Chemical synthesis, characterisation, and biocompatibility of nanometre scale porous anodic aluminium oxide membranes for use as a cell culture substrate for the vero cell line: a preliminary study.

In this preliminary study we investigate for the first time the biomedical potential of using porous anodic aluminium oxide (AAO) membranes as a cell substrate for culturing the Cercopithecus aethiops (African green monkey) Kidney (Vero) epithelial cell line. One advantage of using the inorganic AAO membrane is the presence of nanometre scale pore channels that allow the exchange of molecules and nutrients across the membrane. The size of the pore channels can be preselected by adjusting the controlling parameters of a temperature controlled two-step anodization process. The cellular interaction and response of the Vero cell line with an in-house synthesised AAO membrane, a commercially available membrane, and a glass control were assessed by investigating cell adhesion, morphology, and proliferation over a 72 h period. The number of viable cells proliferating over the respective membrane surfaces revealed that the locally produced in-house AAO membrane had cells numbers similar to the glass control. The study revealed evidence of focal adhesion sites over the surface of the nanoporous membranes and the penetration of cellular extensions into the pore structure as well. The outcome of the study has revealed that nanometre scale porous AAO membranes have the potential to become practical cell culture scaffold substrates with the capability to enhance adhesion and proliferation of Vero cells.

A description of a new "Amoebozoan" isolated from the American lobster, Homarus americanus.

Our knowledge of the diversity of amoeboid protists is rapidly expanding as new and old habitats are more fully explored. In 2003, while investigating the cause of an amoeboid disease afflicting lobsters on the East Coast, samples were examined for the presence of amoebae from the carapace washings of the American lobster, Homarus americanus. During this survey a unique community of gymnamoebae was discovered. Among the new taxa discovered was a small Thecamoeba-like organism with a single posteriorly directed pseudopodium. Although resembling Parvamoeba rugata, this amoeba displayed distinctive morphology from that isolate or any other amoebozoan. Phylogenetic analysis shows this amoeba is distantly related to the Thecamoebidae. In this paper we describe the unique morphology of a second species of Parvamoeba and discuss its phylogenetic position with respect to the "Amoebozoa."

Interaction with the IQ3 motif of myosin-10 is required for calmodulin-like protein-dependent filopodial extension.

Calmodulin-like protein (CLP) is a specific light chain of unconventional myosin-10 (Myo10) and enhances Myo10-dependent filopodial extension. Here we show that phenylalanine-795 in the third IQ domain (IQ3) of Myo10 is critical for CLP binding. Remarkably, mutation of F795 to alanine had little effect on calmodulin binding to IQ3. Fluorescence microscopy and time-lapse video microscopy showed that HeLa cells expressing CLP and transiently transfected with GFP-Myo10-F795A exhibited significantly shorter filopodia and decreased intrafilopodial motility compared to wildtype GFP-Myo10-transfected cells. Thus, F795 represents a unique anchor for CLP and is essential for CLP-mediated Myo10 function in filopodial extension and motility.

Differentiation of human ameloblast-lineage cells in vitro.

Previous studies have shown that ameloblast-like cells can be selectively cultured from the enamel organ in a serum-free medium with low calcium concentrations. The purpose of this study was to further characterize this culture system to identify differentiated ameloblast-lineage cells. Tooth organs from 19-24-wk-old fetal cadavers were either frozen and cryosectioned for immunostaining, or digested in collagenase/dispase for cell culture. The cells were grown in keratinocyte media supplemented with 0.05 mM calcium, and characterized by morphology and immunofluorescence. Epithelial clones with two distinct morphologies, including smaller cobblestone-shaped cells and larger (5-15 times in size) rounded cells, began to form between day 8 and day 12 after culture. The cobblestone-shaped cells continued to proliferate in culture, while the larger cells proliferated slowly or not at all. These larger cells formed filopodia, usually had two or more nuclei and a radiating cytoplasm at the cell margin, and were more abundant with increasing time in culture. Both cell types stained for cytokeratin 14, and the larger cells appeared more differentiated, showing stronger staining for amelogenin and ameloblastin. Immunofluorescence of the tooth bud sections showed staining for these matrix proteins as ameloblasts differentiated from the inner enamel epithelium. These results show the successful culture of differentiating ameloblast-lineage cells, and lay a foundation for use of these cells to further understand ameloblast biology with application to tooth enamel tissue engineering.

A novel role for p75NTR in subplate growth cone complexity and visual thalamocortical innervation.

In cortical development, subplate axons pioneer the pathway from neocortex to the internal capsule, leading to the proposal that they are required for subsequent area-specific innervation of cortex by thalamic axons. A role for p75 neutrophin receptor (NTR) in area-specific thalamic innervation of cortex is suggested by the observation that p75NTR expression is restricted to subplate neurons in a low-rostral to high-caudal gradient throughout the period of thalamocortical innervation. In vitro, neurotrophin 3 binding to p75NTR increases neurite length and filopodial formation of immunopurified subplate neurons, suggesting a role for p75NTR in subplate growth cone morphology and function in vivo. Consistent with this idea, subplate growth cones have markedly fewer filopodia in mice lacking p75NTR than in wild type mice. Despite this gross morphologic defect, many subplate axons in knock-out mice pioneer the projection to the internal capsule as they do in wild-type mice. However a few subplate axons in the knock-out mice make ectopic projections rostral in the intermediate zone and frontal cortex. Concomitant with the altered morphology of subplate growth cones, mice lacking p75NTR have diminished innervation of visual cortex from the lateral geniculate nucleus, with markedly reduced or absent connections in 48% of knock-out mice. Thalamic projections to auditory and somatosensory cortex are normal, consistent with the gradient of p75NTR expression. Our present results are unusual in that they argue that p75NTR functions in a novel way in subplate neurons, that is, in growth cone morphology and function rather than in axon extension or neuronal survival.

Surface analysis of machined versus sandblasted and acid-etched titanium implants.

Initially, implant surface analyses were performed on 10 machined implants and on 10 sandblasted and acid-etched implants. Subsequently, sandblasted and acid-etched implant cytotoxicity (using L929 mouse fibroblasts), morphologic differences between cells (osteoblast-like cells MG63) adhering to the machined implant surfaces, and cell anchorage to sandblasted and acid-etched implant surfaces were evaluated. Results indicated that acid etching with 1% hydrofluoric acid/30% nitric acid after sandblasting eliminated residual alumina particles. The average roughness (Ra) of sandblasted and acid-etched surfaces was about 2.15 microns. Cytotoxicity tests showed that sandblasted and acid-etched implants had non-cytotoxic cellular effects and appeared to be biocompatible. Scanning electron microscopic examination showed that the surface roughness produced by sandblasting and acid etching could affect cell adhesion mechanisms. Osteoblast-like cells adhering to the machined implants presented a very flat configuration, while the same cells adhering to the sandblasted and acid-etched surfaces showed an irregular morphology and many pseudopodi. These morphologic irregularities could improve initial cell anchorage, providing better osseointegration for sandblasted and acid-etched implants.

dl-alpha-tocopherol, a potent inhibitor of phorbol ester induced shape change of erythro- and megakaryoblastic leukemia cells.

Synthetic vitamin E, dl-alpha-tocopherol, added to a human erythroleukemia HEL and a megakaryoblastic leukemia, Meg-01, cell culture produced potent dose-dependent inhibition of phorbol ester-induced adhesion and of the morphologic changes accompanying it. The inhibition was reversible by withdrawal of supplemental vitamin E from the medium. dl-alpha-Tocopherol also inhibited protein kinase C activity both at baseline and after phorbol ester stimulation. Arachidonic acid stimulated protein kinase C activity of erythroleukemia cells and promoted their adhesion, an effect that was also inhibited by dl-alpha-tocopherol. Introduction of a protein kinase C-neutralizing antibody or a protein kinase C-inhibitor substrate into permeabilized HEL cells inhibited phorbol ester-induced adhesion and shape change. dl-alpha-Tocopherol also affected the cellular distribution of protein kinase C, shifting the major portion of the enzyme to the cytosol fraction and reducing phorbol ester-induced membrane association of the enzyme. Thus, protein kinase C appears to mediate shape change and adhesion, both of which are strongly inhibited by dl-alpha-tocopherol.

Pseudopodia formation by neurosecretory granules.

Ultrastrucal studies of the mouse neurohypophysis, under various experimental conditions, revealed a number of neurosecretory granules (NSG) bearing single pseudopodia-like protrusions. Some NSG adhered to the axolemma via pseudopodia; other NSG, distant from the axolemma, budded electron lucent microvesicles from the tip of the pseudopod. Pseudopodia counts were made on electron micrographs, and calculated as a percentage of the NSG population. In neural lobes from intact mice, small numbers of pseudopodia were observed (0.3%); the count increased significantly after injections of large doses of horseradish peroxidase (HRP) (9.4--14.5%); hypertonic saline augmented the count, as did histamine. In vitro incubation experiments with isolated neural lobes in Krebs Ringer revealed concomitant pseudopodia formation and elevated vasopressin release (measured by antidiuretic bioassay) in the presence of HRP and di-butyryl cyclic AMP respectively. Histamine and excess potassium also increased hormone secretion, but did not induce pseudopodia formation in vitro; pseudopodia were observed neither in controls, nor in the presence of ineffective secretagogues. It is suggested that the pseudopod may represent the active site on the granule membrane. Different ultrastructural images of granule release suggest that several modes of hormone release may be operative in the neurohypophysis. The role of HRP in pseudopodia formation and vasopressin release is enigmatic.

Supraependymal cells of hypothalamic third ventricle: identification as resident phagocytes of the brain.

Cells lying on the ventricular surface of the hypothalamic ependyma of the tegu lizard exhibit the pseudopodial and flaplike processes characteristic of macrophages found elsewhere. Since they ingest latex beads, they may be considered a resident phagocytic system of the brain. The importance of ependyma and ventricular phagocytes as a first line of defense against viral invasion of the brain, as well as their role in the pathogenesis of certain virus-related diseases, is suggested by a number of experimental and clinical observations.