bluuebunny/crossref_metadata_2025_split huggingface dataset:
Matrix metalloproteinase-9 (92 kDa gelatinase/type IV collagenase) from U937 monoblastoid cells: correlation with cellular invasion
Authors: Watanabe Hideto , Nakanishi Isao , Yamashita Kyoko , Hayakawa Taro , Okada Yasunori 
Published: April 2021

ABSTRACT
The role of matrix metalloproteinase-9 (MMP-9, 92 kDa gelatinase/type IV collagenase) in invasion of mononuclear phagocytes was studied with U937 monoblastoid cells. 12-o-tetradecanoyl 13-phorbol acetate (TPA) differentiated them to macrophage-like cells with induction of MMP-9, and tumor necrosis factor TNF and interleukin-1α (IL-1α) stimulated the production of MMP-9 by TPA-treated cells. TNF also induced the production of MMP-9 by TPA-untreated U937 cells without morphological differentiation. Other agents including dimethyl sulfoxide (DMSO), all-trans-retinoic acid (all-trans-RA), platelet-derived growth factor and 3′ ;5′-cyclic monophosphate had no effects on MMP-9 production by TPA-treated or-untreated cells, but all-trans-RA and DMSO did have a morphological effect on the differentiation of the cells. These data suggest that MMP-9 production by U937 cells is regulated by a mechanism independent of the differentiation to macrophage-like cells. MMP-9 was purified to homogeneity as an inactive zymogen with Mr 92,000 (proMMP-9) from TPA-differentiated U937 cells treated with TNF. ProMMP-9 was activated by p-aminophenylmercuric acetate (APMA) generating an active species of Mr 67,000. Trypsin and cathepsin G also attained activation of the zymogen to its full activity obtained by APMA activation, but plasmin, leukocyte elastase, thrombin and plasma kallikrein had no ability to activate it. APMA-activated MMP-9 degraded type I gelatin readily and cleaved native collagen types III, IV and V. Invasion assays using reconstituted basement membrane coupled with a type IV collagenolysis assay showed good correlations between invasiveness, type IV collagenolysis and proMMP-9 production. Invasion was significantly inhibited by EDTA, 2-macroglobulin and tissue inhibitor of metalloproteinases-1, but not by inhibitors of cathepsin G and leukocyte elastase. These data suggest that MMP-9 plays an important role in the invasion of mononuclear phagocytes through basement membranes.
A methodology for the systematic and quantitative study of cell contact guidance in oriented collagen gels Correlation of fibroblast orientation and gel birefringence
Authors: Guido Stefano , Tranquillo Robert T. 
Published: April 2021

ABSTRACT
Despite the likely role of contact guidance in every physiological process involving cell migration, its study in a three-dimensional tissue-equivalent environment has been precluded, heretofore, by inherent difficulties in systematically preparing well-defined contact guidance fields and quantifying the resultant contact guidance. Here, we describe a novel use of a magnetic field to orient collagen fibrils during fibrillogenesis, entrapping cells dispersed in the collagen solution. Using computer-controlled staging and image analysis, we show from automated birefringence measurements of the resultant slab of cell-populated gel contained in a specially designed observation chamber that the fibril orientation is biased along the long axis of the chamber uniformly throughout the chamber. Further, we show that the degree of fibril orientation, and consequently the elicited contact guidance, can be controlled by independently varying the magnetic field strength or temperature during fibrillogenesis. We characterize the contact guidance response to the imposed contact guidance field by measuring cell orientation relative to the axis of fibril orientation from still images obtained in time-lapse via automated image analysis. We present the first quantitative correlation of contact guidance (based on cell orientation) with collagen fibril orientation (based on birefringence) for human foreskin fibroblasts cultured in a collagen gel, by using gels of varying orientation resulting from different magnetic field strengths and temperatures during fibrillogenesis, and by using sufficiently low cell concentrations and early observation times.
Modulation of Muc-1 mucin expression in the mouse uterus during the estrus cycle, early pregnancy and placentation
Authors: Braga Vania M. M. , Gendler Sandra J. 
Published: April 2021

ABSTRACT
The Muc-1 protein is an integral membrane protein that is expressed apically by simple secretory epithelia in many different organs. We present in this paper a study on Muc-1 protein expression in the mouse uterus during early pregnancy, placentation and the estrus cycle. Muc-1 immunopositive reaction is found in the decidua by day 8 of pregnancy onwards. The observed pattern was unusual, since Muc-1 protein was present in spherical cytoplasmic granules in granular metrial gland cells. Both the intracellular pattern of expression and the lymphoid origin of these cells were striking results. Muc-1 is thought to be an epithelial differentiation marker, and this is the first report of its expression by non-epithelial cells. Our results on Muc-1 expression in the uterus of cycling mice showed that higher levels of Muc-1 mRNA and protein correlate with higher levels of plasma estrogen in the estrus and proestrus phases. However, in ovariectomized mice without hormone replacement, the endometrium expressed high levels of this protein. These levels could not be substantially changed by estrogen, although progesterone reduced the levels of Muc-1 protein associated with the epithelium. These data together with the normal expression in the cycling mice suggest that progesterone might repress Muc-1 expression during the metestrus and diestrus phases. In cycling mice, when plasma progesterone is at its nadir and the estrogen level is elevated in estrus and proestrus phases, Muc-1 concentration would increase to its basal level, not because of estrogen stimulation, but due to lack of progesterone repression. The low level of expression is also observed in the endometrium during early pregnancy, where reduced levels of Muc-1 protein are seen at the time of implantation.
Controlled induction of focal adhesion disassembly and migration in primary fibroblasts
Authors: Dunlevy Jane R. , Couchman John R. 
Published: April 2021

ABSTRACT
Fibroblast migration is an integral component of biological processes such as wound healing and embryogenesis. Previous experiments examining fibroblast locomotion from tissue explants have shown that migrating fibroblasts lack, or contain only transient, focal adhesions (focal contacts). Focal adhesions are specialized regions of tight cell-matrix interaction, assembled by a complex process of transmembrane signalling. Although the explant model has been used for studying several aspects of fibroblast locomotion, it is limited by the lack of control over migration, and only a small percentage of the cells actually locomoting. Therefore, we have developed an in vitro model for cultured fibroblast strains where the presence or absence of focal adhesions can be manipulated, and in the latter case 70% of these cells become locomotory.
The stimulus used to decrease the percentage of cells containing focal adhesions, and hence enhance locomotion, was newborn rat heart-conditioned medium (HCM). Addition of HCM to rat embryo fibroblasts induced both chemokinesis and chemotaxis. Cells disassembled focal adhesions on a variety of extracellular matrix substrates after approximately 6 h of stimulation with HCM; conversely, removal of HCM promoted reformation of focal adhesions within 12-24 h. HCM-stimulated fibroblasts which lacked focal adhesions concomitantly lacked F-actin stress fibers and focal concentrations of vinculin and talin. Therefore, fibroblast migration can be readily controlled in an on-off manner through conditioned medium, which influences the absence or presence of focal adhesions.
Topoisomerase II inhibition prevents anaphase chromatid segregation in mammalian cells independently of the generation of DNA strand breaks
Authors: Clarke Duncan J. , Johnson Robert T. , Downes C. Stephen 
Published: April 2021

ABSTRACT
Yeast temperature-sensitive mutants of DNA topoisomerase II are incapable of chromosome condensation and anaphase chromatid segregation. In mammalian cells, topoisomerase II inhibitors such as etoposide (VP-16-123) have similar effects. Unfortunately, conclusions drawn from work with mammalian cells have been limited by the fact that the standard inhibitors of topoisomerase II also generate DNA strand breaks, which when produced by other agents (e.g. ionizing radiation) are known to affect progression into and through mitosis. Here we show that the anti-tumour agent ICRF-193, recently identified as a topoisomerase II inhibitor operating by a non-standard mechanism, generates neither covalent complexes between topoisomerase II and DNA, nor adjacent DNA strand breaks, in mitotic HeLa. However, the drug does prevent anaphase segregation in HeLa and PtK2 cells, with effects similar to those of etoposide. We therefore conclude that topoisomerase II function is required for anaphase chromosome segregation in mammalian cells, as it is in yeast.
Regulation of p53 protein expression in human breast cancer cell lines
Authors: Vojtěšek Bořivoj , Lane David P. 
Published: April 2021

ABSTRACT
Mutation of the p53 gene is a common occurrence in human breast cancers but is by no means universal. However, even in tumours where the gene is not mutated altered levels of p53 protein are often detected. This is also observed in cell lines derived from human breast cancers. By transfecting such cell lines containing either wild type or mutant p53 genes with a temperature-sensitive mutant mouse p53 gene we have established that the cellular environment plays a critical role in the regulation of p53 protein expression. The results suggest that tumours that aberrantly express wild-type p53 may have lost the normal growth regulatory response to the protein and thus be functionally similar to those expressing the mutant protein.
Differential localization patterns of myristoylated and nonmyristoylated c-Src proteins in interphase and mitotic c-Src overexpresser cells
Authors: David-Pfeuty Thérèse , Bagrodia Shubha , Shalloway David 
Published: April 2021

ABSTRACT
Myristoylation of pp60src is required for its membrane attachment and transforming activity. The mouse monoclonal antibody, mAb327, which recognizes both normal, myristoylated pp60c-src and a nonmyristoylated mutant, pp60c-src/myr−, has been used to compare the effects of preventing myristoylation on the localization of c-Src in NIH 3T3-derived overexpresser cells using immunofluorescence microscopy. During interphase, pp60c-src partitions between the plasma membrane and the centrosome, while pp60c-src/myr− is predominantly cytoplasmic but also partly nuclear. The cytoplasmic, but not the nuclear, staining can be readily washed out by brief pretritonization of the cells before fixation, indicating that the cytoplasmic pool of pp60c-src/myr−, in contrast with the nuclear one, does not associate tightly with structures that are insoluble in the presence of nonionic detergents. We have previously shown that during G2 phase, pp60c-src leaves the plasma membrane and is redistributed diffusely throughout the cytoplasm and to two clusters of patches surrounding the two separating centriole pairs. In contrast, we now find that pp60c-src/myr− translocates to the nucleus in late G2 or early prophase prior to there being any clear evidence of nuclear membrane breakdown or nuclear lamina disassembly. Similar nuclear translocation of pp60c-src/myr−, but not of pp60c-src, is also observed when cells are arrested in G0 or at the G1/S transition. Furthermore, during mitosis, pp60c-src is found primarily in diffuse and patchy structures dispersed throughout the cytoplasm while pp60c-src/myr− more specifically associates with the main components of the spindle apparatus (poles and fibers) and inside the interchromosomal space.
These results suggest that a possible role for myristoylation might be to prevent unregulated nuclear transport of proteins whose nonmyristoylated counterparts are readily moved into the nucleus. They also raise the possibility that a subfraction of wild-type pp60c-src may behave, at specific times, like its nonmyristoylated counterpart, and may translocate to the nucleus and exert specific functions in that location.
Binding of the α-fodrin SH3 domain to the leading lamellae of locomoting chicken fibroblasts
Authors: Meriläinen J. , Palovuori R. , Sormunen R. , Wasenius V.-M. , Lehto V.-P. 
Published: April 2021

ABSTRACT
Fodrin (nonerythroid spectrin) is a membrane skeletal protein that plays an important role in the establishment and maintenance of the cell shape and polarity. We have identified in α-fodrin an src homology 3 (SH3)-related region, a small domain that is present in a large number of proteins that are involved in signal transduction, cell polarization and membrane-cytoskeleton interactions. In this study we have explored the function of the α-fodrin SH3 by incubating fixed and permeabilized cultured chicken fibroblasts with the αfodrin SH3 peptide, expressed in bacteria as a fusion protein with glutathione S-transferase. Immunofluorescence and immunoelectron microscopy showed that αfodrin SH3 binds to the cytoplasmic face of the plasma membrane in the leading lamellae and the pseudopodial lobes of the spreading and locomoting cells. No, or only minimal, binding was seen in immotile cells, or in the stationary trailing ends of the locomoting cells. SH3 binding was also seen in cytochalasin-D-treated cells, suggesting that actin filaments are not responsible for the binding. These findings suggest that α-fodrin SH3 interacts with plasma membrane components that are present in the leading lamellae exclusively or are modulated in a manner specific to the leading lamellae.
Distribution of acidic and basic fibroblast growth factors in ovine skin during follicle morphogenesis
Authors: Cros D. L. du , Isaacs K. , Moore G. P. M. 
Published: April 2021

ABSTRACT
Acidic and basic fibroblast growth factors (aFGF and bFGF) have been localized by immunochemistry in ovine skin during wool follicle morphogenesis. At 40 days of gestation, prior to the appearance of follicle pri-mordia, bFGF immunoreactivity was detected in the intermediate and periderm layers of the epidermis and at the dermal-epidermal junction. Antibodies to aFGF did not bind to skin at this age. During early follicle formation, at 76 days of gestation, both FGFs were found in the epidermis and associated with the follicle primordia. Antibodies to aFGF, in particular, bound to the basal cells of the epidermis and the follicle cell aggregations. With the development of epidermal plugs, bFGF was confined to the intermediate layers of the epidermis and the dermal-epidermal junction, whereas aFGF staining was associated with the cells of the epidermis and the plugs. At 90 days, when many different stages of follicle development were in evidence, immunoreactivity for both FGFs was associated with the cells of the elongating epidermal column, particularly those adjacent to the dermal-epidermal junction. During follicle maturation, bFGF was found in the suprabasal layer of the epidermis, in the outer root sheath of the follicle and in the basement membrane zone surrounding the bulb matrix. Conversely, strong staining for aFGF was observed in the epidermis and pilary canal contiguous with the epidermis, and in cells of the upper bulb matrix of the follicle in the region of the keratogenous zone. Western blotting of extracts of mature follicles that had been isolated from the skin showed the presence of a major aFGF immunoreactive band with an apparent molecular mass of 27 kDa.
The distributions of aFGF and bFGF, particularly around the dermal-epidermal junction during follicle development, demonstrate that these growth factors may have related functions in local tissue remodelling during follicle morphogenesis. However, in adult skin, the presence of bFGF adjacent to the proliferative zone of the follicle suggests its involvement in regulating the mitotic activity in the follicle bulb. By contrast, the localization of aFGF to the cells of the upper follicle bulb, in the zone of keratinization, implicates this growth factor in cellular differentiation.
Redundant enhancer elements guide β1 tubulin gene expression in apodemes during Drosophila embryogenesis
Authors: Buttgereit Detlev 
Published: April 2021

ABSTRACT
During Drosophila embryogenesis, the β1 tubulin gene ( Tub56D) is expressed in the CNS and PNS as well as in the apodemes. In this report we determine the regulation of β1 tubulin gene expression during formation of the attachment sites of the somatic muscles, the apodemes. The process was analysed in transgenic flies carrying β1 enhancer-hsp70 promoter-lacZ fusion constructs. Expression is first detected at late stage 13 and remains until hatching. By deletion analysis of the intron we identified a 14-bp element present in three copies. This element represents a classical enhancer, as it acts on a heterologous promoter. Separate fragments containing the respective elements yield nearly identical expression patterns, and no cooperativity was observed between the three copies. Thus, the expression of the β1 tubulin gene in the apodemes is under control of redundant enhancer elements. Double staining for β1 tubulin gene expression in apodemes and for β3 tubulin gene expression in muscles allowed us to correlate apodeme and muscle formation. Cells of the apodemes that are in contact with their corresponding muscles show expression of the reporter gene as monitored by antibody staining.
Expression of high molecular weight tau in the central and peripheral nervous systems
Authors: Georgieff Irene S. , Liem Ronald K. H. , Couchie Dominique , Mavilia Carmelo , Nunez Jacques , Shelanski Michael L. 
Published: April 2021

ABSTRACT
Using a novel PCR approach, we have cloned a cDNA encoding the entire high molecular weight tau molecule from rat dorsal root ganglia. The resulting 2080 bp cDNA differs from low molecular weight rat brain tau by the insertion of a novel 762 bp region (exon 4a) between exons 4 and 5. This cDNA clone is identical in sequence with a high molecular weight tau (HMW) cDNA from rat PC12 tumor cells and is closely related to a HMW tau cDNA from mouse N115 tumor cells. In vitro transcription/translation produces a protein that migrates on SDS-PAGE with the same apparent molecular weight as HMW tau purified from rat sciatic nerve. The HMW tau protein is generated from an 8 kb mRNA, which can be detected by northern blots in peripheral ganglia, but not in brain. A more sensitive assay using PCR and Southern blot analysis demonstrates the presence of exon 4a in spinal cord and in retina. In combination with immunohistochemical studies of spinal cord, these data suggest that HMW tau, though primarily in the peripheral nervous system, is also expressed in limited areas of the central nervous system, although its presence cannot be detected in the cerebral cortices.
Endothelial cell attachment and spreading on human tenascin is mediated by α2β1 and αVβ 3 integrins
Authors: Sriramarao P. , Mendler Markus , Bourdon Mario A. 
Published: April 2021

ABSTRACT
Human umbilical vein endothelial cells were found to attach and partially spread on human tenascin. The attachment of endothelial cells to tenascin results in elongated cells with interconnecting processes and is distinct from the flattened appearance of endothelial cells on fibronectin, collagen, vitronectin or laminin substrata, suggesting a role for tenascin in modulating cell adhesion and motility. Endothelial attachment to tenascin was partially inhibitable by the SRRGDMS peptide derived from human tenascin and completely inhibitable by anti-integrin antibodies to α2β1 and αvβ3. Endothelial cell attachment to tenascin could be inhibited up to 80% with anti-x2 and anti-β1 monoclonal antibodies P1E6 and P4C10, respectively, and this was associated with a complete loss in cell spreading. In contrast, pretreatment of endothelial cells with the anti-(XVβ3 monoclonal antibody LM609, resulted in a 35% inhibition in cell attachment but did not alter cell spreading. In combination the anti-α2 and anti-œVβ3 antibodies, could completely abrogate cell spreading and attachment to tenascin-coated surfaces. Affinity purification of 125I-labeled endothelial cell extract on a tenascin matrix column followed by immunoprecipitation with monoclonal antibodies to different integrin ex and β subunits resulted in the identification of α2β1 and (XVβ3 integrins, respectively, as tenascin binding receptors. Collagen affinity-purified ot2β1 receptor from endothelial cells bound not only to collagen and laminin but also to tenascin in a radio receptor binding assay. The results demonstrate that (X2β1 and αVβ3 mediate distinct endothelial cell interactions with tenascin; cell spreading and cell binding, respectively. Binding by cxVβ3 is mediated by the SRRGDMS site on tenascin, whereas the α2β1 binding site remains undefined. The interaction of oc2β1 and cxVβ3 with tenascin may be regulated in a cell typespecific manner as evidenced by the binding of endothelial cell oc2β1 and (XVβ3 to tenascin, and the lack of binding by the same receptors on osteosarcoma MG63 to tenascin.
Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells
Authors: Berryman Mark , Franck Zsofia , Bretscher Anthony 
Published: April 2021

ABSTRACT
Ezrin and moesin are two cytoskeletal proteins originally purified from human placenta that are 74% identical in overall protein sequence. They are believed to be membrane-cytoskeletal linking proteins because they share sequence homology with erythrocyte band 4.1 and colocalize with actin specifically in microvilli and membrane ruffles in cultured cells. To determine if ezrin and moesin share similar distributions in vivo, we studied their localizations with respect to F-actin in tissue sections. Surprisingly, ezrin and moesin exhibited very different cellular distributions. Ezrin was highly concentrated and colocalized with actin on the apical surface of many epithelial cell types. During enterocyte differentiation, the pattern of expression and redistribution of ezrin was consistent with it performing a role in microvillus assembly. Immunoelectron microscopy in differentiated cells revealed that ezrin was restricted mainly to the plasma membrane of microvilli and other actin-rich surface projections. Moesin was found in endothelial cells and was also enriched in the apical microvilli of a restricted set of epithelial cells. All polarized cell types with abundant microvilli contained one or both proteins, suggesting that ezrin and moesin perform related functions. However, the differential expression of ezrin and moesin indicates that they have distinct properties, which are uniquely adapted to specific cell types.