effects on the process of cholesterol crystallization and the resultant crystal mass without influencing the crystal observation time of bile. These findings suggest that biliary proteins, in addition to being crystallization effectors by themselves, may further influence cholesterol crystallization and gallstone formation by interacting with calcium and possibly other elements that coexist in bile.

Crystallization and melt behaviour of isotactic poly((4-α,α-dimethyl-benzyl) phenyl methacrylate)

The crystallization and melting behaviour of practically 100% isotactic poly((4-α,α-dimethylbenzyl) phenyl methacrylate) has been studied by d.s.c. and light microscopy. Crystallization from the melt seemed to be non-spherulitic. The maximum crystallization rate, which could only be determined by d.s.c., was found to be 2% min −1 at a crystallization temperature T of 155°C. Assuming that the growth rate theory for small supercooling is valid and by taking the appropriate value 51.6 K for the cosntant c 2 of the WFL equation, the chain-folded surface free energy σ e was estimated at 89 × 10 −2. The melting curves showed two melting endotherms. Crystallization from the melt produced a small melting endotherm just above T c. This peak may originate from secondary crystallization of tie molecules melt trapped within the crystalline phase. The peak temperature of the second melting endotherm increased linearly with T c, yielding an extrapolated value for the equilibrium melting temperature T m O of 282°C. Avrami analysis showed for the exponent n noninteger values between 2 and 3, indicating at least a non-spherulitic growth process which is confirmed by the ‘rod-like’ morphology of the crystalline phase as determined by phase-contrast light microscopy.

Densification of diamond tool segments by electro-consolidation : W.M. Goldberger, B. Merkle. (Superior Graphite Co., USA.)

We recently found that polyclonal antibodies to laminin, a basement membrane-related glycoprotein, inhibited murine lung morphogenesis when added to organ cultures of mouse embryonic lung. Using a series of monoclonal antilaminin antibodies with previously characterized subunit specificity (termed AL-1, AL-2, AL-3, AL-4, and AL-5), the deposition and functional involvement of different laminin domains in the developing lung were investigated. By immunohistochemistry the antibodies' reactivity was largely localized to the basement membrane, but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were cultured for 3 days in the presence of 50–100 μg/ml of each antibody or in the presence of the same concentration of immunoglobulins G and M, laminin-neutralized antibody, or medium alone. Cultures were monitored by phase-contrast microscopy, light microscopy, and immunofluorescence. Although all antibodies penetrated the tissues in culture, only two of them inhibited branching activity. These two antibodies were AL-1, which binds on or near the cross region of laminin, and AL-5, which binds to the lateral short arms at the globular end regions of the B chain of laminin. Inhibition of branching with these two antibodies was dose-dependent and statistically significant for the two concentrations used. AL-2, AL-3, AL-4, laminin-neutralized antibodies and control immunoglobulins did not alter lung morphogenesis. The two domains of laminin that promote lung branching morphogenesis have been reported by others to promote the attachment of a variety of cells and/or bind heparin. These domains of laminin may promote branching morphogenesis by facilitating cell attachment and, consequently, cell proliferation.