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PubMed1996

Stabilizing role of the basement membrane and dermal fibers during newt limb regeneration.

BACKGROUND: Following amputation of a newt limb, tissues at the amputation site undergo histolysis to give rise to a growth bud, or blastema, but they also provide a base on which the regenerate is constructed. Studies suggest that dermal tissues may differentially resist histolysis. METHODS AND RESULTS: To examine stability of tissues at the amputation site, more than 80 preblastemal staged regenerating limbs were examined histologically. Initially, all soft tissues not attached to bone retracted and were covered by migrating epithelium. The dermis was seen to be stable during the first week postamputation. Muscle dedifferentiated and was heavily stained with anti-tenascin antibodies, but the intact overlying dermis was unstained. Fiber bundles, revealed by staining with phosphotungstic acid hematoxylin, isolated the dermis from dedifferentiating deeper tissues during the first week postamputation, but partially broke down during the second week. However, the basement membrane (BM) remained as the distalmost intact structure at the amputation site in all limbs examined. The BM was the foundation for new BM synthesis which preceded dermis synthesis in the base of the blastema during the second week, even while undifferentiated cells were accumulating centrally. CONCLUSIONS: We suggest that the dermis resists histolysis long enough for new BM to form in continuity with that of the stump. Dermis formation (dermogenesis) distal to the amputation plane begins early as in mammalian healing but is not completed until after blastema formation. Thus, factors that inhibit dermal closure appear to distinguish regenerating from non-regenerating appendages.

Amputation, Surgical↗
PubMed1996

Perspective: a suggested role for basement membrane structures during newt limb regeneration.

BACKGROUND: Interactions between epithelium and mesenchyme, which occur across a basement membrane (BM) zone, are essential to generate a growth bud, or blastema, from which a new limb regenerates. An intact BM at that interface is believed to inhibit regeneration, but that mechanism of inhibition is not understood. METHODS: Interference contrast microscopy and antibodies to laminin have been used to describe reformation of the BM and the basal lamina (BL) and their relationships to wound epithelium and mesenchyme in successive stages of blastema formation. RESULTS: The BL is initially absent from the amputation surface and is reestablished to continuity by the late bud stage of regeneration. It forms generally from base to apex, precedes reticular lamina (RL) formation, and is absent beneath most of the wound epithelium. Our inability to correlate mesenchymal cell accumulation exclusively with the area lacking BL apically and postaxially prompted rethinking of the significance of the BL. CONCLUSIONS: Consistent with these and other observations, we suggest that the BL, when it forms during blastema formation, appears to function as in other developing systems to stabilize the phenotype of adjacent cells. Thus, epithelium becomes epidermis and adjacent mesenchyme synthesizes RL and becomes dermis. Accordingly, the feature that distinguishes regenerating from nonregenerating appendages is the ability of regenerating appendages to delay BL closure until after a critical mass of mesenchymal cells has accumulated.

Animals↗
PubMed1997

Sensitive immunofluorescence detection of the expression of P-glycoprotein in malignant cells.

Because reversal of multidrug resistance increases chemotoxicity, early detection of low P-glycoprotein expression is clinically relevant for justifying early treatment of those patients that might benefit most from reversal therapy. We elected to score P-glycoprotein in single tumor cells, because the gene is rarely amplified, mRNA levels do not necessarily correlate with protein levels, and many normal hematopoietic or stroma cells within tumors and leukemic marrows also express P-glycoprotein. We enhanced the "signal-to-noise" ratio for detecting low P-glycoprotein levels by a novel complex made by pre-incubating mouse peroxidase-antiperoxidase, used solely to provide a stable framework for attaching multiple DTAF-labeled F(ab')2 fragments of rabbit antimouse IgG. We improved specificity by using both C219 and C494, which are directed against separate internal P-glycoprotein epitopes. We standardized staining with two series of negative and positive controls, in which P-glycoprotein was quantified by immunoblot, and confirmed sensitivity by staining a low-expression cell line and "mixed" samples containing small numbers of positive cells. We measured P-glycoprotein by flow cytometry, examining aliquots by differential interference contrast microscopy to identify malignant cells, in which we confirmed P-glycoprotein staining by fluorescence microscopy. We detected low P-glycoprotein expression in clinical samples of leukemic blasts, distinguishing them from normal P-glycoprotein-expressing hematopoietic cells. This assay may be valuable for early diagnosis of low, but potentially important expression of P-glycoprotein, thereby allowing early application of reversal therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Quantitative assessment of leading edge adhesion: reattachment kinetics modulated by injury-derived intracellular calcium predict wound closure rates in endothelial monolayers.

Migrating cells continually develop new substrate attachments at the leading edge (LE) in order to maintain traction for movement. This study evaluates the relationship between LE adhesion and wound closure by modulating injury-derived intracellular free Ca2+ ([Ca2+]i) signaling in endothelial cell (EC) monolayers following scrape-wounding. These data show that brief treatment with increased extracellular Ca2+ ([Ca2+]e) during wounding accelerated wound area closure rates by 50-65%, while brief treatments with calcium influx inhibitors reduced rates by 30-50%. Fura-2 studies in wounded monolayers indicated supranormal [Ca2+]e during wounding increased (by 52%), while influx-inhibitors decreased (by 36%) the percentage of cells exhibiting elevated plateau [Ca2+]i levels. Quantitative time-lapse interference reflection microscopy (IRM) together with indirect alphavbeta3 integrin immunofluorescence was used to measure the effects of 100 microM Gd3+ and 5 mM [Ca2+]e treatment on fractional LE adhesion after wounding. Influx inhibition blocked development of increased injury-derived LE adhesion. Measurements indicated a linear relationship (r2 = 0.99, 0.98) between LE adhesion, development rates (quantified as an association rate constant) and steady state wound closure rates. Changes in filopodial activity, as indicated by phase contrast microscopy, did not correlate with changes in wound closure rates, but an association existed between the percentile peak [Ca2+]i response and the initiation of filopodial activity, suggesting a role for filopodia in mediating Ca2+-sensitive acceleration. Taken together, our data suggest that injury-derived [Ca2+]i signaling may regulate wound closure rates by an adhesion-mediated mechanism.

Animals↗
PubMed1997

A novel action of collapsin: collapsin-1 increases antero- and retrograde axoplasmic transport independently of growth cone collapse.

Chick collapsin-1, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Collapsin-1 induces growth cone collapse via a pathway which may include CRMP-62 and heterotrimeric G proteins. CRMP-62 protein is related to UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. Mutations in unc-33 affect neural microtubules, the basic cytoskeletal elements for axoplasmic transport. Using computer-assisted video-enhanced differential interference contrast microscopy, we now demonstrate that collapsin-1 potently promotes axoplasmic transport. Collapsin-1 doubles the number of antero- and retrograde-transported organelles but not their velocity. Collapsin-1 decreases the number of stationary organelles, suggesting that the fraction of time during which a particle is moving is increased. Collapsin-1-stimulated transport occurs by a mechanism distinct from that causing growth cone collapse. Pertussis toxin (PTX) but not its B oligomer blocks collapsin-induced growth cone collapse. The holotoxin does not affect collapsin-stimulated axoplasmic transport. Mastoparan and a myelin protein NI-35 induce PTX-sensitive growth cone collapse but do not stimulate axoplasmic transport. These results provide evidence that collapsin has a unique property to activate axonal vesicular transport systems. There are at least two distinct pathways through which collapsin exerts its actions in developing neurons.

Animals↗
PubMed2000

"Micrognathozoa: A new class with complicated jaws like those of rotifera and Gnathostomulida:" journal of morphology, volume 246, number 1, october 2000

The cover caption for the October issue of Journal of Morphology (Volume 246, Number 1) incorrectly listed the size of the cover specimen, Limnognathia maerski, as 128 cm in length. This is incorrect. The correct length is 128 &mgr;m. The correct cover caption is: The type species, Limnognathia maerski nov. gen. et sp. of a new group of animals, Micrognathozoa, from Greenland was photographed using differential interference contract microscopy, Nomarski technique. Anterior of the specimen is at the top of the figure. Different internal structures such as the pharyngeal apparatus with jaws and the ovaria with eggs are seen in three dimensions. The long, stiff sensoria are best seen when the animal is living as here in this photomicrograph. This specimen is only 128 &mgr;m in length. The color in this image is not the true color of the transparent animal but is a reflection of the Nomarski technique. Photomicrograph by Reinhardt M. Kristensen. See R. M. Kristensen and P. Funch, page 1. The Editor regrets this error.

Journal Article↗
PubMed2001

Selective adhesion of endothelial cells to artificial membranes with a synthetic RGD-lipopeptide.

A constrained cyclic ArgGly-Asp-D-Phe-Lys, abbreviated as cyclo(-RGDfK-), lipopeptide has been synthesized and incorporated into artificial membranes such as giant vesicles with DOPC and solid-supported lipid bilayers. The selective adhesion and spreading of endothelial cells of the human umbilical cord on solids functionalized by membranes with this RGD-lipopeptide have been observed. Furthermore, we have demonstrated strong selective adhesion of giant vesicles to endothelial cells through local adhesion domains by combined application of hydrodynamic flow field and reflection interference contrast microscopy (RICM). The adhesion can be inhibited by competition with a water-soluble RGD peptide. We suggest that this strategy could improve the efficiency of liposomes targeting used as vectors or as drug carriers to cells.

Cell Adhesion↗
PubMed1984

Mitochondrial reorganization during resumption of arrested meiosis in the mouse oocyte.

Correlated nuclear and cytoplasmic reorganizations during the 14 hr of reactivated meiosis in vivo and in vitro were examined in the laboratory mouse. Observations of living oocytes by differential interference contrast microscopy, and by fluorescent microscopy with nontoxic mitochondrial and DNA-specific probes, enabled us to determine that the major cytoplasmic reorganization involved two mitochondrial translocations associated with two stages of nuclear maturation. These observations were confirmed at the fine structural level by parallel transmission electron microscopy. Mitochondria translocate to the perinuclear region during formation of the first metaphase spindle and subsequently disperse during abstriction of the first polar body. Determinations of frequency of maturation in more than 2,900 normal oocytes, and in more than 1,100 oocytes in which germinal vesicle breakdown was reversibly inhibited, indicated that mitochondrial redistributions are a normal and probably necessary feature of reactivated meiosis in the laboratory mouse. We suggest that these two rapid translocations serve to concentrate mitochondria for localized activities that require elevated levels of adenosine triphosphate.

Animals↗
PubMed1988

Physiologic formation of intracellular vesicles in mature erythrocytes.

The ability of mature erythrocytes to spontaneously form intracellular vesicles has been implied from clinical studies but has not been examined experimentally. An in vitro model was developed to demonstrate whether mature erythrocytes are capable of spontaneously forming intracellular vesicles. Normal human erythrocytes were incubated in vitro at 37 degrees C for 144 hr in a synthetic medium. During the course of these incubations, approximately 4% of erythrocytes developed intracellular vesicles which were quantitated by using interference contrast microscopy. Electron microscopic studies confirmed the intracellular nature of these vesicles. Incubation of erythrocytes in autologous plasma produced similar results. The rate of vesicle acquisition in vivo was measured by quantitating erythrocyte vesicles immediately prior to and following splenectomy. The rates of vesicle acquisition in vivo and in vitro were comparable. This in vitro model confirms the ability of mature erythrocytes to spontaneously form intracellular vesicles and strongly supports the concept that this is a physiologic process.

Adult↗
PubMed1975

Spatial distribution of mitosis in mouse epidermis.

The cells of the upper strata of mammalian epidermis are flattened and aligned to form regular columnar units. It has been suggested that the position of the smaller underlying basal cells is related to the overlying cell columns. Examination of the position of metaphase figures in sheets of mouse epidermis indicated that mitosis occurs principally in cells lying just within the periphery of the cell columns but that there is no alignment of interphase basal cells within the columnar peripheries which could account for this position of mitosis.

Animals↗
PubMed2002

Co-development of Encarsia formosa (Hymenoptera: Aphelinidae) and the greenhouse whitefly, Trialeurodes vaporariorum (Homoptera: Aleyrodidae): a histological examination.

Using histological techniques, we have simultaneously examined the co-development of the Aphelinid parasitoid Encarsia formosa and its host the greenhouse whitefly, Trialeurodes vaporariorum. Previously we have determined that regardless of the whitefly instar parasitized, parasitoid larvae would not molt to their final instar until the whitefly reaches its maximum dimensions. In unparasitized T. vaporariorum, this point in development corresponds to the initiation of the adult molt. In part, this study was conducted to determine the developmental state of parasitized whiteflies at the time they achieve their maximum dimensions. It was found that parasitized final instar T. vaporariorum do, in fact, undergo a final molt and that E. formosa larvae will not molt to their final instar until this has occurred. The timing of the final whitefly molt appears unaffected by parasitization. The commonly observed melanization of parasitized whiteflies appears to be a consequence of this molt. In addition, we have discovered that the adult wasp oviposits within the ventral ganglion of the whitefly, and that major organ systems of the whitefly persist very late into parasitoid development. We also report the presence of possible endosymbiotic bacteria residing in the fatbody of E. formosa.

Animals↗
PubMed2003

Effects of silkworm paralytic peptide on in vitro hematopoiesis and plasmatocyte spreading.

Bombyx mori paralytic peptide (BmPP), a multifunctional cytokine-like molecule, is expressed in the hematopoietic organ-wing imaginal disc complex, suggesting that BmPP is involved in both immune response and the hematopoietic process. We studied the effects of BmPP on plasmatocytes and hematopoietic organs of the silkworm. BmPP (1 microM) stimulated spreading of circulating plasmatocytes, but the percentage of spread plasmatocytes was only 20%. Over 10 nM of BmPP, however, elicited prominent spreading in 70% of young plasmatocytes discharged from cultured hematopoietic organs. Cells in hematopoietic organs that were enzymatically dispersed did not spread even after adding 100 nM of BmPP, indicating that plasmatocytes acquired BmPP-sensitivity immediately after discharge. When cultured in a medium containing larval plasma, hematopoietic organs grew markedly and discharged a large number of hemocytes, over 95% of which were morphologically plasmatocytes. The hemocyte discharge was blocked in the medium containing BmPP dose-dependently, although hematopoietic organ growth was not suppressed. These results suggest that BmPP plays important roles both in hematopoietic regulation and in the hemocyte immune reaction of the silkworm.

Animals↗
PubMed1984

The altered biomechanical state of human femoral head osteoarthritic articular cartilage.

In this study, the biomechanical characteristics of normal and osteoarthritic human femoral head articular cartilage are compared using both microcompression and notch propagation techniques, combined with simultaneous differential interference contrast microscopy. The latter permits examination of the process of matrix rupture occurring in a controlled manner at the notch root. The results provide new biomechanical insights into the differences between normal and osteoarthritic cartilage and draw particular attention to some areas of fundamental structural breakdown associated with the loss of load-bearing function in this tissue.

Adult↗
PubMed1991

Chondrons from articular cartilage. III. Morphologic changes in the cellular microenvironment of chondrons isolated from osteoarthritic cartilage.

Chondrons were isolated from human and canine osteoarthritic cartilage using low-speed homogenization techniques. Changes in chondron morphology were evaluated using differential interference-contrast microscopy, phase-contrast microscopy, and histochemical and ultrastructural methods. Chondrocyte viability was assessed using fluorescein diacetate staining, and chondron metabolism was investigated using autoradiography. The results suggest that initial changes in the collagen and proteoglycan distribution within the chondron are followed by chondrocyte proliferation to form clusters. These techniques offer the potential to study cell matrix interactions in degenerative osteoarthritis.

Aged↗
PubMed2005

Identification of the epidermal growth factor-TM7 receptor EMR2 and its ligand dermatan sulfate in rheumatoid synovial tissue.

OBJECTIVE: EMR2 and CD97 are closely related members of the epidermal growth factor (EGF)-TM7 family of adhesion class 7-span transmembrane (TM7) receptors. Chondroitin sulfates (CS) have recently been identified as ligands for EMR2 and CD97. CS have been implicated in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine the expression of EMR2 and the distribution of EMR2 and CD97 ligands within RA synovial tissue (ST). METHODS: ST samples were obtained by arthroscopy from 19 patients with RA, 13 patients with inflammatory osteoarthritis (OA), and 13 patients with reactive arthritis (ReA). Immunohistochemistry was performed with a monoclonal antibody against EMR2, and stained STs were analyzed by digital image analysis. Coexpression of EMR2 with cell lineage- and activation-specific markers was determined by double immunofluorescence microscopy. To evaluate the expression of EMR2 and CD97 ligands in RA synovium, binding assays were performed using EMR2- and CD97-specific multivalent fluorescent probes. RESULTS: EMR2 expression in the synovial sublining was found to be significantly higher in RA patients compared with OA and ReA control patients. Most EMR2+ cells were macrophages and dendritic cells expressing costimulatory molecules and tumor necrosis factor alpha. Dermatan sulfate was shown to be the ligand of the largest isoforms of EMR2 and CD97 in rheumatoid synovium. In addition, the smaller isoforms of CD97, but not those of EMR2, bound CD55 on fibroblast-like synoviocytes. CONCLUSION: The EGF-TM7 receptors EMR2 and CD97 are abundantly expressed on myeloid cells in ST of RA patients where their cognate ligands dermatan sulfate and CD55 are detected. These results suggest that these interactions may facilitate the retention of activated macrophages in the synovium.

Aged↗
PubMed2004

Dynamic imaging of cell, extracellular matrix, and tissue movements during avian vertebral axis patterning.

Vertebrate axis patterning depends on cell and extracellular matrix (ECM) repositioning and proper cell-ECM interactions. However, there are few in vivo data addressing how large-scale tissue deformations are coordinated with the motion of local cell ensembles or the displacement of ECM constituents. Combining the methods of dynamic imaging and experimental biology allows both cell and ECM fate-mapping to be correlated with ongoing tissue deformations. These fate-mapping studies suggest that the axial ECM components "move" both as a composite meshwork and as autonomous particles, depending on the length scale being examined. Cells are also part of this composite, and subject to passive displacements resulting from tissue deformations. However, in contrast to the ECM, cells are self-propelled. The net result of cell and ECM displacements, along with proper ECM-cell adhesion, is the assembly of new tissue architecture. Data herein show that disruption of normal cell-ECM interactions during axis formation results in developmental abnormalities and a disorganization of the ECM. Our goal in characterizing the global displacement patterns of axial cells and ECM is to provide critical information regarding existing strain fields in the segmental plate and paraxial mesoderm. Deducing the mechanical influences on cell behavior is critical, if we are to understand vertebral axis patterning. Supplementary material for this article is available online at http://www.mrw.interscience.wiley.com/suppmat/1542-975X/suppmat/72/v72.266.html.

Animals↗
PubMed1984

Use of microscopic interferometry for measuring changes in water content of small samples of tissue.

Oedema following periods of ischaemic arrest and subsequent reperfusion has been shown experimentally and clinically to affect the functional state of the heart. Tissue water content has been measured in myocardial sections by microscopic interferometry and densitometry, and the results correlated with those obtained by wet and dry weight analysis (r = 0.87; p less than 0.001). Microscopic interferometry also revealed the distribution of the water in the tissue. Experimentally induced ischaemic arrest in isolated rat hearts resulted in predominantly intra-fibrillar oedema, whilst subsequent reperfusion resulted in interfibrillar oedema. Microscopic interferometry facilitates accurate measurement of water content in tissue samples as small as 2 mg wet weight and shows (as conventional wet/dry weight analysis cannot) the distribution of the water in the tissue.

Animals↗