bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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.
The response to thrombin of human neutrophils: Evidence for two novel receptors
Authors: Jenkins Alison L. , Howells Gareth L. , Scott Emma , Bonniec Bernard F. Le , Curtis Michael A. , Stone Stuart R. 
Published: April 2021

ABSTRACT
Human α-thrombin was a chemoattractant for human neutrophils yielding a maximal response of similar magnitude to that observed with formyl-Met-Leu-Phe. The observed chemotaxis was not due to stimulation of the proteolytically activated thrombin receptor since: (1) this receptor was not detected by flow cytometry; (2) the inactive thrombin mutant Ser195→Ala elicited a chemotactic response indistinguishable from that caused by wild-type thrombin; (3) antibodies to the cleavage site of the proteolytically activated receptor did not affect thrombin-induced chemotaxis; (4) a thrombin receptor activating peptide (TRAP) failed to stimulate chemotaxis. These data indicate the existence of a thrombin receptor for neutrophil chemotaxis which is not activated by proteolysis. In addition, although wild-type and Ser195→Ala thrombin did not cause an increase in intracellular Ca2+, a Ca2+ response to TRAP was observed with neutrophils from some donors. The TRAP-induced increase in Ca2+ was reproducible, dose dependent and specific. The use of alanine-substituted peptides demonstrated that the Ca2+ response was due to TRAP stimulation of a receptor other than the proteolytically activated thrombin receptor. Thus, it is necessary to re-evaluate the assumption made in previous studies that responses to TRAP are mediated by the proteolytically activated thrombin receptor.
Distinct heparin-binding and neurite-promoting properties of laminin isoforms isolated from chick heart
Authors: Brandenberger Ralph , Chiquet Matthias 
Published: April 2021

ABSTRACT
Laminin isolated from chick heart is composed of several heterotrimeric variants of 800 and 700 kDa. Here, we used monoclonal antibodies against chick laminin to purify different laminin isoforms from this mixture. Antibody 8D3 specifically removed laminin containing α2 chain from chick heart laminin preparations, leaving behind 700 kDa variants. Using antibody C4 against the laminin β2 chain, α2 chain containing variants were further separated into α2β1γ1 and α2β2γ1 laminin, respectively. Laminins containing α2 chain and recognized by antibody 8D3 are crossshaped molecules. Their expression during embryogenesis is tightly regulated. In 5-day embryos staining with monoclonal antibody 8D3 is restricted to the dermamyotome. Older embryos (8 days) express α2 chain containing variants at myotendinous junction primordia of skeletal muscle, and only late in development these variants are generally expressed in skeletal and heart muscle basement membranes. The 700 kDa laminin variants contain β1, β2, and γ1 subunits affiliated with an immunologically distinct, shorter ax chain and appear to be T-shaped in the electron microscope. Whereas laminins with an α2 subunit bind to heparin, variants with the novel ax chain do not. Experiments using cultured sympathetic neurons showed that laminins with ax chain are less potent than α2 chain containing variants in promoting neurite outgrowth. In contrast, sympathetic neurons cannot discriminate between α2β1γ1 and α2β2γ1 laminin substrates, respectively, and show identical high rates of neurite formation.
CD44 exhibits a cell type dependent interaction with Triton X-100 insoluble, lipid rich, plasma membrane domains
Authors: Neame Stephen J. , Uff Caroline R. , Sheikh Humma , Wheatley Susan C. , Isacke Clare M. 
Published: April 2021

ABSTRACT
CD44 is an abundant, widely expressed transmembrane glycoprotein which can act as a receptor for the extracellular matrix glycosaminoglycan, hyaluronan. Biochemical and morphological studies have demonstrated that in fibroblasts a significant proportion of the CD44 population is resistant to Triton X-100 extraction and that the detergent insoluble protein is co-localized with components of the cortical cytoskeleton. Surprisingly, this distribution is not abrogated upon deletion of the CD44 cytoplasmic tail indicating that mechanisms other than a direct interaction with the cytoskeleton can regulate CD44. In this manuscript, the mechanisms underlying this detergent-insoluble association are further investigated. There was no evidence that the Triton X-100 insolubility of CD44 resulted from homotypic aggregation, an association with hyaluronan or from a direct, or indirect, association with the cytoskeleton. Instead, evidence is presented that the detergent insolubility of fibroblast CD44 at 4°C results from an association of the CD44 transmembrane domain with Triton X-100 resistant, lipid rich, plasma membrane domains. The proportion of the CD44 found in these Triton X-100 insoluble structures is dependent upon cell type and cannot be altered by changing cell motility or extracellular matrix associations. These studies provide evidence for a novel mechanism regulating this adhesion protein in the plasma membrane.
Intranuclear retention of ribosomal RNAs in response to herpes simplex virus type 1 infection
Authors: Besse Sylvie , Puvion-Dutilleul Francine 
Published: April 2021

ABSTRACT
The localization of ribosomal RNA (rRNA) was investigated at the ultrastructural level in herpes simplex virus type 1 infected HeLa cells using three distinct biotinylated probes which bind in sequence to three different segments of the ribosomal genes. Comparison of the above with the signal levels obtained from non-infected cells reveals information about the effects of HSV-1 infection on ribosome biogenesis. A probe specific for the 5′end portion of prerRNA labeled all nucleoli of both non-infected and infected cells in the same way, that is, it mainly labeled the dense fibrillar component and the border of the fibrillar centers but only slightly labeled the granular component. This indicates that the initial cleavage of pre-rRNA in herpes infection still occurs in the 5′ region of the 5′ external transcribed spacer. However, a probe specific for 18 S rRNA labeled the granular component of the nucleoli more intensely after infection. In addition, significant amounts of rRNA molecules were present within the intranuclear viral region, except over the enclosed viral dense bodies, and within the virus-enlarged clusters of interchromatin granules. The data indicate that the still enigmatic viral dense bodies, which are nucleolus-related structures, are excluded from the marked intranuclear retention of ribosomal RNAs and, in addition, reveal a possible role for the interchromatin granules of infected cells in the regulation of the export of the ribosomal subunits towards the cytoplasm.
Localization of Gαo to growth cones in PC12 cells: role of Gαo association with receptors and Gβγ
Authors: Nüße Oliver , Neer Eva J. 
Published: April 2021

ABSTRACT
The heterotrimeric G protein Go is highly enriched in the growth cones of neuronal cells and makes up 10% of the membrane protein of growth cones from neonatal rat brain. We have used PC12 cells, a cell line that differentiates to a neuron-like phenotype, as a model with which to study the mechanism of G protein localization. First, the role of the βγ-subunit was investigated. The attachment of the βγ-subunit to the membrane depends on the isoprenylation of the γ-subunit. The drug lovastatin blocks isoprenylation by inhibiting a key enzyme in the biosynthetic pathway. After treatment of PC12 cells with 10 μM lovastatin for 48 hours 50% of the βγ-subunits were cytosolic compared with 100% membrane bound βγ in control cells, as determined by cell fractionation, gel electrophoresis and western blot. Addition of 200 μM mevalonic acid reverses this effect. However, lovastatin affects neither the membrane attachment of αo nor its localization to the growth cones as determined by immunohistochemistry. This suggests that the localization and retention of αo are independent of the membrane attachment of the full complement of βγ-subunits. Second, pertussis toxin was used to block the interaction between αo and receptors. PC12 cells were treated with 0.1 μg/ml pertussis toxin prior to and during nerve growth factor-induced differentiation. In vitro [32P]ADP-ribosylation confirmed that αo and αi were completely ADP-ribosylated by this treatment. The ADP-ribosylation by pertussis toxin did not interfere with neurite outgrowth. The localization of αo to the growth cones was indistinguishable from that in untreated cells. We conclude that G protein-receptor interaction is not necessary for the distribution of αo to growth cones.
The gelsolin-related flightless I protein is required for actin distribution during cellularisation in Drosophila
Authors: Straub Kristina L. , Stella Maria Cristina , Leptin Maria 
Published: April 2021

ABSTRACT
We have analysed the developmental defects in Drosophila embryos lacking a gelsolin-related protein encoded by the gene flightless I. Such embryos have previously been reported to gastrulate abnormally. We now show that the most dramatic defects are seen earlier, in actin-dependent events during cellularisation of the syncytial blastoderm, a process with similarities to cytokinesis. The blastoderm nuclei migrate to the periphery of the egg normally but lose their precise cortical positioning during cellularisation. Cleavage membranes are initially formed, but invaginate irregularly and often fail to close at the basal end of the newly formed cells. The association of actin with the cellularisation membranes is irregular, suggesting a role for flightless I in the delivery of actin to the actin network, or in its stabilisation.
Characterization of VIP36, an animal lectin homologous to leguminous lectins
Authors: Fiedler Klaus , Simons Kai 
Published: April 2021

ABSTRACT
VIP36 was isolated from MDCK cells as a component of glycolipid-enriched detergent-insoluble complexes. The protein is localized to the Golgi apparatus and the cell surface, and belongs to a new family of legume lectin homologues in the animal secretory pathway that might be involved in the trafficking of glycoproteins, glycolipids or both. Here we show that VIP36 is N-glycosylated and expressed in organs abundant in epithelial cells as well as in non-epithelial organs. Our studies demonstrate that the recombinant exoplasmic/luminal domain of VIP36 binds Ca2+ and that the protein decorates internal membrane structures of MDCK cells in vitro that are distinct from the Golgi apparatus. This binding requires Ca2+ and can be specifically inhibited by N-acetyl-D-galactosamine. The recombinant protein was used for affinity chromatography. Glycopeptides obtained from [3H]galactose-labelled cells bind to VIP36 and can be eluted with N-acetyl-D-galactosamine. Our data imply that VIP36 functions as a lectin in post-Golgi trafficking.