Ameloblast apoptosis and IGF-1 receptor expression in the continuously erupting rat incisor model

Enamel-producing cells (ameloblasts) pass through several phenotypic and functional stages during enamel formation. In the transition between secretory and maturation stages, about one quarter of the ameloblasts suddenly undergo apoptosis. We have studied this phenomenon using the continuously erupting rat incisor model. A special feature of this model is that all stages of ameloblast differentiation are presented within a single longitudinal section of the developing tooth. This permits investigation of the temporal sequence of gene and growth factor receptor expression during ameloblast differentiation and apoptosis. We describe the light and electron microscopic morphology of ameloblast apoptosis and the pattern of insulin-like growth factor-1 receptor expression by ameloblasts in the continuously erupting rat incisor model. In the developing rat incisor, ameloblast apoptosis is associated with downregulated expression of the insulin-like growth factor-1 receptor. These data are consistent with the hypothesis that ameloblasts are hard wired for apoptosis and that insulin-like growth factor-1 receptor expression is required to block the default apoptotic pathway. Possible mechanisms of insulin-like growth factor-1 inhibition of ameloblast apoptosis are presented. The rat incisor model may be useful in studies of physiological apoptosis as it presents apoptosis in a predictable pattern in adult tissues.

Thylakoid membrane architecture

Confocal scanning laser microscopic observations were made on live chloroplasts in intact cells and on mechanically isolated, intact chloroplasts. Chlorophyll fluorescence was imaged to observe thylakoid membrane architecture. C-3 plant species studied included Spinacia oleracea L., Spathiphyllum sp. Schott, cv. 'Mauna Loa', and Pisum sativum L. C-4 plants were also investigated: Saccharum officinarum L., Sorghum bicolor L. Moench, Zea mays L. and Panicum miliaceum L. Some Spinacia chloroplasts were treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to enhance or sodium dithionite (SD) to reduce the photosystem II fluorescence signal. Confocal microscopy images of C-3 chloroplasts differed from electron microscopy pictures because they showed discrete spots of bright fluorescence with black regions between them. There was no evidence of fluorescence from stroma thylakoids. The thylakoid membrane system at times appeared to be string-like, with brightly fluorescing grana lined up like beads. C-4 bundle sheath chloroplasts were imaged from three different types of C-4 plants. Saccharum and Sorghum bundle sheath chloroplasts showed homogeneous fluorescence and were much dimmer than mesophyll chloroplasts. Zea had rudimentary grana, and dim, homogeneous intergrana fluorescence was visualised. Panicum contained thylakoids similar in appearance and string-like arrangement to mesophyll chloroplasts. Isolated Pisum chloroplasts, treated with a drop of 5 mM MgCl2 showed a thylakoid membrane system which appeared to be unravelling. Spongy mesophyll chloroplasts of Spinacia treated with 5 mM sodium dithionite showed a granal thylakoid system with distinct regions of no fluorescence. A time-series experiment provided evidence of dynamic membrane rearrangements over a period of half an hour.

Encoding combinatorial libraries: A novel application of fluorescent silica colloids

A major challenge associated with using large chemical libraries synthesized on microscopic solid support beads is the rapid discrimination of individual compounds in these libraries. This challenge can be overcome by encoding the beads with 1 mum silica colloidal particles (reporters) that contain specific and identifiable combinations of fluorescent byes. The colored bar code generated on support beads during combinatorial library synthesis can be easily, rapidly, and inexpensively decoded through the use of fluorescence microscopy. All reporters are precoated with polyelectrolytes [poly(acrylic acid), PAA, poly(sodium 4-styrenesulfonate PSSS, polyethylenimine, PEI, and/or poly(diallyldimethylammonium chloride), PDADMAC] with the aim of enhancing surface charge, promoting electrostatic attraction to the bead, and facilitating polymer bridging between the bead and reporter for permanent adhesion. As shown in this article, reporters coated with polyelectrolytes clearly outperform uncoated reporters with regard to quantity of attached reporters per bead (54 +/- 23 in 2500 mum(2) area for PEI/PAA coated and 11 +/- 6 for uncoated reporters) and minimization of cross-contamination (1 red reporter in 2500 mum(2) area of green-labeled bead for PEI/PAA coated and 26 +/- 15 red reporters on green-labeled beads for uncoated reporters after 10 days). Examination of various polyelectrolyte systems shows that the magnitude of the xi -potential of polyelectrolyte-coated reporters (-64 mV for PDADMAC/PSSS and -42 mV for PEI/PAA-coated reporters) has no correlation with the number of reporters that adhere to the solid support beads (21 +/- 16 in 2500 mum(2) area for PDADMAC/PSSS and 54 +/- 23 for PEI/PAA-coated reporters). The contribution of polymer bridging to the adhesion has a far greater influence than electrostatic attraction and is demonstrated by modification of the polyelectrolyte multilayers using gamma irradiation of precoated reporters either in aqueous solution or in polyelectrolyte solution.

An agent-based approach to modelling driver route choice behaviour under the influence of real-time information

This paper presents an agent-based approach to modelling individual driver behaviour under the influence of real-time traffic information. The driver behaviour models developed in this study are based on a behavioural survey of drivers which was conducted on a congested commuting corridor in Brisbane, Australia. Commuters' responses to travel information were analysed and a number of discrete choice models were developed to determine the factors influencing drivers' behaviour and their propensity to change route and adjust travel patterns. Based on the results obtained from the behavioural survey, the agent behaviour parameters which define driver characteristics, knowledge and preferences were identified and their values determined. A case study implementing a simple agent-based route choice decision model within a microscopic traffic simulation tool is also presented. Driver-vehicle units (DVUs) were modelled as autonomous software components that can each be assigned a set of goals to achieve and a database of knowledge comprising certain beliefs, intentions and preferences concerning the driving task. Each DVU provided route choice decision-making capabilities, based on perception of its environment, that were similar to the described intentions of the driver it represented. The case study clearly demonstrated the feasibility of the approach and the potential to develop more complex driver behavioural dynamics based on the belief-desire-intention agent architecture. (C) 2002 Elsevier Science Ltd. All rights reserved.

Modeling of columnar-to-equiaxed transition in solidified Al-Si alloys

A Cellular-Automaton Finite-Volume-Method (CAFVM) algorithm has been developed, coupling with macroscopic model for heat transfer calculation and microscopic models for nucleation and growth. The solution equations have been solved to determine the time-dependent constitutional undercooling and interface retardation during solidification. The constitutional undercooling is then coupled into the CAFVM algorithm to investigate both the effects of thermal and constitutional undercooling on columnar growth and crystal selection in the columnar zone, and formation of equiaxed crystals in the bulk liquid. The model cannot only simulate microstructures of alloys but also investigates nucleation mechanisms and growth kinetics of alloys solidified with various solute concentrations and solidification morphologies.

Characterization of optically driven fluid stress fields with optical tweezers

We present a controlled stress microviscometer with applications to complex fluids. It generates and measures microscopic fluid velocity fields, based on dual beam optical tweezers. This allows an investigation of bulk viscous properties and local inhomogeneities at the probe particle surface. The accuracy of the method is demonstrated in water. In a complex fluid model (hyaluronic acid), we observe a strong deviation of the flow field from classical behavior. Knowledge of the deviation together with an optical torque measurement is used to determine the bulk viscosity. Furthermore, we model the observed deviation and derive microscopic parameters.

Numerical simulation of dendrite growth during solidification

A comprehensive probabilistic model for simulating dendrite morphology and investigating dendritic growth kinetics during solidification has been developed, based on a modified Cellular Automaton (mCA) for microscopic modeling of nucleation, growth of crystals and solute diffusion. The mCA model numerically calculated solute redistribution both in the solid and liquid phases, the curvature of dendrite tips and the growth anisotropy. This modeling takes account of thermal, curvature and solute diffusion effects. Therefore, it can simulate microstructure formation both on the scale of the dendrite tip length. This model was then applied for simulating dendritic solidification of an Al-7%Si alloy. Both directional and equiaxed dendritic growth has been performed to investigate the growth anisotropy and cooling rate on dendrite morphology. Furthermore, the competitive growth and selection of dendritic crystals have also investigated.

Giardia trophozoites in dog hookworm, Ancylostoma caninum: accident or hyperparasitism?

During the course of transmission electron microscopic studies of adult Ancylostoma caninum removed from a dog, several Giardia trophozoites were found in sections of the buccal cavity, oesophagus and intestine of several hookworms. Although the protozoa appeared viable, this unusual finding probably represents accidental uptake by, rather than an established infection of, the hookworm. It is feasible, however, that the trophozoites might have survived and even multiplied in this aberrant site. (C) 2001 Elsevier Science B.V. All rights reserved.

Diagnostic performance of Antineutrophil Cytoplasmic Antibody tests for Idiopathic Vasculitides: Metaanalysis with a focus on antimyeloperoxidase antibodies

Objective. The diagnostic value of tests for antimyeloperoxidase antibodies (anti-MPO) for systemic vasculitis is less established than that for cytoplasmic antineutrophil cytoplasmic antibody (cANCA)/antiproteinase 3 antibodies (anti-PR3). Controversy exists regarding the optimal utilization of indirect immunofluorescence (IIF) ANCA testing versus antigen-specific ANCA testing. To summarize the pertinent data, we conducted a metaanalysis examining the diagnostic value of ANCA testing systems that include assays for anti-MPO. Methods. We performed a structured Medline search and reference list review. Target articles in the search strategy were those reporting the diagnostic value of immunoassays for anti-MPO for the spectrum of systemic necrotizing vasculitides that includes Wegener's granulomatosis, microscopic polyangiitis, the Churg-Strauss syndrome, and isolated pauci-immune necrotizing or crescentic glomerulonephritis, regardless of other types of ANCA tests. Inclusion criteria required specification of a consecutive or random patient selection method and the use of acceptable criteria for the diagnosis of vasculitis exclusive of ANCA test results. Weighted pooled summary estimates of sensitivity and specificity were calculated for anti-MPO alone, anti-MPO + perinuclear ANCA (pANCA), and anti-MPO/pANCA + anti-PR3/cANCA. Results. Of 457 articles reviewed, only 7 met the selection criteria. Summary estimates of sensitivity and specificity (against disease controls only) of assays for anti-MPO for the diagnosis of systemic necrotizing vasculitides were 37.1% (confidence interval 26.6% to 47.6%) and 96.3% (CI 94.1% to 98.5%), respectively. When the pANCA pattern by IIF was combined with anti-MPO testing, the specificity improved to 99.4%, with a lower sensitivity, 31.5%. The combined ANCA testing system (anti-PR3/cANCA + anti-MPO/pANCA) increased the sensitivity to 85.5% with a specificity of 98.6%. Conclusion. These results suggest that while anti-MPO is relatively specific for the diagnosis of systemic vasculitis, the combination system of immunoassays for anti-MPO and IIF for pANCA is highly specific and both tests should be used together given the high diagnostic precision required for these conditions. Because patients with ANCA associated vasculitis have either anti-MPO with pANCA or anti-PR3 with cANCA, and rarely both, a combined ANCA testing system including anti-PR3/cANCA and anti-MPO/pANCA is recommended to optimize the diagnostic performance of ANCA testing. (J Rheumatol 2001;28:1584-90)

Generalized quasi mode theory of macroscopic canonical quantization in cavity quantum electrodynamics and quantum optics II. Application to reflection and refraction at a dielectric interface

The generalization of the quasi mode theory of macroscopic quantization in quantum optics and cavity QED presented in the previous paper, is applied to provide a fully quantum theoretic derivation of the laws of reflection and refraction at a boundary. The quasi mode picture of this process involves the annihilation of a photon travelling in the incident region quasi mode, and the subsequent creation of a photon in either the incident region or transmitted region quasi modes. The derivation of the laws of reflection and refraction is achieved through the dual application of the quasi mode theory and a quantum scattering theory based on the Heisenberg picture. Formal expressions from scattering theory are given for the reflection and transmission coefficients. The behaviour of the intensity for a localized one photon wave packet coming in at time minus infinity from the incident direction is examined and it is shown that at time plus infinity, the light intensity is only significant where the classical laws of reflection and refraction predict. The occurrence of both refraction and reflection is dependent upon the quasi mode theory coupling constants between incident and transmitted region quasi modes being nonzero, and it is seen that the contributions to such coupling constants come from the overlap of the mode functions in the boundary layer region, as might be expected from a microscopic theory.

Receptor-mediated endocytosis of Neisseria gonorrhoeae into primary human urethral epithelial cells: the role of the asialoglycoprotein receptor

Urethral epithelial cells are invaded by Neisseria gonorrhoeae during gonococcal infection in men. To understand further the mechanisms of gonococcal entry into host cells, we used the primary human urethral epithelial cells (PHUECs) tissue culture system recently developed by our laboratory. These studies showed that human asialoglycoprotein receptor (ASGP-R) and the terminal lactosamine of lacto-N-neotetraose-expressing gonococcal lipooligosaccharide (LOS) play an important role in invasion of PHUECs. Microscopy studies showed that ASGP-R traffics to the cell surface after gonococcal challenge. Co-localization of ASGP-R with gonococci was observed. As ASGP-R-mediated endocytosis is clathrin dependent, clathrin localization in PHUECs was examined after infection. Infected PHUECs showed increased clathrin recruitment and co-localization of clathrin and gonococci. Preincubating PHUECs in 0.3 M sucrose or monodansylcadaverine (MDC), which both inhibit clathrin-coated pit formation, resulted in decreased invasion. N. gonorrhoeae strain 1291 produces a single LOS glycoform that terminates with Gal(beta1-4)Glc-Nac(beta1-3)Gal(beta1-4)Glc (lacto-N-neotetraose). Invasion assays showed that strain 1291 invades significantly more than four isogenic mutants expressing truncated LOS. Sialylation of strain 1291 LOS inhibited invasion significantly. Preincubation of PHUECs in asialofetuin (ASF), an ASGP-R ligand, significantly reduced invasion. A dose-response reduction in invasion was observed in PHUECs preincubated with increasing concentrations of NaOH-deacylated 1291 LOS. These studies indicated that an interaction between lacto-N-neotetraose-terminal LOS and ASGP-R allows gonococcal entry into PHUECs.

A high efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors

Lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are emerging as the vectors of choice for in vitro and in vivo gene therapy studies. However, the current method for harvesting lentivectors relies upon ultracentrifugation at 50 000 g for 2 h. At this ultra-high speed, rotors currently in use generally have small volume capacity. Therefore, preparations of large volumes of high-titre vectors are time-consuming and laborious to perform. In the present study, viral vector supernatant harvests from vector-producing cells (VPCs) were pre-treated with various amounts of poly-L-lysine (PLL) and concentrated by low speed centrifugation. Optimal conditions were established when 0.005% of PLL (w/v) was added to vector supernatant harvests, followed by incubation for 30 min and centrifugation at 10 000 g for 2 h at 4 degreesC. Direct comparison with ultracentrifugation demonstrated that the new method consistently produced larger volumes (6 ml) of high-titre viral vector at 1 x 10(8) transduction unit (TU)/ml (from about 3000 ml of supernatant) in one round of concentration. Electron microscopic analysis showed that PLL/viral vector formed complexes, which probably facilitated easy precipitation at low-speed concentration (10 000 g), a speed which does not usually precipitate viral particles efficiently. Transfection of several cell lines in vitro and transduction in vivo in the liver with the lentivector/PLL complexes demonstrated efficient gene transfer without any significant signs of toxicity. These results suggest that the new method provides a convenient means for harvesting large volumes of high-titre lentivectors, facilitate gene therapy experiments in large animal or human gene therapy trials, in which large amounts of lentiviral vectors are a prerequisite.

In situ zymography: topographical considerations

In situ gelatin zymography is a simple technique providing valuable information about the cellular and tissue localization of gelatinases. Until recently, the use of this technique has been confined to soft, relatively homogeneous tissue. In this report in situ zymography has been utilized to assess the sub-lamellar location of gelatinases in the hard, semi-keratinized epidermal layer and the adjacent soft connective tissue matrix of the dermis of the equine hoof. We show that alterations in the orientation at which the tissue is dipped and withdrawn from the emulsion cause profound alterations in emulsion thickness. Microscopic Variations in the surface topography of frozen tissue sections also influence emulsion thickness making interpretation of the results difficult. Given these results, researchers must be aware of potential variations in zymographic analysis may be influenced by physical tissue parameters in addition to suspected gelatinase activity. (C) 2001 Elsevier Science B.V. All rights reserved.

Leptospiral antibodies in Flying Foxes in Australia

The sera of 271 pteropid bats (or flying foxes) collected from Queensland, New South Wales, Western Australia, and the Northern Territory were screened against it reference panel of 21 Leptospira spp. using the microscopic agglutination test (MAT). Sera were collected from December 1997 through August 1999. The MAT panel represented those serovars previously isolated in Australia. as well as exotic serovar found in neighboring countries. Leptospiral antibodies were detected in 75 (28%) of the sera and represented seven serovars, one of which. L. interrogans serovar cynopteri has been regarded as exotic to Australia. Sixty sera were reactive to one serovar, 12 sera were reactive to two serovars, and three sera were reactive to three serovars. The L. kirschneri serovar australis was most frequently identified (60.2%). The findings suggest a previously unrecognized role of pteropid bats in the natural history of leptospirosis. The potential exists for establishment of infection in new host species, the transmission of new serovars to known host species, and for changes in virulence of leptospires as a result of passage through these species.

Growth and hard tissue remodelling in the dentition of the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

The extant lungfish, including three genera, the Australian, South American and African lungfishes, retain a dentition that appeared first in the Devonian, in some of the oldest members of this group. The dentition consists of permanent tooth plates with persistent cusps that appear early in development of the fish. The cusps, separate early in development, form ridges that are arranged in a radiating pattern, and fusion of the cusps to each other and to the underlying jaw bone produces a tooth plate. The lungfish dentition is based on a template of mantle dentine that surrounds bone trabeculae enclosed in the tooth plate. The mantle layer is covered by enamel. In most derived dipnoans, this framework encloses two further forms of dentine, known as interdenteonal and circumdenteonal dentines. The tooth plates grow in area and in depth without evidence of macroscopic resorption of dentines or of enamel. Increase in size and changes in shape of lungfish tooth plates is actually achieved by a process involving microscopic remodelling of the bone contained within the margin of each tooth plate, and the later addition of new dentines and enamel within and around the bone. This is accomplished without creating weakness in the structural integrity of the tooth plate and bone complex, and proceeds in line with growth and remodelling of the jaw bones attached to the tooth plates.