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PubMed1975

Ultrathin coating by plasma polymerization applied to corneal contact lens.

Plasma coating (deposition of polymer under the influence of plasma) is utilized to modify the surface properties of corneal contact lens. An ultrathin layer (thickness of roughly 200 A) of plasma polymer of acetylene/H20/N2 is applied to poly(methyl methacrylate) (PMMA) corneal contact lenses. The surface becomes highly wettable with water compared to uncoated lens. When coated and uncoated contact lenses are placed on rabbit eyes, a remarkable difference in accumulation of mucous matter is observed. With the control lenses, the accumulation of mucous matter in a week is sufficient to affect the optical clarity of the lenses, whereas the coated lenses show no change after three months continuous wearing. The comparative degree of adhesion of the corneal epithelium cells onto glass, modified glass, PMMA, and coated PMMA surfaces is studied using tissue cultures and phase contrast microscopy. The coated PMMA surface exhibits a degree of tissue adhesion lower than that of control PMMA and higher than that of glass surface, and no sign of toxicity of the coated surface is observed by the tissue cultures.

Acetylene↗
PubMed1986

In vitro testing of dental materials by means of macrophage cultures. I: Methodological aspects.

Macrophages play a central role in the pathogenesis of inflammation. In addition, activated macrophages are the first cells to come in contact with foreign particles in tissue. In the present study the applicability of macrophages for in vitro biological screening of dental materials was tested. Monolayers of murine peritoneal macrophages were prepared. After three days culture the macrophages were inoculated with alloy particles prepared from silver and tin (Ag3Sn), the gamma-phase of dental silver-amalgam. After different inoculation periods the macrophages were fixed and examined with phase contrast microscopy, scanning electron microscopy, and energy dispersive x-ray analysis. Comparison with phagocytosis of particles injected intraperitoneally was also performed. Macrophage cultures inoculated with Latex particles served as controls. Ten minutes after inoculation with alloy particles, about 58% of the macrophages had ingested particles. EDAX-analysis indicated that the phagocytized alloy particles contained both silver and tin in the same proportions as in the original alloy. When the cultures had been inoculated for ten days, however, a marked reduction in phagocytosis was observed probably due to cytolysis of those macrophages which initially had phagocytized the alloy particles. The results indicate that, although the Ag3Sn alloy particles at all the time intervals studied were phagocytized more slowly and to a lesser extent than the Latex particles, the method could be of value in studying the biocompatibility of dental materials available in particulate form.

Animals↗
PubMed2006

Extensive H(+) release by bone substitutes affects biocompatibility in vitro testing.

Bone substitutes are widespread in orthopedic and trauma surgery to restore critical bony defects and/or promote local bone healing. Cell culture systems have been used for many years to screen biomaterials for their toxicity and biocompatibility. This study applies a human bone marrow cell culture system to evaluate the toxic in vitro effects of soluble components of different bone substitutes, which are already in clinical use. Different specimens of tricalcium phosphates (TCP) (Vitoss, Cerasorb), nondecalcified bovine bone (Lubboc), demineralized human bone matrices (DBM) (Grafton Flex/Putty), and collagen I/III matrix (ACI-Maix) were tested in Dulbecco's modified Eagle's medium (DMEM) and MesenCult culture solution and compared with a biomaterial-free cell culture. Biocompatibility parameters were cell viability evaluated by phase-contrast microscopy and laser flow cytometry, morphology, and the local H(+) release by bone substitutes. There were significant differences (p < 0.05) between the tested biomaterials and culture solutions. Collagen I/III, non-demineralized bovine bone, and TCP materials showed advantages for cell survival over other tested biomaterials (average values of vital cells/mL MesenCult/DMEM: Collagen I/III: 1090/1083; Vitoss: 893/483; Cerasorb: 471/523; Lubboc: 815/410; Grafton Putty: 61/44; Grafton Flex: 149/57). Especially the DBM materials lead to a significant decrease of pH, which is considered to be a major factor for cell death. DMEM culture solution supports cell survival for those bone substitutes that induce an alkaline reaction, whereas MesenCult media promotes cell vitality in biomaterials, which leads to an acidification of culture solution.

Animals↗
PubMed2006

Effects of 58S sol-gel glasses on the temporal expression of bone markers during mouse osteoblastic differentiation.

Previous studies have shown that bioactive glasses can support osteoblastic growth and differentiation in vitro as well as in vivo. More recently, a new category of sol-gel glasses has been developed with enhanced bioactivity and open pores enclosed in a mesoporous matrix. In our study, we investigated the effect of 58S sol-gel glasses on the growth and differentiation of mouse calvaria osteoblasts. Two types of granules were used: 58S sol-gel granules and 60S inert glasses used as control. Phase contrast microscopy showed that cells proliferated and formed mineralized bone nodules in both cultures. However, this phenomenon occurred earlier and to a higher degree in cultures with 58S sol-gel glasses. Northern blot analysis of the expression of osteoblastic markers revealed that osteoblasts retained their phenotype in both types of cultures. Interestingly, stimulation of alkaline phosphatase, bone sialoprotein, and osteocalcin was noticed at day 18 in sol-gel cultures when compared with that in control. These data confirm that 58S bioactive glasses are capable of supporting the growth and maturation of primary mouse osteoblasts. In addition, it was shown that 58S glasses affected the gene-expression profile, causing an up-regulation of the major bone markers. These results indicated that 58S sol-gel glasses appeared as suitable candidates for osteoblast scaffolds in the field of bone tissue engineering.

Animals↗
PubMed1995

Suramin inhibits C6 glioma-induced angiogenesis in vitro.

Aspects of tumor-induced angiogenesis in vitro were examined using an assay involving collagen gel invasion by a surface monolayer of bovine endothelial cells under the influence of serum free conditioned medium produced by C6 cells, an experimentally derived rat glial tumor cell line. The effects of the polyanionic compound suramin, known to interfere with growth factor/cell signaling on this process were evaluated. Collagen gel invasion was quantified by adding C6 conditioned medium with or without various doses of suramin to monolayers of bovine aortic endothelial cells grown on type I collagen gels in transwell inserts. Cultures were monitored with phase-contrast microscopy. After various periods of incubation collagen gels were fixed, embedded in epoxy resin, and 1-micron thick sections were stained with toluidine blue. Additional cultures were used to evaluate the effects of C6 conditioned medium and suramin on endothelial cell proliferation, and on chemotaxis through 8-microns pores. C6 glioma cell conditioned medium induced large vessel endothelial cells to sprout into the underlying collagen matrix and subsequently form networks of capillary like tubes. Conditioned medium was also chemotactic and mitogenic for these cells. The addition of suramin to C6 glioma conditioned medium prevents tube formation in collagen gels, and inhibits both endothelial cell proliferation and chemotaxis in a dose dependent manner. These results suggest that glial tumor cell conditioned medium induces angiogenesis in large vessel endothelial cells in vitro via mechanisms which are disrupted by suramin, most likely involving tumor-derived growth factor release and/or endothelium-mediated matrix proteolysis.

Analysis of Variance↗
PubMed1991

Maintenance and mobility of hemoglobin and water within the human erythrocyte after detergent disruption of the plasma membrane.

Is an intact plasma membrane responsible for keeping hemoglobin and water within the human erythrocyte? If not, what is responsible? How free is Hb to move about within the erythrocyte? To answer these questions erythrocytes were taken for phase contrast microscopy, transmission electron microscopy (TEM), determination of water-holding capacity, and proton NMR studies both before and after membrane disruption with a nonionic detergent (Brij 58). Addition of 0.2% Brij to a D2O saline solution of hemoglobin (Hb) caused particles of Hb to appear and to aggregate. This aggregation of Hb caused the amplitude of the Hb proton NMR spectra to decrease. Thus, the less mobile the Hb the lower the Hb proton spectra amplitude. Erythrocytes washed in D2O saline showed proton NMR spectra of relatively low amplitude. Addition of Brij (0.2%) to these erythrocytes caused increased Hb mobility within these erythrocytes. The TEM of fixed and thin-sectioned erythrocytes treated with Brij showed disruption of the plasma membrane of all erythrocytes regardless of whether or not they had lost Hb. Brij-permeabilized erythrocytes washed in D2O saline or in a D2O K buffer maintained a higher heavy water-holding capacity upon centrifugation as compared to nonpermeabilized erythrocytes. The TEM of Brij-treated and washed erythrocyte "shells" revealed a continuous submembrane lamina but no other evidence of cytoskeletal elements. The water-holding capacity of the erythrocyte can be accounted for by the water-holding capacity of hemoglobin. The evidence favors a relatively immobile state of Hb and of water in the erythrocyte that is not immediately dependent on an intact plasma membrane but is attributed to interactions between Hb molecules and the submembrane lamina.

Body Water↗
PubMed1992

Induction of fibronectin gene expression by transforming growth factor beta-1 is attenuated in bronchial epithelial cells by ADP-ribosyltransferase inhibitors.

Transforming growth factor-beta (TGF-beta) exerts several effects on cultured airway epithelial cells including inhibition of proliferation and stimulation of fibronectin gene expression. ADP-ribosylation is one potential regulatory mechanism of gene expression by TGF-beta. We tested this possibility by exposing cultured bovine bronchial epithelial cells to the chemical inhibitor of ADP-ribosyl transferase enzymes, 3-aminobenzamide (3-AB) and, for comparison, 3-aminobenzoic acid (3-ABA), which is structurally similar to 3-AB but which does not inhibit ADP-ribosyl transferases. Exponential cell growth rate (1.2 doublings/day) or cellular morphology observed by phase contrast microscopy were not affected by 3 mM 3-AB or 3-ABA. Neither compound antagonized inhibition of cell division or induction of squamous morphology by TGF-beta 1. In contrast, the sixfold stimulation of fibronectin production by exposure of cells to 30 pM TGF-beta 1 for 48 h was reduced by 50% in the presence of 3 mM 3-AB, whereas 3 mM 3-ABA had no effect. The antagonistic effect was augmented by administration of 3-AB 24 h prior to induction by TGF-beta 1. Northern blot hybridization analyses demonstrated that 3-AB, but not 3-ABA, attenuated the induction of fibronectin mRNA by TGF-beta 1 by up to 50%. These observations may implicate a role of cellular ADP-ribosylation in the regulation of some gene expression by TGF-beta.

Aminobenzoates↗
PubMed1993

Endothelial cell hypoxia associated proteins are cell and stress specific.

Vascular endothelial cells (EC) are one of the initial cells exposed to decreases in blood oxygen tension. Bovine EC respond not only by altering secretion of vasoactive, mitogenic, and thrombogenic substances, but also by developing adaptive mechanisms in order to survive acute and chronic hypoxic exposures. EC exposed to hypoxia in vitro upregulate a unique set of stress proteins of Mr 34, 36, 39, 47, and 56 kD. Previous studies have shown that these proteins are cell associated, upregulated in a time and oxygen-concentration dependent manner, and are distinct from heat shock (HSPs) and glucose-regulated proteins (GRPs). To further characterize these hypoxia-associated proteins (HAPs), we investigated their upregulation in human EC from various vascular beds and compared this to possible HAP upregulation in other cell types. Human aortic, pulmonary artery, and microvascular EC upregulated the same set of proteins in response to hypoxia. In comparison, neither lung fibroblasts, pulmonary artery smooth muscle cells, pulmonary alveolar type II cells, nor renal tubular epithelial cells upregulated proteins of these Mr. Instead, most of these cell types induced synthesis of proteins of Mrs corresponding to either HSPs, GRPs, or both. Further studies demonstrated that exposure of EC to related stresses such as cyanide, 2-deoxyglucose, hydrogen peroxide, dithiothreitol, and glucose deprivation did not cause upregulation of HAPs. Evaluation of cellular damage during hypoxia using phase-contrast microscopy, trypan blue exclusion, chromium release, and adherent cell counts showed that EC survived longer with less damage than any of the above cell types. The induction of HAPs, and the lack of induction of HSPs or GRPs, by EC in response to hypoxia may be related to their unique ability to tolerate hypoxia for prolonged periods.

Adult↗
PubMed2005

Experimental and mathematical study of the influence of growth factors on the growth kinetics of adult human articular chondrocytes.

This study aimed at determining how kinetic parameters of adult human articular chondrocytes (AHAC) growth are modulated by the growth factor combination TGFbeta1, FGF-2, and PDGF BB (TFP), recently shown to stimulate AHAC proliferation. AHAC, isolated from cartilage biopsies of three individuals, were cultured in medium without (CTR) or with TFP. For growth curves, AHAC were seeded at 1,000 cells/cm(2) and cultured for 12 days, with cell numbers measured fluorimetrically in the same wells every 12 h. For microcolony tests, AHAC were seeded at 2.5 cells/cm(2) and cultured for 6 days, with cell numbers determined for each microcolony by phase contrast microscopy every 8 h. A mathematical model combining delay and logistic equations was developed to capture the growth kinetic parameters and to enable the description of the complete growth process of the cell culture. As compared to CTR medium, the presence of TFP increased the number of cells/well starting from the fifth day of culture, and a four-fold larger cell number was reached at confluency. For single microcolonies, TFP reduced the time for the first cell division by 26.6%, the time for subsequent cell divisions (generation time) by 16.8%, and the percentage of quiescent cells (Q(c)) by 42.5%. The mathematical model fitted well the experimental data of the growth kinetic. Finally, using both microcolony tests and the mathematical model, we determined that prolonged cell expansion induces an enrichment of AHAC with shorter first division time, but not of those with shorter generation time.

Aging↗
PubMed1991

Stage- and cell-specific gene expression and hormone regulation of the seminiferous epithelium.

The regulation of spermatogenesis seems to involve complex cell interactions in the testis. Little is known about these cellular communication events. Advances in molecular technology and cell or cell group separation methods have made it possible to analyze function of defined spermatogenic and Sertoli cells, thereby giving some insights into the paracrine regulation of spermatogenesis. In this review we will describe how seminiferous tubule segments with distinct cell associations can be rapidly isolated and how the cell composition can be modified by high-energy X-irradiation. Results of the recent studies performed using these techniques will be briefly summarized. Spermatogenic cells at defined stages of their development can be isolated in living condition for morphological and biochemical studies by the transillumination technique. For accurate identification of the stages of the seminiferous epithelial cycle, phase contrast microscopy of live cell squashes has been used. The criteria described by Leblond and Clermont (Am. NY Acad. Sci., 55:548-573, 1952) can be used for accurate recognition of most of the stages of the cycle. However, stages I and II and substages of VII that are important in several studies are difficult to distinguish. Therefore, in addition to the morphology of early spermatids, development of the flagella at step 16 of spermiogenesis and the changing morphology of the cytoplasmic lobes (residual bodies) at stage VII of the cycle were used as criteria for rapid identification and isolation (preparative) of the seminiferous tubule segments. Expression of nucleoprotein and heat shock protein 70-related protein genes was analyzed with Northern blot, slot blot, and in situ hybridization techniques in accurately staged seminiferous tubules. Accurate stage-dependent timing of the onset of transcription, followed by storage and disappearance of the messages was demonstrated. The chromatoid body (cb) has been proposed to have a specific function in storage of the long-lived mRNAs in the spermatids. It is an actively moving cytoplasmic organelle that interacts with Golgi complex during formation of the acrosomic system. The chromatoid body is apparently also dependent on cytoplasmic microtubules, since its movements are inhibited and its structure becomes abnormal in the presence of vincristin, an inhibitor of tubulin polymerization. Follicle-stimulating hormone (FSH) is an important regulator of Sertoli cell function. Since both basal and FSH-dependent cyclic AMP (cAMP) production by seminiferous tubules showed marked stage dependency, Sertoli cells are apparently influenced by spermatogenic cells. Thus, Sertoli cell function varies cyclically depending on the stage of the seminiferous epithelial cycle to provide an optimal microenvironment for spermatogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

The culture of chick embryo mesoderm cells in hydrated collagen gels.

Chick embryo mesoderm cells are various stages of differentiation were cultured in three-dimensional matrices of hydrated collagen. The tissues used were: stage 5 mesoderm from regions adjacent to the primitive streak; stage 12 mesoderm, comprising somitic, unsegmented (segmental plate) and lateral plate mesoderm; and stage 18 sclerotome. Explants were examined by phase contrast microscopy, including time-lapse, and scanning and transmission electron microscopy. The cells showed an increased ability to adhere to, and move in, the collagen gel with advancing stage. Of the stage 12 tissues, the unsegmented mesoderm was initially the slowest to grow out of the explant. Sclerotome cells showed by far the greatest ability to move within the gel. Where the collagen fibrils were randomly oriented, the cell morphology was polypodial and advancing lamellipodia showed clear undulations at their leading edges. A distinction was drawn between these undulations and the classical major ruffles which are seen in two-dimensional culture to uplift and pass back along the cell surface. The latter were not seen in the collagen matrix and were presumably suppressed by the three-dimensional culture configuration while the leading edge undulations were not. Ultrastructural examination showed that the cells possessed patches of amorphous material on their surface, which was sometimes interposed between the plasma membrane and collagen fibrils. Addition of hyaluronic acid (2 mg/ml) had an effect only the segmented mesoderm, where outgrowth was enhanced. Although the addition of plasma fibronectin (50 micrograms/ml) to the cultures did not affect any of the tissues, the removal of this substance, by antifibronectin antiserum or by the use of fibronectin depleted serum, inhibited outgrowth in most cases. The only tissue not reproducibly inhibited in this way was sclerotome. Alignment of the collagen fibres by the explants was observed, accompanied by an elongation of the outgrowing cells which, in bipolar form, preferentially moved up and down the aligned tracts. Scanning electron microscopy suggested that cell processes attached to, and presumably exerted tension on, bundles of fibrils thereby pulling them into line. Cell-to-cell contact was not accompanied by contact paralysis as judged by time-lapse micrography.

Animals↗
PubMed1986

Glycosaminoglycans stimulate the acrosome reaction of previously capacitated hamster sperm.

The glycosaminoglycans (GAGs) hyaluronic acid and heparin were added (10 micrograms and 100 micrograms/ml to golden hamster sperm suspensions previously incubated for 4.5 h under capacitating conditions. After additions, sperm were incubated for 5-15 min and acrosome reactions (AR) assayed in motile sperm by phase contrast microscopy. Hyaluronic acid and heparin significantly stimulated AR over control levels. Hyaluronic acid did not stimulate AR 15 min after addition to sperm previously incubated for only 2.5 h. Pre-incubation of hyaluronic acid with streptomyces hyaluronidase destroyed the ability of that GAG to stimulate the AR. These results indicate that GAGs (at least one of which, hyaluronic acid, is present in the oocyte cumulus oophorous) can rapidly stimulate the acrosome reaction in motile previously capacitated hamster sperm.

Acrosome↗
PubMed1993

Rate of establishment of a fertilising population of spermatozoa in the sheep cervix after a single mating at the onset of oestrus.

In order to study how soon a population of spermatozoa capable of fertilisation could progress into the cervical canal, the vaginal contents were flushed with a detergent solution at different intervals after mating at the onset of oestrus. Eggs recovered 1 or 2 days later from the oviducts were stained and examined by phase-contrast microscopy. Flushing at 3 minutes after mating did not prevent fertilisation in 44% of instances, whereas similar flushes performed at 6 or 9 minutes completely prevented fertilisation in a total of 26 animals. In the groups flushed at 12 and 15 minutes, the proportion of eggs fertilised was 21% and 24%, respectively, and this increased to 37% after flushing at 18 minutes. An explanation is offered for the paradoxical finding at 3 minutes, and it is concluded that at least 30-60 minutes would be required for adequate colonisation of the cervix with a fertilising population of spermatozoa under the conditions employed in the experiment. However, multiple matings or mating closer to the time of ovulation might act to reduce this interval.

Animals↗
PubMed1994

Reactivated movement of decondensed rat sperm models and a description of their ultrastructure.

This study aimed at finding optimal conditions to decondense rat sperm nuclear chromatin with minimal damage. This was judged by the ability of the sperm-tail axoneme in the partially decondensed sperm models to be reactivated. Decondensation was assessed by phase contrast microscopy. Partial decondensation was judged to occur when the bright refractive appearance of the sperm nucleus turned black, and full decondensation when the nucleus turned pale and increased in volume. Demembranation was shown to have occurred by electron microscopy. With 0.03% Triton X-100 rat caudal epididymal sperm were partially demembranated to produce sperm models. Demembranation using a 0.1% solution of Triton X-100 was complete, but as with the solution of 0.05% Triton X-100, resulted in poorer reactivation of the partially decondensed sperm models. Reactivated movement of decondensed sperm models was used to assess the effect of the decondensing agents DTT and heparin. We were only able to achieve reactivation of sperm models that had undergone partial decondensation. Optimal reactivation was obtained after rat sperm models had decondensed in the decondensation solution containing 5 mM DTT, 6 mM EDTA, and 27.3 or 34.1 USP/ml heparin. Concentrations of heparin above or below these values resulted in a decrease in the number of sperm models reactivated. Ultrastructurally, sperm partially decondensed with 5 mM DTT, 6 mM EDTA, and 34.1 USP/ml heparin had their plasma membrane further extracted compared with sperm treated with 0.03% Triton X-100 alone. Decondensation was greatest in the peripheral regions of the nucleus with extraction of the acrosome but not of the perforatorium. The decondensed regions had a filamentous appearance. This procedure will allow access to sperm nuclear chromatin for experimental manipulation in rat sperm models.

Animals↗
PubMed1986

Phenylhydrazine-induced leukocytosis in the rat.

Rats were injected with phenylhydrazine (PHZ) for periods of up to 6 months, during which time a marked leukocytosis was induced. The highest leukocyte counts occurred within 4-5 days following injection. An initial injection of 4 mg/100 gm body weight evoked a mean total leukocyte count of 129 X 10(3) cells/microliter. Successive weekly injections of 2 mg/100 gm resulted in a mean total leukocyte count of 70 X 10(3) cells/microliter compared to a mean total leukocyte count of 12.5 X 10(3) cells/microliter in saline-injected rats. Lymphocytes and monocytes accounted for approximately 75% of the total cell counts in both the PHZ-treated and control rats. The presence of increased numbers of mononuclear cells was confirmed by Percoll gradient separation and by phase-contrast microscopy. Although a leukocytosis was evident when using the automated Coulter electronic cell counter, it was not discernible when blood samples were counted manually in a hemocytometer by light microscopy. Histological examination of the thymus, lymph nodes, and spleen of the PHZ-treated rats indicated that lymphocytes and monocytes were mobilized from these sites. Lymphocyte depletion was evident, and germinal centers were found in all these lymphoid organs, indicating that PHZ induced a lymphopoietic response. A possible autoimmune etiology for PHZ-induced red blood cell destruction is discussed.

Animals↗
PubMed1994

HIV infection of monocyte-derived macrophages in vitro reduces phagocytosis of Candida albicans.

HIV-1 infection of peripheral blood monocyte-derived macrophages (MDMs) is unrelated to the level of CD4 expression on the surface of the cell, is associated with considerable donor variability, causes minimal cytopathology, and results in peak viral antigen production after 2 weeks of infection. Phagocytosis of opsonized Candida albicans by MDMs infected in vitro with several strains of HIV was compared with that of uninfected cells from the same donors; the proportion of MDMs containing the fluorescein isothiocyanate-labeled yeast was determined by flow cytometry and phase contrast microscopy. The intracellular localization of C. albicans was confirmed by confocal microscopy. Using paired MDMs from nine donors, 81% of uninfected and 53% of HIV-infected MDMs phagocytosed C. albicans. In addition, the number of yeast per cell was significantly higher in uninfected MDMs than in HIV-infected cells (mean 6.1 versus 2.5). These findings may partially explain the high incidence of mucocutaneous candidiasis in HIV-infected patients with advanced disease.

AIDS-Related Opportunistic Infections↗
PubMed1995

Stability of nuclear segments in human neutrophils and evidence against a role for microfilaments or microtubules in their genesis during differentiation of HL60 myelocytes.

The nucleus of the mature human neutrophil is segmented into three to five interconnected lobes. The physiological purpose of this segmentation is unknown, as is the mechanism by which the lobes are formed during differentiation. Using video observation of migrating human neutrophils simultaneously illuminated for fluorescence and phase-contrast microscopy, we analyzed nuclear movements with respect to cell shape changes. The number of nuclear lobes and their relative size remained constant during observation (up to 1 h). The thin connecting segments between the lobes elongated and attenuated extensively but never separated. Electron microscopic analysis of neutrophil nuclei revealed no specialized nuclear or cytoplasmic structures in the vicinity of connecting segments. With fluorescence in situ hybridization of whole chromosome probes, we determined that chromosomes are randomly distributed among neutrophil nuclear lobes. HL60 cells are a human myelocytic line that, with retinoic acid treatment, segment their nuclei and differentiate into neutrophil-like cells over several days. Using a rapidly responding variant line termed HL60/S4 (Cancer Res. 52, 949-954), we found that segmentation could be induced within 24 h. We tested the role of cytoskeletal elements in the process of nuclear segmentation. Neither the microtubule inhibitor nocodazole nor the microfilament inhibitor cytochalasin D prevented nuclear segmentation. Together, our studies suggest that nuclear lobes in neutrophils are relatively stable structures that are not generated by microtubule- or microfilament-dependent forces.

Actin Cytoskeleton↗
PubMed2004

Matrix metalloproteinase-9 promotes nerve growth factor-induced neurite elongation but not new sprout formation in vitro.

Matrix metalloproteinase-9 (MMP-9) is a basal-lamina-degrading protease that we have recently shown to be localized in regenerating sciatic nerve. We now demonstrate that MMP-9 colocalizes with growth-associated protein GAP-43 in regenerating nerves in vivo and is involved in vitro in axonal sprouting. By using a PC12 cell model for neuronal sprouting, we analyzed the effects of recombinant MMP-9, MMP-9-neutralizing antibody, and a broad-spectrum MMP inhibitor (Ro 31-9790) on sprout formation, elongation, and branching. Quantitative phase-contrast microscopy showed that MMP-9 elongated neuronal sprouts by 67% and increased their branching by 14% but did not change the number of sprouts relative to nerve growth factor (NGF) treatment. Double immunofluorescence for GAP-43, a marker for growth cones, and alpha-tubulin, a marker for axonal microtubules, showed that MMP-9-treated cells had increased distribution of alpha-tubulin but no effect on GAP-43. Western blot analyses of cell lysates demonstrated that the NGF-induced increase in GAP-43 was unchanged with MMP-9 treatment or inhibition, confirming that MMP-9 had no effect on new sprout formation. However, Ro 31-9790 reduced GAP-43 levels to those seen in untreated cells, suggesting that an MMP other than MMP-9 is important for sprout formation. Finally, phosphorylated neurofilament M (NFM-p), a marker for regenerative elongation, was induced with MMP-9 treatment and was inhibited by the anti-MMP-9 antibody treatment, confirming the role of MMP-9 in axonal elongation. NFM-p colocalized with MMP-9 in regenerating sciatic nerve fibers. These findings suggest that MMP-9 regulates neurite extension in regenerating peripheral nerve fibers and, therefore, might be of therapeutic value in promoting regeneration in vivo.

Animals↗