bluuebunny/crossref_metadata_2025_split huggingface dataset:
In vitro p53 and/or Rb antisense oligonucleotide treatment in association with growth factors induces the proliferation of peripheral hematopoietic progenitors
Authors: Mahdi Tarek , Brizard André , Millet Christine , Doré Pierre , Tanzer Joseph , Kitzis Alain 
Published: April 2021

ABSTRACT
In this work we intended to determine whether p53 and/or retinoblastoma (Rb) tumor suppressor genes are involved at specific stages in the process of in vitro human peripheral stem cell hematopoiesis. Mononuclear peripheral blood cells were depleted of adherent cells and T lymphocytes (A-T-PMCs). Cells were then cultured in semisolid medium, under conditions that favor the growth of specific progenitor cell types. A-T-PMCs were exposed to p53 and/or Rb sense, scrambled DNA and antisense oligodeoxynucleotides. p53 and/or Rb antisenses (but not their senses or scrambled DNA) treatment of A-T-PMCs resulted in a significant increase in the number of granu-locyte/macrophage colony-forming units (CFU-GM) in the presence of interleukin-3 (IL-3) and/or granulocyte/macrophage colony-stimulating factor (GM-CSF). After antisense treatment, blast forming units/erythroblasts (BFU-E) derived from A-T-PMCs cultured in the presence of IL-3 + erythropoietin (Epo) were also increased whereas colony forming units/erythroblasts (CFU-E) were not markedly affected in the presence of Epo only. Megakary-ocytic colony (CFU-Meg) formation from A-T-PMCs in the presence of interleukin-6 (IL-6) + IL-3 + Epo was also increased after antisense oligodeoxynucleotide treatment. These results are consistent with the hypothesis that p53 and Rb tumor suppressor gene products are involved in the control of distinct signal pathways in different peripheral progenitor cells.
Selective action of uncoating atpase towards clathrin-coated vesicles from brain
Authors: Buxbaum Engelbert , Woodman Philip G. 
Published: April 2021

ABSTRACT
Clathrin-coated vesicles from brain are primarily involved in synaptic vesicle recycling and are substrates for the con-stitutively expressed heat shock cognate hsc70 protein (uncoating ATPase). To investigate the regulation of clathrin coat turnover in other tissues the activity of hsc70 towards coated vesicles from other sources was examined. Concentrations of hsc70 which caused near-complete removal of clathrin from brain coated vesicles effected only partial uncoating of vesicles prepared from other tissues. The selective action of hsc70 could not be accounted for by tissue or species specificities of hsc70, but rather reflected differences in coat structure. Selective action was associated with two differences in the hsc70-dependent ATPase cycle. Firstly, uncoating of brain, but not placental vesicles, could occur under circumstances where ATP hydrolysis was prevented. Secondly, only brain coated vesicles could support multiple rounds of hsc70-dependent ATP hydroly-sis. Implications of these findings for the mechanism of hsc70-dependent vesicle uncoating in non-neuronal cells and the organisation of the endocytic pathway in the axon are discussed.
Cysteine-to-arginine point mutation in a ‘hybrid’ eight-cysteine domain of FBN1: consequences for fibrillin aggregation and microfibril assembly
Authors: Kielty Cay M. , Rantamäki Terhi , Child Anne H. , Shuttleworth C. Adrian , Peltonen Leena 
Published: April 2021

ABSTRACT
Mutations in the FBN1 gene encoding the microfibrillar glycoprotein fibrillin cause Marfan syndrome, a relatively common autosomal dominant connective tissue disease. Causative FBN1 mutations appear to be dispersed throughout the coding frame, and to date no predictable genotype: phenotype correlations have emerged. We have identified a point mutation within an eight-cysteine ‘hybrid’ motif of the fibrillin polypeptide which results in the substitution of an arginine for a cysteine, in a patient severely affected in the cardiovascular, skeletal and ocular systems.
We have utilised cell cultures from various tissues of this patient to investigate the effects of this mutation on fibrillin expression and deposition, and the consequences in terms of microfibril assembly and organisation. We have established that there is no difference in the expression of normal and mutant alleles, and fibrillin synthesis, secretion and deposition are also normal. However, the rate of fibrillin aggregation is reduced and microfibrillar assemblies are both remarkably scarce and morphologically abnormal.
These data clearly demonstrate that the mutated allele interferes with normal assembly, and strongly implicate this particular region of the fibrillin-1 molecule in stabilising microfibrillar assemblies.
Fission yeast TPR-family protein nuc2 is required for G1-arrest upon nitrogen starvation and is an inhibitor of septum formation
Authors: Kumada Kazuki , Su Sophia , Yanagida Mitsuhiro , Toda Takashi 
Published: April 2021

ABSTRACT
Fission yeast nuc2+ gene encodes a protein of a tetratri- copeptide repeat (TPR) family which is conserved through- out evolution. We previously showed that nuc2 is required for exit from the mitotic metaphase. In this study, we present evidence which shows that nuc2 has two additional roles in the cell cycle. We showed that the nuc2 mutant is sterile even at the permissive temperature and septation occurs in the absence of chromosome separation at the restrictive temperature. The nuc2 mutant fails to arrest at the G1 phase upon nitrogen starvation at the permissive temperature which is a prerequisite for conjugation. Upon starvation, however, the nuc2 mutant ceased division normally and induced starvation-dependent gene expression. Therefore, the nuc2 mutant is deficient only for failure to block DNA replication upon starvation. At the lower restrictive temperature, the nuc2 mutant showed a ‘cut’ phenotype where septation and cytokinesis takes place without the completion of mitosis. Ectopic overex- pression of the nuc2+ gene caused multiple rounds of S and M phases in the complete absence of septum formation. We propose that nuc2 is a novel cell cycle regulator essential for three events; firstly for exit from mitosis, secondly for DNA replication restraint under nutrient starvation and thirdly for inhibition of septation and cytokinesis until the completion of mitosis.
Targeted expression of SV40 T antigen in the hair follicle of transgenic mice produces an aberrant hair phenotype
Authors: Powell R. Keough, B. , Rogers G. 
Published: April 2021

ABSTRACT
Directed expression of SV40 large T antigen (TAg) in trans-genic mice can induce tissue-specific tumorigenesis and useful cell lines exhibiting differentiated characteristics can be established from resultant tumor cells. In an attempt to produce an immortalised mouse hair follicle cortical cell line for the study of hair keratin gene control, SV40 TAg expression was targeted to the hair follicles of transgenic mice using a sheep hair gene promoter. Expression of SV40 TAg in the follicle cortex disrupted normal fiber ultra-structure, producing a marked phenotypic effect. Affected hairs were wavy or severely kinked (depending on the severity of the phenotype) producing an appearance ranging from a ruffled coat to a stubble covering the back of the mouse. The transgenic hairs appeared to be weakened at the base of the fibers, leading to premature hair-loss and a thinner pelage, or regions of temporary nudity. No follicle tumors or neoplasia were apparent and immortalisation of cortical cells could not be established in culture. In situ hybridisation studies in the hair follicle using histone H3 as a cell proliferation marker suggested that cell proliferation had ceased prior to commencement of K2.10-TAg expression and was not re-established in the differentiating cortical cells. Hence, TAg was unable to induce cell immortalisation at that stage of cortical cell differentiation. However, transgenic mice developed various other abnormalities including vertebral abnormal-ities and bladder, liver and intestinal tumors, which resulted in reduced life expectancy.
Reappearance and long-term maintenance of connexin32 in proliferated adult rat hepatocytes: use of serum-free L-15 medium supplemented with EGF and DMSO
Authors: Kojima Takashi , Mitaka Toshihiro , Paul David L. , Mori Michio , Mochizuki Yohichi 
Published: April 2021

ABSTRACT
Intercellular communication, especially gap junctional communication, is thought to be one of the highly differentiated functions of hepatocytes. In primary cultures of rat hepatocytes, it has been considered that the maintenance and the reinduction of differentiated functions is very difficult. In the present study, we succeeded in inducing the gap junctional protein connexin32 (Cx32) in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor (EGF) and dimethylsulfoxide (DMSO). When the hepatocytes were cultured in L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2:95% air incubator, the cells proliferated. Fluorescence immuno-cytochemistry showed spots immunoreactive to Cx32 on the cell membranes between adjacent cells until day 3, but only a few Cx32-positive spots were found after day 4. Western and northern blot analyses also showed that the amounts of both the protein and mRNA of Cx32 in the cells decreased with time in culture. However, when the cells were treated with 2% DMSO from day 4, the immunoreactive spots reappeared on the cell membranes from day 6 and both their number and intensity gradually increased. The reappearance of Cx32 was accompanied by increases in both the protein and mRNA of Cx32. Futhermore, the expression of Cx32 was well maintained, together with extensive gap junctional intercellular communication, for more than 4 weeks. In addition, ultrastructually, many gap junctional structures were observed between the hepatocytes, and the antibodies to Cx32 were shown to bind to those structures. This culture system may be useful for studies of the reconstruction of the gap junctional structure, the intracellular pathways of the proteins, and the regulation of synthesis and processing in differentiated hepatocytes.
Adhesion-induced tyrosine phosphorylation of the p130 SRC substrate
Authors: Petch Leslie A. , Bockholt Susanne M. , Bouton Amy , Parsons J. Thomas , Burridge Keith 
Published: April 2021

ABSTRACT
Adhesion of cells to the extracellular matrix leads to an increase in the tyrosine phosphorylation of a specific set of proteins, three of which have now been identified as the focal adhesion proteins pp125FAK, paxillin and tensin. In addition, we have previously noted the adhesion-induced tyrosine phosphorylation of a fourth protein, with an apparent molecular mass of 130. As in the case of FAK, paxillin and tensin, a 130 kDa protein is also found to be highly tyrosine phosphorylated in Rous sarcoma virus (RSV)-transformed cells. This protein forms a stable complex with pp60src and is directly phosphorylated by activated forms of c-src. Using a monoclonal antibody (mAb 4F4) specific for the src-associated p130 we show that p130 is also phosphorylated in response to cell adhesion. Immunoprecipitation of p130 followed by an anti-phosphotyrosine immunoblot revealed that adhesion of rat embryo fibroblasts (REF52) to fibronectin (FN) led to a sig-nificant increase in the phosphotyrosine content of p130. Furthermore, a comparison of cell lysates before and after immunoprecipitation confirmed the absence of tyrosine phosphorylated p130 from lysates immunoprecipitated with mAb 4F4. Immunofluorescence staining of REF52s revealed that p130 is found in focal adhesions as well as along stress fibers in a pattern reminiscent of that exhibited by α-actinin. In addition, in many cells, we found significant staining in the nucleus, but evidence is presented that the nuclear staining is not due to tyrosine phosphorylated p130. Finally, unlike pp125FAK, p130 does not appear to be itself a kinase as evidenced by immune-complex kinase assays carried out in the presence or absence of exogenous substrates.
Expression, phosphorylation and nuclear localization of the human P1 protein, a homologue of the yeast Mcm 3 replication protein
Authors: Schulte Dorothea , Burkhart Roland , Musahl Christine , Hu Bing , Schlatterer Christina , Hameister Horst , Knippers Rolf 
Published: April 2021

ABSTRACT
The human protein P1 belongs to a newly discovered class of mammalian nuclear proteins with high sequence homology to yeast replication proteins. We present the entire amino acid sequence of the human protein P1 as predicted from the cDNA sequence, and show that P1 shares three central regions of high sequence similarity (about 75%) and a highly hydrophilic carboxy-terminal region with the yeast Mcm3 replication protein. The human genome most probably contains one P1 gene which is activated when HeLa cells progress to S phase, as shown by a several-fold increase in P1-specific mRNA. However, the amounts of P1 protein do not detectably change during this period, but P1 protein becomes phosphorylated at the beginning of S phase. In contrast to the yeast Mcm proteins, which disappear from nuclei after initiation of DNA replication, protein P1 remains in the nucleus during and after S phase. P1 is dispersed in mitotic cells and may be excluded from binding to chromosomes.
The mutant not enough muscles (nem) reveals reduction of the Drosophila embryonic muscle pattern
Authors: Burchard Susanne , Paululat Achim , Hinz Uwe , Renkawitz-Pohl Renate 
Published: April 2021

ABSTRACT
In a search for mutations affecting embryonic muscle development in Drosophila we identified a mutation caused by the insertion of a P-element, which we called not enough muscles (nem). The phenotype of the P-element mutation of the nem gene suggests that it may be required for the development of the somatic musculature and the chordotonal organs of the PNS, while it is not involved in the development of the visceral mesoderm and the dorsal vessel. Mutant embryos are characterized by partial absence of muscles, monitored by immunostainings with mesoderm-specific anti-β3 tubulin and anti-myosin heavy chain anti-bodies. Besides these muscle distortions, defects in the peripheral nervous system were found, indicating a dual function of the nem gene product. Ethyl methane sulfonate-induced alleles for the P-element mutation were created for a detailed analysis. One of these alleles is characterized by unfused myoblasts which express β3 tubulin and myosin heavy chain, indicating the state of cell differentiation.
In vitro motility of immunoadsorbed brain myosin-V using a Limulus acrosomal process and optical tweezer-based assay
Authors: Wolenski Joseph S. , Cheney Richard E. , Mooseker Mark S. , Forscher Paul 
Published: April 2021

ABSTRACT
To facilitate functional studies of novel myosins, we have developed a strategy for characterizing the mechanochemical properties of motors isolated by immunoadsorption directly from small amounts of crude tissue extracts. In this initial study, silica beads coated with an antibody that specifically recognizes the tail of myosin-V were used to immunoadsorb this motor protein from brain extracts. The myosin-containing beads were then positioned with optical tweezers onto actin filaments nucleated from Limulus sperm acrosomal processes and observed for motility using high resolution video DIC microscopy. The addition of brush border spectrin to the motility chamber enabled the growth of stable actin filament tracks that were ∼4-fold longer than filaments grown in the absence of this actin crosslinking protein. The velocity of myosin-V immunoadsorbed from brain extracts was similar to that observed for purified myosin-V that was antibody-linked to beads or assessed using the sliding actin filament assay. Motile beads containing myosin-V immunoadsorbed from brain extracts bound poorly to nucleated actin filaments and were incapable of linear migrations following the addition of a different antibody that specifically recognizes the motor-containing head domain of myosin-V. Myosin-V motility was most robust in the absence of Ca2+. Interestingly, skeletal muscle tropomyosin and brush border spectrin had no detectable effect on myosin-V mechanochemistry. Myosin-V containing beads were also occasionally observed migrating directly on acrosomal processes in the absence of exogenously added actin. The use of laser tweezers to trap and position particles on the elongate actin filament tracks should expedite mechanochemical analysis of novel immunoadsorbed myosins that resist isolation using preparative techniques. Moreover, this assay should facilitate studies of myosin-dependent organelle/vesicle motility.
Monoclonal antibody TeM 106 reacts with a tonoplast intrinsic protein of 106 kDa from Brassica oleracea L.
Authors: Dozolme Pascale , Marty-Mazars Danièle , Clémencet Marie-Claude , Marty Francis 
Published: April 2021

ABSTRACT
A monoclonal antibody, designated TeM 106, that recognizes an intrinsic protein from the vacuole membrane (tonoplast) of cauliflower (Brassica oleracea L. var. botrytis) is described. Mice were immunized with a tonoplast fraction that had been purified from differentiating meristematic cells from the cauliflower head. Hybridomas were generated and screened by means of Enzyme Linked Immuno Sorbent Assays for differential reactivity to tonoplast over non-related proteins (bovine serum albumin). One out of 14 reactive murine clones was selected on the basis of its stability, secretory efficiency, and high affinity of the secreted antibodies. TeM 106 is an IgM which was shown by indirect immunofluorescence microscopy of frozen thin sections to bind specifically to the tonoplast of highly vacuolated cells as well as to the tonoplast of small vacuoles in meristematic cells. The molecular specificities of TeM 106 were preliminarily determined using electrophoretic transfer procedures (immunoblotting). TeM 106 reacted with a single protein band of 106,000 Mr from the tonoplast of cauliflower. Using two-dimensional gel electrophoresis, it was shown that the epitope is borne by a single polypeptide. The antigen is a glycopeptide containing mannose and/or glucose residues in the oligosaccharide side chain but the epitope, resistant to the metaperiodate oxidation, is contained in the polypeptide backbone. Salt elution experiments indicated that the antigen, unlike several proteins from the tonoplast, is not eluted from the membrane by KCl treatments and is, therefore, tentatively considered as a tonoplast intrinsic protein, designated TIP 106.
Motility and substratum adhesion of Dictyostelium wild-type and cytoskeletal mutant cells: a study by RICM/bright-field double-view image analysis
Authors: Weber Igor , Wallraff Eva , Albrecht Richard , Gerisch Günther 
Published: April 2021

ABSTRACT
To investigate the dynamics of cell-substratum adhesion during locomotion, a double-view optical technique and computer-assisted image analysis has been developed which combines reflection interference contrast microscopy (RICM) with bright-field imaging. The simultaneous recording of cell-substratum contact and cell body contour has been applied to aggregation-competent cells of Dictyostelium discoideum. These cells are distinguished from cells at earlier stages of development by small areas of contact to a substratum.
Three questions have been addressed in analysing the locomotion of aggregation-competent cells. (1) What is the relationship between changes in the shape of cells and their contact to a substratum during a chemotactic response? (2) What is the relationship between protrusion and retraction of the cell body, and between local attachment and detachment? (3) Are there differences between wild-type and mutant cells that lack certain cytoskeletal proteins?
During a chemotactic response the front region of the amoeba can bend towards the gradient of attractant without being supported by its contact with a surface, which excludes the necessity for gradients of adhesion for the response. The finding that in locomoting cells protrusion of the leading edge often precedes retraction establishes a pioneer role for the front region. The finding that gain of contact area precedes loss provides evidence for the coordination of interactions between the cell surface and a substratum. For comparison with wild-type, aggregation-competent triple mutant cells have been used that lack two F-actin crosslinking proteins, α-actinin and 120 kDa gelation factor, and an actin filament fragmenting protein, severin. Disturbances in the spatial and temporal control of cytoskeletal activities have been unravelled in the mutant by RICM and quantified by cross-correlation analysis of attachment and detachment vectors. In order to detect these disturbances, it was essential to analyse cell locomotion on the weakly adhesive surface of freshly cleaved mica.
Localization of the small GTP-binding protein rab1p to early compartments of the secretory pathway
Authors: Saraste Jaakko , Lahtinen Ulla , Goud Bruno 
Published: April 2021

ABSTRACT
We have studied the localization of the small GTPase rab1p in different cell types using polyclonal antibodies prepared against the rab1A isoform of the protein. Immunofluorescence microscopy of normal rat kidney (NRK) and mouse myeloma cells showed the association of the protein with the Golgi complex and peripheral sites where it colocalized with p58, a pre- and cis-Golgi marker protein. Rab1p and p58 also had similar distributions in membrane fractions derived from rat pancreas microsomes. Both were concentrated in two intermediate density subfractions between the rough endoplasmic reticulum and trans-Golgi, whereas rab6p, previously localized to middle and trans-Golgi, was enriched in the light density trans-Golgi fraction. Immunoperoxidase electron microscopy of NRK and myeloma cells revealed the association of rab1p with 1-2 cisternae, vacuolar, and tubulovesicular membranes in the cis-Golgi region. The rab1p-specific staining typically covered the entire lateral surface of the cisternae but, in weakly stained cells, local labeling between closely opposed membranes could also be seen. The rab1p-positive pre-Golgi compartment had a predominantly tubulovesicular appearance in NRK cells whereas in myeloma cells it consisted of vacuoles surrounded by rab1p-positive vesicles and tubules of heterogenous size. In both cell types the rough ER cisternae and the nuclear envelope contained negligible labeling and no continuities between these and the rab1p-positive membranes were observed. In addition, in myeloma cells the smooth ER subcompartment, containing endogenous retrovirus particles, was devoid of rab1p-labeling. These results indicate that the pre-Golgi (intermediate) compartment consists of different membrane domains and its morphology can vary considerably between different cell types. Further, they suggest that the recruitment of rab1p to membranes occurs pre-dominantly in a post-ER location and that the protein functions in targeting/fusion events within the pre- and cis-Golgi membranes.