bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycolipid migration from the apical to the equatorial subdomains of the sperm head plasma membrane precedes the acrosome reaction Evidence for a primary capacitation event in boar spermatozoa
Authors: Gadella B. M. , Lopes-Cardozo M. , Golde L. M. G. van , Colenbrander B. , Gadella T. W. J.
Published: April 2021

In order to extend the static information of immunola-belling sulphogalactolipids in fixed boar spermatozoa, a fluorescent sulphogalactolipid analogue, galactose(3-sulphate)-β1-1′[(N-lissamine rhodaminyl)-12-aminodode-canoyl]-sphingosine, was incorporated into plasma membranes of living spermatozoa and its lateral distribu-tion over the sperm head was studied. The fluorescent lipid was enriched in the apical ridge subdomain of freshly ejac-ulated sperm cells. After sperm binding to the zona pellucida the lipid redistributed to the equatorial segment of the sperm surface. A similar shift occurred during capacitation in vitro with 2 mM CaCl2 or with 4% (w/v) bovine serum albumin. The desulphated derivative galactose-β1-1′[(N-lissamine rhodaminyl)-12-aminodo-decanoyl]-sphingosine was also incorporated into the plasma membrane of freshly ejaculated sperm cells and clearly stained the apical ridge subdomain and the (pre)-equatorial subdomains of the sperm heads. The desulphogalactolipid analogue showed a slightly faster migration to the equatori-al segment of the sperm plasma membrane than did its sulphated counterpart. The measured fluorescence intensity distributions correlated linearly with the spatial probe dis-tribution, which was checked by fluorescence lifetime imaging microscopy. The observed migration of the incor-porated glycolipids precedes the acrosome reaction and is one of the underlying molecular events likely to be important in the process of sperm capacitation. The results of this study suggest that lipid phase segregation is an important driving force for the organization of the sperm head plasma membrane into subdomains.
Cerebral microenvironment influences expression of the vitronectin gene in astrocytic tumors
Authors: Gladson Candece L. , Wilcox Josiah N. , Sanders Luraynne , Gillespie G. Yancey , Cheresh David A.
Published: April 2021

Expression of the vitronectin gene was detected in advanced human astrocytoma by in situ hybridization, whereas vitronectin mRNA was undetectable in low grade tumors or in normal adult brain, indicating that vitronectin is a marker of malignant astrocytoma. We established a model of human astrocytoma by transplanting U-251MG human astrocytoma cells intracerebrally into scid mice (C.B.17 severe combined immunodeficient mice). In this model, tumors progressed rapidly and vitronectin mRNA was preferentially detected at the invading tumor margins, i.e. where tumor cells were adjacent to the normal brain tissue. Surprisingly, when U-251MG cells were injected subcutaneously into scid mice, vitronectin mRNA was undetectable throughout the tumor. Moreover, vitronectin mRNA or protein could not be detected among these cells in culture under a wide variety of growth conditions. These findings demonstrate that the cerebral microenvironment influences the expression of the vitronectin gene in malignant astrocytoma. Importantly, the vitronectin binding integrins αvβ3 and αvβ5 localized to distinct sites within these tumors, with β3 mRNA synthesized among invading cells, and αv and β5 mRNAs detected throughout the tumor. In vitro, both of these receptors were capable of promoting adhesion and invasion of astrocytoma cells on a vitronectin substratum. These findings implicate the expression of the vitronectin gene as a contributing factor to the biological behavior of astrocytomas within the cerebral microenvironment.
Nuclear import of glycoconjugates is distinct from the classical NLS pathway
Authors: Duverger Eric , Pellerin-Mendes Christina , Mayer Roger , Roche Annie-Claude , Monsigny Michel
Published: April 2021

The nuclear import of many proteins depends on a short peptide sequence called the nuclear localization signal. However, glycosylated proteins, which lack such a nuclear localization signal, upon their injection into the cytosol by electroporation, enter the nucleus in a sugar-dependent manner. This paper brings new insights on the mechanism of this process, based on a study of neoglycoprotein nuclear uptake by digitonin-permeabilized cells. The nuclear import of neoglycoproteins is energy dependent: it does not occur when cells are maintained at 4°C or when cells are ATP-depleted by treatment with apyrase. The nuclear import of neoglycoproteins occurs through the nuclear pore: it is inhibited by preincubation of cells with wheat germ agglutinin, a lectin which binds the nuclear pore gly-coproteins and blocks the translocation step of nuclear localization signal bearing proteins through the nuclear pore. Furthermore, the nuclear import of neoglycoproteins does not use the pathway of nuclear localization signal bearing proteins: nuclear import of nuclear localization signal bearing proteins depends on cytosolic factors and is inhibited by treatment of cells with N-ethylmaleimide, while the nuclear import of neoglycoproteins neither requires added cytosolic factors nor is sensitive to alkylation by N-ethylmaleimide. In addition, upon incubation in the presence of a large excess of nuclear localization signal bearing protein, the nuclear import of neoglycoproteins is not inhibited.
Formation of two microtubule-nucleating sites which perform differently during centrosomal reorganization in a mouse cochlear epithelial cell
Authors: Tucker John B. , Mogensen Mette M. , Paton Calum C. , Mackie John B. , Henderson Craig G. , Leckie Lorna M.
Published: April 2021

This report provides evidence for the formation of a cell surface-associated centrosome with two spatially discrete microtubule-nucleating sites that perform differently; the minus ends of microtubules remain anchored to one site but escape from the other. Centrosomal reorganization in the cells in question, outer pillar cells of the organ of Corti, indicates that its pericentriolar material becomes intimately associated with the plasma membrane at the two nucleating sites.

Two large microtubule bundles assemble in each cell. A beam which includes about 1,300 microtubules spans most of the cell apex. It is positioned at right angles to a pillar with about 4,500 microtubules which is oriented parallel to the cell’s longitudinal axis.

The beam’s microtubules elongate from, and remain attached to, a centrosomal region with two centrioles which acts as a microtubule-nucleating site. However, the elon-gating microtubules do not radiate from the immediate vicinity of the centrioles. During beam assembly, the minus ends of the microtubules are concentrated together close to the plasma membrane (less than 0.2 μm away in many cases) at a site which is located to one side of the cell apex. High concentrations of the pillar’s microtubules elongating from one particular site have not been detected. Analyses of pillar assembly indicate that the following sequence of events occurs. Pillar microtubules elongate from an apical cell surface-associated nucleating site, which becomes more distantly separated from the centriolar locality as cell morphogenesis progresses. Microtubules do not accumulate at this apical nucleating site because they escape from it. They migrate down to lower levels in the cell where the mature bundle is finally situated and their plus ends are captured at the cell base.
Cyclical bending movements induced locally by successive iontophoretic application of ATP to an elastase-treated flagellar axoneme
Authors: Shingyoji Chikako , Takahashi Keiichi
Published: April 2021

To elucidate the mechanism of oscillatory bending in cilia and flagella, we studied the effect of protease digestion on the response of axonemes to localized application of ATP. When the axonemes were treated with elastase and then reactivated locally by ATP iontophoresis, a pair of local bends were formed due to localized unidirectional sliding in the vicinity of the ATP pipette. Upon repeated application of ATP, the direction of bending with respect to the sperm head axis changed cyclically from side to side over several cycles. The bends were planar and similar to those observed in axonemes that had not been treated with elastase. In trypsin-treated axonemes, in contrast, repetitive local reactivation did not induce such cyclical bending; instead, it induced a bend that grew only in one direction upon repeated application of ATP. Moreover, the bends were not planar. Electron microscopy of these protease-digested axonemes showed that both the interdoublet (nexin) links and the radial spokes were disrupted, but the effects of these proteases were different; trypsin disrupted 60-70% of these structures whereas elastase disrupted 20-30% of them. In both cases, spokes no. 3 and no. 8 (and no. 7) were more resistant to digestion than the others, although they tended to be more resistant to elastase than to trypsin. The importance of radial spokes and interdoublet links in the generation of cyclical bending and the determination of the bending plane is discussed.
Single stranded nucleic acid binding structures on chicken lampbrush chromosomes
Authors: Solovei Irina , Macgregor Herbert , Gaginskaya Elena
Published: April 2021

In chicken oocytes, proteins of the K/J family or their analogs, such as are known to be involved in mRNA processing in humans, are closely associated with nascent C-rich RNA transcripts on the loops of lampbrush chromosomes. Using labelled single stranded nucleotide probes and an antibody to protein K, these C-rich transcripts have been mapped to six different pairs of lampbrush loops situated on 3 macrochromosomes, the sex bivalent (ZW) and certain microchromosomes. Each of these loop pairs has a distinctive morphology. The observations represent cytological evidence of the connection between K-proteins and C-rich RNA transcripts. Another structure, the spaghetti marker of macrochromosome II, also preferentially binds C-rich homonucleotides. This spaghetti marker has a highly distinctive fine structural organization that is quite unlike that of lampbrush loops. Its proteins are not recognised by anti-bodies to protein K. Homonucleotide binding loops are recommended as potentially extremely valuable as markers on physical maps of chicken chromosomes.
Heterochromatin protein 1 is required for correct chromosome segregation in Drosophila embryos
Authors: Kellum Rebecca , Alberts Bruce M.
Published: April 2021

Heterochromatin protein 1 is associated with centromeric heterochromatin in Drosophila, mice, and humans. Loss of function mutations in the gene encoding heterochromatin protein 1 in Drosophila, Suppressor of variegation2-5, decrease the mosaic repression observed for euchromatic genes that have been juxtaposed to centromeric heterochromatin. These heterochromatin protein 1 mutations not only suppress this position-effect variegation, but also cause recessive embryonic lethality. In this study, we analyze the latter phenotype in the hope of gaining insight into heterochromatin function.

In our analyses of four alleles of Suppressor of variegation2-5, the lethality was found to be associated with defects in chromosome morphology and segregation. While some of these defects are seen throughout embryonic development, both the frequency and severity of the defects are greatest between cycles 10 and 14 when zygotic transcription of the Suppressor of variegation2-5 gene apparently begins. By this time in development, heterochromatin protein 1 levels are diminished by four-fold in a quarter of the embryos produced by parents that are both heterozygous for a null allele (Suppressor of variegation2-505).

In a live analysis of the phenotype, we find prophase to be lengthened by more than two-fold in Suppressor of variegation2-505 mutant embryos with subsequent defects in chromosome segregation. The elongated prophase suggests that the segregation phenotype is a consequence of defects in events that occur during prophase, either in chromosome condensation or kinetochore assembly or function. Immunostaining with an antibody against a centromere-specific antigen indicates that the kinetochores of most chromosomes are functional. The immunostaining results are more consistent with defects in chromosome condensation being responsible for the segregation phenotype.
Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture
Authors: Buschmann Michael D. , Gluzband Yehezkiel A. , Grodzinsky Alan J. , Hunziker Ernst B.
Published: April 2021

This study focuses on the effect of static and dynamic mechanical compression on the biosynthetic activity of chondrocytes cultured within agarose gel. Chondrocyte/agarose disks (3 mm diameter) were placed between impermeable platens and subjected to uniaxial unconfined compression at various times in culture (2-43 days). [35S]sulfate and [3H]proline radiolabel incorporation were used as measures of proteoglycan and protein synthesis, respectively. Graded levels of static compression (up to 50%) produced little or no change in biosynthesis at very early times, but resulted in significant decreases in synthesis with increasing compression amplitude at later times in culture; the latter observation was qualitatively similar to that seen in intact cartilage explants. Dynamic compression of ∼3% dynamic strain amplitude (≃30 μm displacement amplitude) at 0.01-1.0 Hz, superimposed on a static offset compression, stimulated radiolabel incorporation by an amount that increased with time in culture prior to loading as more matrix was deposited around and near the cells. This stimulation was also similar to that observed in cartilage explants. The presence of greater matrix content at later times in culture also created differences in biosynthetic response at the center versus near the periphery of the 3 mm chondrocyte/agarose disks.The fact that chondrocyte response to static compression was significantly affected by the presence or absence of matrix, as were the physical properties of the disks, suggested that cell-matrix interactions (e.g. mechanical and/or receptor mediated) and extracellular physicochemical effects (increased [Na+], reduced pH) may be more important than matrix-independent cell deformation and transport limitations in determining the biosynthetic response to static compression. For dynamic compression, fluid flow, streaming potentials, and cell-matrix interactions appeared to be more significant as stimuli than the small increase in fluid pressure, altered molecular transport, and matrix-independent cell deformation. The qualitative similarity in the biosynthetic response to mechanical compression of chondrocytes cultured in agarose gel and chondrocytes in intact cartilage further indicates that gel culture preserves certain physiological features of chondrocyte behavior and can be used to investigate chondrocyte response to physical and chemical stimuli in a controlled manner.
MHC class I molecules are an essential cell surface component involved in Theileria parva sporozoite binding to bovine lymphocytes
Authors: Shaw Michael K. , Tilney Lewis G. , Musoke Antony J. , Teale Alan J.
Published: April 2021

The major histocompatibility complex (MHC) class I molecules are ubiquitous cell surface molecules involved in the cell-mediated immune response. We show here, using a number of different, independent approaches, that these proteins are an essential component of the host cell surface receptor involved in Theileria parva sporozoite invasion. Monoclonal antibodies (mAbs) reactive with common determinants on MHC class I molecules and with β-2 microglobulin inhibited sporozoite entry by specifically preventing the initial binding event. However, in experiments using lymphocytes from heterozygous cattle in which at least four MHC class I gene products are expressed, mAbs which reacted with only one of these products did not inhibit entry. Using a series of bovine deletion mutant cell lines from which one or both MHC class I haplotypes had been lost, sporozoite binding and entry clearly correlated with the level of class I surface expression. While the level of sporozoite entry into cells in which one of the MHC class I haplotypes was lost was only slightly lower than into the parent cells, in a double deletion cell line having less than 5% of the class I expression of the parent cells the level of infection was only 4.3% of that into the parent cells. Furthermore, sporozoite entry into cells from a spontaneously arising mutant cell line exhibiting low levels of class I expression was correspondingly low. Treatment of lymphocytes with IL-2 produced a significant increase in host cell susceptibility and sporozoite entry and this increase correlated with either an increase in the number of target molecules per host cell, or in the binding of bovine MHC class I molecules to the mAbs. In particular, a significant increase in the level of reactivity with mAb W6/32 was observed. Lastly, we show that parasite entry can be competitively inhibited with an isolated sporozoite surface protein, p67. However, p67 binds weakly to lymphocyte surface molecules and initial attempts to use p67 to isolate the relevant host cell molecule(s) have not been successful.
Nuclear distribution of Drosophila DNA topoisomerase II is sensitive to both RNase and DNase
Authors: Meller Victoria H. , Fisher Paul A.
Published: April 2021

The nuclear distribution of Drosophila DNA topoisomerase II was determined by immunoblot analysis after nuclease digestion and cell fractionation. About 60% of DNA topoisomerase II could be removed from nuclei by RNase A, about 70% by DNase I, and about 90% by incubation with both enzymes together or with micrococcal nuclease. Nuclease treatment of nuclei did not affect the distribution of lamins Dm1 and Dm2 or other nuclear proteins similarly. Nuclease-mediated solubilization of DNA topoisomerase II from Drosophila nuclei was also dependent on NaCl concentration. Solubilization was not efficient below 100 mM NaCl. Sucrose velocity gradient ultracentrifugation demonstrated that DNA topoisomerase II solubilized from nuclei by either RNase A or DNase I migrated at about 9 S, as expected for the homodimer. Results of chemical crosslinking supported this observation. We conclude that DNA topoisomerase II has both RNA- and DNA-dependent anchorages in Drosophila embryo nuclei.
The role of the microtubular system in the cell response to HGF/SF
Authors: Dugina V. B. , Alexandrova A. Y. , Lane K. , Bulanova E. , Vasiliev J. M.
Published: April 2021

The effects of the microtubular drugs colcemid and taxol on the morphological changes induced by hepatocyte growth factor/scatter factor (HGF/SF) in MDCK cells were studied. Dynamic changes in the area and shape of individual cells were assessed by morphometric methods whereas alterations of the cytoskeleton were assessed by immunomorphological methods. The results suggest that there are two components in the response to HGF/SF: (a) activation of the extension of lamellae leading to cell spreading; and (b) reorganization of microtubules leading to polarization of cell shape. The latter response is highly sensitive to microtubular drugs, especially taxol. HGF/SF induced spreading in taxol-treated MDCK cells but these cells retained a non-polarized discoid shape and a pattern of actin microfilament bundles characteristic of the untreated cells. Colcemid and taxol did not prevent HGF/SF-induced migration of cells in Boyden chambers but completely inhibited the outgrowth of multicellular strands and tubules from cell aggregates in collagen gels. These results show that enhanced lamella formation in response to HGF/SF without polarization of cell shape is sufficient to induce cell motility. In contrast, microtubule-dependent polarization is essential for complex morpho-genetic responses such as tubulogenesis in collagen gels.
Modulation of microtubule dynamic instability in vivo by brain microtubule associated proteins
Authors: Dhamodharan Rama , Wadsworth Patricia
Published: April 2021

Heat-stable brain microtubule associated proteins (MAPs) and purified microtubule associated protein 2 (MAP-2) were microinjected into cultured BSC-1 cells which had been previously injected with rhodamine-labeled tubulin. The dynamic instability behavior of individual micro-tubules was then examined using low-light-level fluorescence microscopy and quantitative microtubule tracking methods. Both MAP preparations suppressed microtubule dynamics in vivo, by reducing the average rate and extent of both growing and shortening events. The average duration of growing events was not affected. When measured as events/unit time, heat-stable MAPs and MAP-2 did not significantly alter the frequency of rescue; the frequency of catastrophe was decreased approximately two-fold by heat-stable MAPs and MAP-2. When transition frequencies were calculated as events/unit distance, both MAP preparations increased the frequency of rescue, without altering the frequency of catastrophe. The percentage of total time spent in the phases of growth, shrink and pause was determined. Both MAP-2 and heat-stable MAPs decreased the percentage of time spent shortening, increased the percentage of time spent paused, and had no effect on percentage of time spent growing. Heat-stable MAPs increased the average pause duration, decreased the average number of events per minute per microtubule and increased the probability that a paused microtubule would switch to growing rather than shortening. The results demonstrate that addition of MAPs to living cells reduces the dynamic behavior of individual microtubules primarily by suppressing the magnitude of dynamic events and increasing the time spent in pause, where no change in the microtubule length can be detected. The results further suggest that the expression of MAPs directly contributes to cell type-specific microtubule dynamic behavior.
Regulation of growth and dissemination of a human lymphoma by CD44 splice variants
Authors: Bartolazzi Armando , Jackson David , Bennett Kelly , Aruffo Alejandro , Dickinson Richard , Shields John , Whittle Nigel , Stamenkovic Ivan
Published: April 2021

CD44 is a polymorphic cell surface glycoprotein, currently proposed to be the principal cell surface receptor for hyaluronan. However, different isoforms of CD44, expressed in human lymphoid tumor cells, appear to have distinct effects on the ability of the cells to attach to hyaluronan-coated surfaces and on their capacity to form tumors in vivo. In the present study, we address the mechanisms that may regulate CD44 isoform-dependent adhesion to hyaluronan. We use a human Burkitt lymphoma, stably transfected with six different alternatively spliced human CD44 isoforms, to determine their potential hyaluronan binding and tumor growth promoting roles. We show that transfectants expressing CD44 splice variants that contain variable exons 6-10, 7-10 and 8-10 adhere to hyaluronan-coated surfaces weakly and that corresponding tumor formation in vivo is delayed with respect to CD44-negative parental cell-derived tumors. Abundant shedding of these three isoforms may play a significant role in determining the rate of tumor development. Transfectants expressing variable exon 3, on the other hand, fail to display CD44-mediated adhesion to hyaluronan, but form bone marrow tumors rapidly following intravenous injection. These observations suggest that different mechanisms regulate CD44-mediated adhesion and tumor growth, and provide evidence that expression of exon v3 may confer novel ligand-binding properties.
Rapid intracellular assembly of tenascin hexabrachions suggests a novel co-translational process
Authors: Redick Sambra D. , Schwarzbauer Jean E.
Published: April 2021

Tenascin, an extracellular matrix protein that modulates cell adhesion, exists as a unique six-armed structure called a hexabrachion. The human hexabrachion is composed of six identical 320 kDa subunits and the structure is stabilized by inter-subunit disulfide bonds between amino-terminal segments. We have examined the biosynthesis of tenascin and its assembly into hexabrachions using pulsechase labeling of U-138 MG human glioma cells. Newly synthesized tenascin hexamers are secreted within 60 minutes of translation initiation. Intracellularly, as early as full length tenascin can be detected in pulse-labeled cell lysates, it is already in hexameric form. No precursors, such as monomers, dimers, or trimers, were identified that could be chased into hexamers. This lack of assembly intermediates suggests that nascent tenascin polypeptides associate prior to completion of translation. In contrast, fibronectin monomers in the same lysates are gradually formed into disulfide-bonded dimers. Although hexamer assembly is rapid, the rate-limiting step in secretion appears to be transport to the medial Golgi as endoglycosidase H-resistance was not detected until after a 30 minute chase. These results provide evidence for a novel co-translational mechanism of tenascin assembly which would be facilitated by its length and by the amino-terminal location of the assembly domain.
Morphological polarization of human polymorphonuclear leucocytes in response to three different chemoattractants: an effector response independent of calcium rise and tyrosine kinases
Authors: Lepidi H. , Zaffran Y. , Ansaldi J. L. , Mege J. L. , Capo C.
Published: April 2021

Chemoattractants such as interleukin-8, C5a and N-formylmethionyl-leucyl-phenylalanine induce a cytosolic calcium rise involved in triggering the secretory functions of human polymorphonuclear leucocytes. We studied the possible role of calcium rise in membrane ruffling, actin polymerization, filamentous actin distribution, and morphological polarization, which are all events contributing to chemotaxis. Membrane ruffling was assessed by right-angle light-scatter changes, the cellular content of polymerized actin by fluorescence of bodipy phallacidin, the intracellular distribution of filamentous actin by fluorescence microscopy and image digitization, and morphological polarization by scanning electron microscopy. Pretreatment of polymorphonuclear leucocytes with 50 μM BAPTA/AM, an intracellular calcium chelator, lowered the basal level in cell calcium and inhibited the transient calcium rise stimulated by 2 nM interleukin-8, 2 nM C5a, and 10 nM N-formylmethionyl-leucyl-phenylalanine. However, BAPTA pretreatment of polymorphonuclear leucocytes did not modify membrane ruffling, actin polymerization, filamentous actin distribution, and morphological polarization stimulated by chemoattractants. Downstream effectors may be protein tyrosine kinases. However, the tyrosine kinase inhibitor tyrphostin did not affect the cytoskeletal characteristics elicited by chemoattractants. Taken together, our results suggest that the transductional pathway leading to cytoskeleton organization and morphological polarization of polymorphonuclear leucocytes is different from that leading to secretion.
On the possible role of cytoskeletal filamentous networks in intracellular signaling: An approach based on percolation
Authors: Forgacs G.
Published: April 2021

Signaling in multicellular organisms is a challenging problem, for it requires the understanding of processes at drastically varying scales, at molecular, cellular and multicellular levels. As an embryo develops it must coordinate cell division, cellular adhesion and migration, and ultimately form a properly functioning organism. Extracellular morphogens and direct cell-to-cell interactions are used to establish and redefine the pattern of the developmental process. The associated extracellular signals must be sensed by the cells and transmitted eventually to the nucleus to coordinate with internal signals to induce the appropriate changes in gene activities. The cell-to-cell or environment-to-cell communication requires intricate signal transduction mechanisms whose importance is clearly not restricted to development.
Regulation of mesenchymal extracellular matrix protein synthesis by transforming growth factor-β and glucocorticoids in tumor stroma
Authors: Talts Jan Fredrik , Weller Andreas , Timpl Rupert , Ekblom Marja , Ekblom* Peter
Published: April 2021

We have here studied the composition and regulation of stromal extracellular matrix components in an experimental tumor model. Nude mice were inoculated with WCCS- 1 cells, a human Wilms’ tumor cell line. In the formed tumors the stroma was found to contain mesenchymal extracellular matrix proteins such as tenascin-C, fibulins-1 and 2 and fibronectin, but no nidogen. Nidogen was confined to basement membranes of tumor blood vessels. Since glucocorticoids have been shown to downregulate tenascin-C expression in vitro, we tested whether dexamethasone can influence biosynthesis of extracellular matrix components during tumor formation in vivo. A downregulation of tenascin-C mRNA and an upregulation of fibronectin mRNA expression by dexamethasone was noted. Transforming growth factor-β1 mRNA levels were unaffected by the dexamethasone treatment. Glucocorticoids can thus downregulate tenascin-C synthesis although local stimulatory growth factors are present. The competition between a negative and a positive extrinsic factor on synthesis of stromal extracellular matrix components was studied in a fibroblast/preadipocyte cell line. Transforming growth factor-β1 stimulated tenascin-C synthesis but did not affect fibronectin or fibulin-2 synthesis. Dexamethasone at high concentrations could completely suppress the effect of transforming growth factor-β1 on tenascin-C mRNA expression. Transforming growth factor-β1 could in turn overcome the downregulation of tenascin-C mRNA expression caused by a lower concentration of dexamethasone. We therefore suggest that the limited expression of tenascin-C in part is due to a continuous suppression by physiological levels of glucocorticoids, which can be overcome by local stimulatory growth factors when present in sufficient amounts.
Emergence of salivary gland cell lineage diversity suggests a role for androgen-independent epidermal growth factor receptor signaling
Authors: Durban Elisa M. , Nagpala Pablito G. , Barreto Patricia D. , Durban Egon
Published: April 2021

Diversity of cell lineages within glandular organs is generated postnatally by differentiation of committed progenitor cells. Fundamental regulatory aspects of this process are not understood. The mouse submandibular salivary gland (SSG) served as model to assess the role of epidermal growth factor (EGF) receptor signaling during emergence of cell lineage diversity. Temporal fluctuations in EGF receptor mRNA levels coincident with crucial differentiative cell lineage transitions were revealed by RNase protection analyses. Between days 2 and 5, when proacinar cells are maturing and striated duct cells emerge, EGF receptor mRNA levels were highest and all differentiating cells exhibited EGF receptor immunoreactivity. EGF receptor mRNA levels then declined sharply and immunoreactivity became confined to ductal cells. At day 11 in male mice, and days 11 and 16 in females, a second increase in EGF receptor mRNA was detected coincident with emergence of granular convoluted tubule (GCT) cells. With completion of androgen-dependent GCT cell differentiation at the onset of puberty, EGF receptor mRNA levels and intensity of immunoreactivity decreased. Androgen effects on EGF receptor mRNA or immunoreactivity could not be detected. These temporally distinct patterns of EGF receptor expression suggest that this signaling pathway is a mechanism of potential importance in emergence of cell lineage diversity in a glandular organ.
Focal adhesion formation by f9 embryonal carcinoma cells after vinculin gene disruption
Authors: Volberg Tova , Geiger Benjamin , Kam Zvi , Pankov Roumen , Simcha Inbal , Sabanay Helena , Coll Jean-Luc , Adamson Eileen , Ben-Ze’ev Avri
Published: April 2021

The assembly of focal adhesions was investigated in F9 embryonal carcinoma cells in which the expression of vinculin was eliminated by a targeted disruption of the vinculin gene. Vinculin-deficient F9 cells were capable of adhering to fibronectin-coated surfaces, though they displayed a reduced spreading compared to the parental cells. Transmission electron microscopy as well as inter-ference reflection microscopy of live cells showed that vinculin-null F9 cells formed focal adhesions that were indistinguishable from those of the control cells. Fluores-cent labeling for actin, talin, α-actinin, paxillin and phos-photyrosinated components indicated that the organization of all these focal contact-associated components was essen-tially identical in the vinculin-containing and vinculin-null cells. However, quantitative, digitized microscopy indicated that the intensity of fluorescence labeling in focal adhesions for α-actinin, talin and paxillin was significantly higher in cells lacking vinculin. The results suggest that there are multiple molecular mechanisms for the formation of focal adhesions in the absence of vinculin.
Differential expression of bcl-2 in intestinal epithelia correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia
Authors: Merritt Anita J. , Potten Christopher S. , Watson Alastair J. M. , Loh Dennis Y. , Nakayama Kei-ichi , Nakayama Keiko , Hickman John A.
Published: April 2021

The cell-positional incidence of both spontaneous and damage-induced apoptosis of epithelial cells was assessed in longitudinal sections of the crypts of small intestine and colon of BDF1 mice. This was compared, using immuno-histochemistry, with the pattern of expression of bcl-2, a suppressor of apoptosis. In the small intestine, apoptosis was maximal around cell position 4 from the base of the crypt; this closely corresponds to the position considered to contain the stem cells. In the colon, however, apoptosis was not confined to the area considered to harbour the stem cells (position 1 and 2). Instead, apoptosis was attenuated and distributed along the length of the crypt. Some cells at the base of murine colonic crypts expressed bcl-2 protein, whereas bcl-2 was absent in the crypts of the small intestine. Most pertinently, bcl-2 was absent from small intestinal crypt cells at positions 4-5 (the stem cell region). The importance of the expression of bcl-2 to the attenuation of apoptosis in stem cells was confirmed by analysis of the levels of both spontaneous and induced apoptosis in homozygously bcl-2 null C57BL/6 mice: in colonic crypts the level of spontaneous apoptosis rose significantly, and selectively at the base of the crypt, in comparison with crypts from wild-type animals. In contrast, there was no rise in spontaneous apoptosis in the small intestinal crypts from the bcl-2 null animals. Analysis of sections of human colon and small intestine also showed that expression of bcl-2 was confined to the base of the colonic crypt. The attenuation of apoptosis by bcl-2 in the region of the stem cells of the colonic crypts may dispose these to neoplastic transformation. Indeed, analysis of human carcinomas revealed expression of bcl-2, which in some samples was reciprocal with the expression of p53.
The small gtp-binding protein, RHO p21, is involved in bone resorption by regulating cytoskeletal organization in osteoclasts
Authors: Zhang Ding , Udagawa Nobuyuki , Nakamura Ichiro , Murakami Hiroshi , Saito Shigeru , Yamasaki Kenichi , Shibasaki Yoshinobu , Morii Narito , Narumiya Shuh , Takahashi Naoyuki , Suda Tatsuo
Published: April 2021

Rho protein (rho p21), a p21ras-related small guanine nucleotide binding protein, regulates cytoskeletal organization in a number of different types of cells. Evidence has indicated that Clostridium botulinum-derived ADP-ribosyltransferase (C3 exoenzyme) specifically ADP-ribosylates rho p21 at Asn41 and renders it functionally inactive. In this study, we examined the involvement of rho p21 in osteoclastic bone resorption using the C3 exoenzyme. When osteoclast-like multinucleated cells obtained from cocultures of mouse osteoblastic cells and bone marrow cells were placed on dentine slices, they formed ringed structures of podosomes containing F-actin (corresponding to the clear zone) within 8 hours. Many resorption pits were formed on dentine slices after culture for 24 hours. The C3 exoenzyme at 0.15-10 μg/ml added to the culture medium disrupted the ringed structure of podosomes in osteoclastlike cells in a dose-dependent manner. Correspondingly, pit formation by osteoclast-like cells on dentine slices was dose- dependently inhibited also by adding the C3 exoenzyme.

Microinjection of the C3 exoenzyme into osteoclast-like cells placed on culture dishes completely disrupted the ringed podosome structure within 20 minutes. The amount of the rho p21 which was ADP-ribosylated by the C3 exoenzyme in vitro was much greater in purified osteoclastlike cells than in osteoblastic cells. Prior exposure of the purified osteoclast-like cell preparation to the C3 exoenzyme in vivo markedly decreased the amount of unribosylated rho p21. This indicated that the C3 exoenzyme incorporated into osteoclast-like cells effectively ADP-ribosylates rho p21 in vivo. Isoelectric focusing and immunoprecipitation studies revealed that the preferentially expressed rho p21 in osteoclast-like cells was rhoA. These results suggested that rho p21, probably rhoA, plays an important role in bone resorption by regulating cytoskeletal organization in osteoclasts.
β2-microglobulin co-distributes with the heavy chain of the intestinal igg-fc receptor throughout the transepithelial transport pathway of the neonatal rat
Authors: Berryman Mark , Rodewald Richard
Published: April 2021

Maternal IgG crosses the proximal small intestine of the suckling rat by receptor-mediated endocytosis and transepithelial transport. The Fc receptor resembles the major his-tocompatibility complex class I antigens in that it consists of two subunits: a transmembrane glycoprotein (gp50) in association with β2-microglobulin. We used immunofluorescence microscopy and quantitative immunogold cyto-chemistry to study the subcellular distribution of the two subunits. In mature absorptive cells both subunits were colocalized in each of the membrane compartments that mediate transcytosis of IgG. IgG administered in situ apparently caused both subunits to concentrate within endocytic pits of the apical plasma membrane, suggesting that ligand causes redistribution of receptors at this site. These results support a model for transport in which IgG is transferred across the cell as a complex with both subunits. During absorptive cell differentiation, gp50 and β2-microglobulin showed nearly identical patterns of increased expression that accompanied the development of the apical endocytic apparatus and terminal web. However, absorptive cells in weanling rats expressed no detectable gp50 and only low levels of β2-microglobulin in the Golgi region and on the basolateral plasma membrane where class I antigens would likely reside. Thus, β2-microglobulin has a novel distribution unrelated to its function as a subunit of the class I antigens. The co-expression of the two receptor subunits is restricted to neonatal epithelial cells engaged in IgG transport and is coordinately regulated during absorptive cell differentiation and during postnatal intestinal development.
Monoclonal antibody hfd9 identifies a novel 28 kda integral membrane protein on the Cis-golgi
Authors: Subramaniam V. Nathan , Krijnse-Locker Jacomine , Tang Bor Luen , Ericsson Maria , Yusoff Abdul Rashid bin Mohd , Griffiths Gareth , Hong Wanjin
Published: April 2021

We have raised a monoclonal antibody (mAb) (HFD9) that detects a 28 kDa protein (p28) enriched in the Golgi membrane. p28 was localized to the perinuclear Golgi region in all cell lines thus far examined. Its Golgi localization was confirmed by its colocalization with Golgi markers using indirect immunofluorescence microscopy. Immunogold labelling demonstrates that the majority of p28 was localized on the cis-Golgi and its associated structures. Two independent experiments demonstrate that the p28 epitope recognized by mAb HFD9 is exposed to the cytosol. Extraction of Golgi membranes with a variety of reagents revealed that p28 behaves like an integral membrane protein. mAb HFD9 thus defines a novel 28 kDa integral membrane protein on the cis-Golgi. To our knowledge, p28 represents the first integral membrane protein of the Golgi system identified via the antibody approach whose epitope is cytoplasmically-oriented and highly-conserved. Monoclonal antibody HFD9 will thus provide a useful tool for further studies on the cis side of the Golgi, which is not well characterised due to the lack of good markers.
Production and characterisation of monoclonal and polyclonal antibodies to different regions of the cystic fibrosis transmembrane conductance regulator (cftr): Detection of immunologically related proteins
Authors: Walker Jenny , Watson Judy , Holmes Christopher , Edelman Aleksander , Banting George
Published: April 2021

We have raised mouse monoclonal antibodies to eight synthetic peptides corresponding to different regions of the human cystic fibrosis transmembrane conductance regulator (CFTR) and rabbit polyclonal antisera to β-galactosidase fusion proteins which encompass three different regions of CFTR. Immunoblot, immunoprecipitation, immunofluorescence and immunocytochemical experiments demonstrate that, in addition to recognising CFTR, these antibodies recognise one or more immunologically related proteins with a similar molecular mass, calcium responsiveness and tissue distribution to CFTR.
XCL100, an inducible nuclear MAP kinase phosphatase from Xenopus laevis: Its role in MAP kinase inactivation in differentiated cells and its expression during early development
Authors: Lewis Tom , Groom Linda A. , Sneddon Alan A. , Smythe Carl , Keyse Stephen M.
Published: April 2021

We have cloned the Xenopus laevis homologue (XCL100) of the human CL100 (Thr/Tyr) MAP kinase phosphatase. Expression of the XCL100 mRNA and protein is inducible by serum stimulation and oxidative/heat stress in a X. laevis kidney cell line. In contrast, XCL100 is constitutively expressed in growing Xenopus oocytes. Recombinant XCL100 protein is able to dephosphorylate both tyrosine and threonine residues of activated p42 MAP kinase in vitro and both the Xenopus and human CL100 proteins were localised predominantly in the nucleus in transfected COS-1 cells. As nuclear translocation of activated MAP kinase is necessary for some of its essential functions in proliferation and cell differentiation our results indicate a role for CL100 in the regulation of these nuclear signalling events. In Xenopus kidney cells both heat shock and serum stimulation lead to transient activation of MAP kinase. However, in contrast to results previously reported from studies on mammalian fibroblasts the inactivation of MAP kinase in these epitheloid cells is rapid and is not dependent on synthesis of new protein. These results indicate that the induction of CL100 (or CL100-like enzymes) may not be required for MAP kinase inactivation in all cell types. Finally, during early embryogenesis, levels of XCL100 mRNA are greatly increased at the mid-blastula transition, suggesting that this enzyme may be involved in the regulation of MAP kinase activity during early development.
Metastasis-associated 5T4 oncofoetal antigen is concentrated at microvillus projections of the plasma membrane
Authors: Carsberg Catherine J. , Myers Kevin A. , Evans Gareth S. , Allen Terence D. , Stern Peter L.
Published: April 2021

The 5T4 oncofoetal antigen is a 72 kDa glycoprotein defined by a monoclonal antibody raised against human placental trophoblast and is expressed in many different carcinomas but detected only at low levels in some normal epithelia. Immunohistochemical analysis of the patterns of expression in colorectal carcinomas has indicated a significant association between the presence of the antigen in tumour cells and metastatic spread. A cDNA encoding the 5T4 molecules has been isolated and the extracellular portion contains several leucine-rich repeats which have been implicated in cellular interactions. To study the cell biological role of 5T4 molecules, murine L cells (A9 derivative) were stably transfected with 5T4 cDNA under the control of the CMV immediate-early promoter. The 5T4-expressing cells exhibited a more spindle-shaped morphology compared to the vector alone transfected cells. Confocal immunofluorescence microscopy revealed a ‘polkadot’ pattern of 5T4 antigen expression, heterogeneous in intensity between cells, but distributed over the entire cell surface. Transmission and scanning electron microscopy showed that the 5T4 antigen is concentrated at microvillus projections of the plasma membrane both in the transfected A9 cells and in various carcinoma cell lines. Such projections express an array of surface molecules which function in cell adhesion and motility. This association of 5T4 antigen with microvillus projections was also observed in various carcinoma cell lines. 5T4 expression in A9 cells was also associated with an altered pattern of cell division, decreased cell-substratum adhesion and increased cellular motility. These results support the hypothesis that 5T4 molecules may have a direct function in trophoblast and tumour cell invasion processes.
Phosphorylation of the tight junction protein cingulin and the effects of protein kinase inhibitors and activators in MDCK epithelial cells
Authors: Citi Sandra , Denisenko Natasha
Published: April 2021

In previous studies we have shown that protein kinase inhibitors and extracellular calcium can affect dramatically the assembly of tight junctions (TJ) and the localization of the TJ protein cingulin at sites of cell-cell contact in renal epithelial (MDCK) cells. To characterize in more detail the relationships between kinase activity and junction organization, we have studied the effects of the protein kinase C agonist phorbol myristate acetate (PMA) on the intracellular localization of cingulin, E-cadherin, desmoplakin and actin microfilaments in confluent MDCK monolayers. To study cingulin phosphorylation, MDCK cells were metabolically labelled with [32P]orthophosphate and immunoprecipitates were prepared with anti-cingulin antiserum. We show here that cingulin is phosphorylated in vivo on serine, and its specific phosphorylation is not significantly changed by treatment of confluent MDCK mono-layers with PMA, with the protein kinase inhibitor H-7, or with the calcium chelator EGTA. Metabolic labeling with a pulse of [35S]methionine/cysteine showed that at normal extracellular calcium net cingulin biosynthesis was not affected by PMA or H-7. During junction assembly by calcium switch, H-7 did not change the specific phosphorylation of the immunoprecipitated cingulin, however, it prevented the increase in the amount of cingulin in the immunoprecipitates, suggesting that H-7 may block tight junction assembly by interfering with cellular processes that lead to the accumulation and stabilization of TJ proteins at sites of cell-cell contact.
A transient association of γ-tubulin at the midbody is required for the completion of cytokinesis during the mammalian cell division
Authors: Shu Hong-Bing , Li Zhiqiang , Palacios Monica J. , Li Qingqin , Joshi Harish C.
Published: April 2021

γ-Tubulin, a relatively new member of the tubulin gene family, is localized primarily at the centrosome throughout the mammalian cell cycle and may play a key role in nucleation of cellular microtubule assembly. A transient association of γ-tubulin at the cytoplasmic bridge of telophase mammalian cells, the midbody, is recently documented. Using immunogold electron microsocopy and serial section reconstruction analysis, we show here that the transiently associated midbody γ-tubulin is localized at the minus ends of microtubules in the midbody structure. Using antisense RNA methods we also demonstrate that a selective depletion of transiently associated midbody γ-tubulin causes an abortive cytokinesis due to a failure in the morphogenesis of the midbody structure.