bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of mucins and proteoglycans synthesized by a mucin-secreting HT-29 cell subpopulation
Authors: Huet G. , Kim I. , Bolos C. de , Lo-Guidice J. M. , Moreau O. , Hemon B. , Richet C. , Delannoy P. , Real F. X. , Degand P.
Published: April 2021

HT-29 cells selected by adaptation to 10−5 M methotrexate (HT-29 MTX) are a homogeneous cell population producing high amounts of mucin. Intracellular mucins and proteoglycans were isolated from these cells by ultra-centrifugation of cell lysates on a cesium bromide gradient and further separated by anion-exchange high perfomance liquid chromatography. The major mucin fraction isolated was characterized by a high hydroxy amino acid content (40%), a Thr/Ser ratio of 1.52, a high sialic acid content, and a low sulfate content. When the same procedure was applied to undifferentiated HT-29 cells, a minor mucin fraction was isolated which appeared less sialylated and more sulfated. The major proteoglycan species identified in HT-29 MTX cells showed less acidic behavior than the pro-teoglycan isolated from HT-29 cells. The effect of brefeldin A and the sugar analog GalNAc-α-O-benzyl on the synthesis and biochemical properties of mucins synthesized by HT-29 MTX cells was examined. Brefeldin A induced the synthesis of more-sulfated mucins. GalNAc-α-O-benzyl treatment resulted in mucins with an increased content of T antigen and a 13-fold lower sialic acid content. We show that GalNAc-α-O-benzyl was metabolized by the cells to Galβ1-3GalNAc-α-O-benzyl, which, in turn, was a potent competitive inhibitor of the O-glycan α-2,3-sialyltrans-ferase. These results illustrate the suitability of HT-29 MTX cells as a model to analyse mucin synthesis and sia-lylation.
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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Stimulation of calcium influx by platelet activating factor in Dictyostelium
Authors: Schaloske Ralph , Sordano Concetta , Bozzaro Salvatore , Malchow Dieter
Published: April 2021

Platelet activating factor (PAF) induces Ca2+ influx in Dictyostelium discoideum. In this investigation we used this activity to analyze the mechanism of PAF action. We found that PAF activity was confined to the period of spike-shaped oscillations and suggest that the role of PAF is to augment cAMP relay. PAF seems to act only a few times during this time period of two hours, since Ca2+ entry adapted to a subsequent stimulus for about 30 minutes. PAF showed a reduced response in the G protein β− strain LW14 and was unable to induce Ca2+ influx in the Gα2− strains HC85 and JM1. The latter expresses the cAMP receptors cAR1 constitutively, and exhibits cAMP-induced Ca2+ influx, albeit at a reduced level. In order to decide whether the inability of PAF to elicit a Ca2+ response in JM1 cells was due to the lack of differentiation and/or the lack of Gα2, we inhibited the IP3-dependent pathway with compound U73122 and found that Ca2+ entry was blocked, whereas a closely related inactive compound, U73343, did not alter the response. In agreement with this, NBD-Cl, an inhibitor of Ca2+ uptake into the IP3-sensitive store in Dictyostelium, also abolished PAF activity. The latter was not inhibited by the plasma membrane antagonists BN-52021 or WEB 2170. Therefore PAF seems to operate intracellularly via the IP3-signalling pathway at or upstream of the IP3-sensitive store.
Regulated secretion of multi-lamellar vesicles leads to formation of a tubulo-vesicular network in host-cell vacuoles occupied by Toxoplasma gondii
Authors: Sibley L. David , Niesman Ingrid R. , Parmley Steve F. , Cesbron-Delauw Marie-France
Published: April 2021

Toxoplasma gondii is an obligate intracellular parasite that actively invades virtually all types of nucleated cells, surviving within a specialized vacuole called the para-sitophorous vacuole. Shortly after invasion, the parasite modifies this vacuole by secreting a variety of proteins from electron-dense storage granules. Additionally, the parasite forms a network of membranous tubules within the lumen of the vacuole and connecting with the vacuolar membrane. We have used immunolabeling and cell fractionation to examine the secretion of two dense granule proteins, GRA1 and GRA2, which are involved in formation of the intravacuolar network. Following host-cell invasion, GRA1 was secreted into the lumen of the vacuole as a soluble protein that subsequently became peripherally associated with the network. In addition to being secreted as a soluble protein from dense granules, GRA2 was secreted within multi-lamellar vesicles released from a specialized posterior invagination of the parasite. The multi-lamellar vesicles assemble to form the intravacuolar network, which contains an integral membrane form of GRA2. These findings indicate that Toxoplasma has a highly developed regulated exocytosis pathway that modifies the para-sitophorous vacuole by secretion of soluble proteins and by a novel process of membrane secretion.
The expression of carbohydrate blood group antigens correlates with heat resistance
Authors: Ménoret Antoine , Otry Caroline , Labarrière Nathalie , Breimer Michael E. , Piller Friedrich , Meflah Khaled , Pendu Jacques Le
Published: April 2021

Recent data indicate that cells may resist heat shock via more than one route: heat shock protein synthesis and other still ill-defined mechanisms. We investigated this phenomenon using four types of cells derived from a single rat colon carcinoma: clones REGb and PROb; PRO A+, a glycosylation variant of PROb selected for its high expression of blood group A antigen; and Ph8, a thermoresistant variant of PROb selected by repeated sublethal heat treatments. Basal heat resistance was clearly associated with the level of cell surface expression of blood group H and A antigens. Biosynthesis of these carbohydrate structures requires two glycosyltransferases, H and A enzymes, whose activities are also correlated with basal heat resistance. In addition, heat sensitive REGb cells were rendered more resistant by transfection with the gene encoding for H enzyme, allowing expression of H antigen. Thus, these terminal glycosylations could play a role as cellular protectors against heat treatment. Blood group carbohydrate antigens were mainly located on O-linked carbohydrate chains of a major glycoprotein of 200 kDa and to a lesser extent on N-linked chains. Only trace amounts were present as glycolipids.
The inductive effect of a human DNA sequence (HALF1) on the differentiation of a variant rat hepatoma cell (C2) is restricted to episomal forms of the molecule
Authors: Ng Kim Heng , Maigné Joèlle , Deschatrette Jean
Published: April 2021

HALF1, a 4.3 kb human DNA sequence, was originally identified as a double-stranded, closed-circular DNA molecule in revertants from a dedifferentiated rat hepatoma cell (C2) transfected with human liver DNA. Here we report its specific properties in inducing the transition to the hepatic phenotype. (i) In vitro recircularized HALF1 induces reversion after a minimum time lag of 7 days post-transfection. (ii) After induction, the presence of HALF1 is not required for maintaining the induced hepatic state. (iii) HALF1 is detected as a sequence integrated in high molecular mass DNA of human liver. (iv) HALF1 monomer or dimer plasmid constructs do not induce reversion when integrated into the genome of transfectants. (V)Short ubiquitous RNA transcripts (∼400 bases) are detected with specific HALF1 probes.

These results indicate that the reversion process is linked to the presence of HALF1 extrachromosomal molecules.
Maturation of early endosomes and vesicular traffic to lysosomes in relation to membrane recycling
Authors: Thilo Lutz , Stroud Evelyn , Haylett Thomas
Published: April 2021

The controversy whether endocytic processing occurs by organellar maturation or by vesicular traffic has not been resolved. It is also not clear whether maturation continues to the stage of lysosomes, to what extent it involves a decrease in organellar fusogenicity, and how it relates to membrane recycling. Maturation and vesicular traffic imply distinct kinetics for the intermingling of endocytic markers after sequential endocytic uptake. We have studied the kinetics of intermingling of fluid-phase markers (fluorescein-labelled dextran and horseradish peroxidase) and cell surface-derived membrane (labelled by galactosylation) in organelles at early and late stages of the endocytic pathway in macrophage-like P388D1 cells. Intermingling declined by sigmoid kinetics, indicating that endosomes matured within about 3 minutes to become non-fusogenic towards early endosomes. During maturation about 60% of internalized membrane was recycled with TG ≈2 minutes. Whereas matured endosomes were non-fusogenic towards early endosomes and towards each other, a second phase of intermingling was observed upon delivery to lysosomes. This intermingling occurred by a first-order process (TG ≈4 minutes), concurrent with recycling of the remaining 40% of internalized membrane marker. These kinetic observations suggest a model for endocytic processing which reconciles maturation of early endosomes with the known function of carrier vesicles: Endocytic carrier vesicles do not bud off from permanent early endosomes as proposed for vesicular traffic, but are derived, together with recycling vesicles, from the maturation of early endosomes which are consumed by this process; these carrier vesicles subsequently mediate delivery to lysosomes by vesicular traffic during which the remaining surface-derived membrane is recycled.
Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extracts
Authors: Wangh Lawrence J. , DeGrace Diane , Sanchez J. Aquiles , Gold Alex , Yeghiazarians Yerem , Wiedemann Karen , Daniels Susan
Published: April 2021

Rapid genome replication is one of the hallmarks of the frog embryonic cell cycle. We report here that complete reactivation of quiescent somatic cell nuclei in Xenopus egg extracts depends on prior restructuring of the nuclear substrate and prior preparation of cytoplasmic extract with the highest capacity to initiate and sustain DNA synthesis. Nuclei from mature erythrocytes swell, replicate their DNA efficiently, and enter mitosis in frozen/thawed extracts prepared from activated Xenopus eggs, provided the nuclei are first treated with trypsin, heparin, and an extract prepared from unactivated, meiotically arrested, eggs. Optimal replicating extracts are prepared from large batches of unfertilized eggs that are synchronously activated into the cell cycle for 28 minutes (at 20°C). Because the Xenopus cell cycle progresses so rapidly, extracts prepared just a few minutes before or after this time have substantially lower DNA synthetic capacities. At the optimal time and temperature, eggs have just reached the G1/S boundary of the first cell cycle. This fact was revealed by injecting and replicating an SV40 plasmid in intact unfertilized eggs as described previously. We estimate that under optimal conditions approximately 6.14×109 base pairs of DNA/per nucleus are synthesized in 30-40 minutes, a rate that rivals that observed in the zygotic nucleus.

The findings reported here are one step in our long term effort to develop a new in vitro/in vivo approach to nuclear transplantation. Nuclear transplantation in amphibian embryos has been used to establish that the genomes of many types of differentiated somatic cells are pluripotent. But very few such nuclei have ever developed into advanced tadpoles or adult frogs, probably because somatic nuclei injected directly into activated eggs fail to reactivate quickly enough to avoid being damaged during first mitosis. We have already shown that unfertilized eggs can be injected prior to activation of the first cell cycle. Future experiments will reveal whether in vitro reactivated somatic cell nuclei transplanted into such eggs reliably reach advanced stages of development.
Co-localization of hydrolytic enzymes with widely disparate ph optima: Implications for the regulation of lysosomal ph
Authors: Butor Cécile , Griffiths Gareth , Aronson Nathan N. , Varki Ajit
Published: April 2021

Lysosomes are traditionally defined by their acidic interior, their content of degradative ‘acid hydrolases’, and the presence of distinctive membrane proteins. Terminal degradation of the N-linked oligosaccharides of glycoproteins takes place in lysosomes, and involves several hydrolases, many of which are known to have acidic pH optima. However, a sialic acid-specific 9-O-acetyl-esterase and a glycosyl-N-asparaginase, which degrade the outer- and inner-most linkages of N-linked oligosaccharides, respectively, both have pH optima in the neutral to alkaline range. By immunoelectron microscopy, these enzymes co-localize in lysosomes with several conventional acid hydrolases and with lysosomal membrane glycoproteins. Factors modifying the pH/activity profiles of these enzymes could not be found in lysosomal extracts. Thus, the function of the enzymes with neutral pH optima must depend either upon their minimal residual activity at acidic pH, or upon the possibility that lysosomes are not always strongly acidic. Indeed, when lysosomes are marked in living cells by uptake of fluorescently labeled mannose 6-phosphorylated proteins, the labeled organelles do not all rapidly accumulate Acridine Orange, a vital stain that is specific for acidic compartments. One plausible explanation is that lysosomal pH fluctuates, allowing hydrolytic enzymes with a wide range of pH optima to efficiently degrade macromolecules.
Actin-dependent light-induced translocation of mitochondria and er cisternae in the photoreceptor cells of the locust Schistocerca gregaria
Authors: Stürmer Karoline , Baumann Otto , Walz Bernd
Published: April 2021

Light-dependent changes in the positioning of organelles in photoreceptor cells of arthropods are a well-known phenomenon. In this study, we examine the role of the cytoskeleton in these light-dependent antagonistic movements. In dark-adapted photoreceptor cells of the locust Schistocerca gregaria, prominent sacs of smooth endoplasmic reticulum (ER) oppose the bases of the photoreceptive microvilli. Light stimulation causes a translocation of the ER elements towards the main cell body, and an aggregation of mitochondria adjacent to the microvilli. Immunofluorescence studies and electron-microscopic examination of chemically fixed or high-pressure-frozen, freeze-substituted specimens demonstrate a lack of microtubules in the submicrovillar region. However, numerous filament bundles are aligned in close association with mitochondria and ER elements, along the track of their movement. Fluorescent phallotoxins and monoclonal antiactin antibodies label filament bundles in the submicrovillar region, indicating that they are composed of F-actin. Finally, depolymerization of the submicrovillar actin filaments by incubation with cytochalasin B results in a blockade of the movement of mitochondria and ER cisternae towards the rhabdom. These results suggest that the light-dependent translocation of both ER cisternae and mitochondria occurs along actin filaments.
Different endocytic compartments are involved in the tight association of class ii molecules with processed hen egg lysozyme and ribonuclease a in b cells
Authors: Escola Jean-Michel , Grivel Jean-Charles , Chavrier Philippe , Gorvel Jean-Pierre
Published: April 2021

The processing of exogenous antigens and the association of peptides with class II molecules both occur within the endocytic pathway. 2A4 B lymphoma cells of the H-2k haplotype were grown in the presence or the absence of two different exogenous antigens (hen egg lysozyme and ribo-nuclease A) internalized by fluid-phase endocytosis. Using subcellular fractionation techniques, we demonstrate that, in the presence of hen egg lysozyme, newly synthesized SDS-stable class II molecules are detected in a dense endocytic compartment which does not have the characteristics of neither early and late endosomes nor lysosomes. In contrast, no SDS-stable class II molecules are observed between ribonuclease A and newly synthesized class II molecules. Interestingly, when class II molecules are analyzed at steady state, SDS-stable class II molecules induced by ribonuclease A are found in a compartment cosedimenting with late endosomes. These results suggest that the tight associations between ribonuclease A or hen egg lysozyme with class II molecules occur in distinct endocytic compartments and that these associations may depend on the sensitivity of antigens to proteolysis.
Subcellular localisation of human wee1 kinase is regulated during the cell cycle
Authors: Baldin Véronique , Ducommun Bernard
Published: April 2021

Wee1 kinase-dependent phosphorylation of cdc2 maintains the cdc2/cyclin B complex in an inert form until it is activated by the cdc25 tyrosine phosphatase at the end of G2. As described for cdc25, cell cycle-linked changes in the intracellular localisation of wee1 may constitute an important aspect of the temporal regulation of cdc2 activity. Here we report that the subcellular distribution of human wee1 changes during the cell cycle in HeLa and IMR90 cells. During interphase, wee1 is found almost exclusively in the nucleus. When the cell enters mitosis, wee1 is relocalised into the cytoplasm. During cell division, wee1 becomes restricted to the mitotic equator and by the end of mitosis it is found exclusively in association with midbody bridges, a phenomenon that is dependent on microtubule assembly. The relocalisations of wee1 and its association with subcellular structures may play key regulatory roles at different stages of the cell cycle and during mitosis.
Involvement of M-cadherin in terminal differentiation of skeletal muscle cells
Authors: Zeschnigk Michael , Kozian Detlef , Kuch Christine , Schmoll Marion , Starzinski-Powitz Anna
Published: April 2021

Cadherins are a gene family encoding calcium-dependent cell adhesion proteins which are thought to act in the establishment and maintenance of tissue organization. M-cadherin, one member of the family, has been found in myogenic cells of somitic origin during embryogenesis and in the adult. These findings have suggested that M-cadherin is involved in the regulation of morphogenesis of skeletal muscle cells. Therefore, we investigated the function of M-cadherin in the fusion of myoblasts into myotubes (terminal differentiation) in cell culture. Furthermore, we tested whether M-cadherin might influence (a) the expression of troponin T, a typical marker of biochemical differentiation of skeletal muscle cells, and (b) withdrawal of myoblasts from the cell cycle (called terminal commitment). The studies were performed by using antagonistic peptides which correspond to sequences of the putative M-cadherin binding domain. Analogous peptides of N-cadherin have previously been shown to interfere functionally with the N-cadherin-mediated cell adhesion. In the presence of antagonistic M-cadherin peptides, the fusion of myoblasts into myotubes was inhibited. Analysis of troponin T revealed that it was downregulated at the protein level although its mRNA was still detectable. In addition, withdrawal from the cell cycle typical for terminal commitment of muscle cells was not complete in fusion-blocked myogenic cells. Finally, expression of M-cadherin antisense RNA reducing the expression of the endogenous M-cadherin protein interfered with the fusion process of myoblasts. Our data imply that M-cadherin-mediated myoblast interaction plays an important role in terminal differentiation of skeletal muscle cells.
Characterization of a 5.4 kb cDNA fragment from the Z-line region of rabbit cardiac titin reveals phosphorylation sites for proline-directed kinases
Authors: Sebestyén Magdolna G. , Wolff Jon A. , Greaser Marion L.
Published: April 2021

Titin is an approximately 3 MDa protein that spans from the M- to the Z-line in the sarcomeres of vertebrate striated muscle. The protein is presumably encoded by unusually large mRNAs of 70-80 kb. Although titin has been studied by several laboratories, barely more than half of the cDNA sequence (≈45 kb) has been published, most of it obtained from the A-band and M-line region (corresponding to the C-terminal half of the molecule). A special cDNA library was constructed using size selected total RNA from adult rabbit cardiac muscle in order to obtain sequence data from titin’s unknown N-terminal region. A monoclonal antibody (T12), which binds to an epitope close to the Z-line, was used to identify initial cDNA clones. Additional overlapping clones were isolated and sequenced yielding a 5.4 kb contig. The encoded polypeptide contains 16 Type-II domains and four unique intervening segments. Poly-clonal sera, raised against an expressed protein fragment encoded by the 5′ end of the contig, strongly stained the Z-line of myofibrils of different species. However, the sequence of this fragment is 83% identical at the amino acid level with the previously reported C-terminal (i.e. M-line) end of chicken embryonic skeletal muscle titin. The expressed protein fragment could be phosphorylated in vitro by embryonic skeletal muscle extract and by the purified proline-directed kinase ERK1, presumably at the xSPxR recognition sites located in the first interdomain segment.
Differential distribution of two cytoplasmic variants of the α6β1 integrin laminin receptor in the ventral plasma membrane of embryonic fibroblasts
Authors: Cattelino Anna , Longhi Renato , Curtis Ivan de
Published: April 2021

The integrin α6β1 is a receptor involved in the adhesion of several cell types to laminin. By using function-blocking antibodies, we have shown that (z60 1 is a functional laminin receptor in chick embryo fibroblasts. We also found that these cells express two variants of the α6 subunit, α6A and a6B, characterized by different cytoplasmic domains. By using indirect immunofluorescence with isoform-specific polyclonal antibodies, we showed that the two isoforms of the α6 subunit distribute differently on the ventral plasma membrane of these cells cultured on laminin-coated substrates. In fact, while the α6A subunit was found codistributing with vinculin in focal contacts, the α6B subunit showed a homogeneously distributed punctate pattern. This difference was particularly evident when preparations of ventral plasma membranes were used for the immunolocalization. Furthermore, when cells were cultured on fibronectin, a substrate not recognized by the α6β1 laminin receptor, the distribution of the two α6 isoforms was similar to that observed on laminin, with α6A still colocalizing with vinculin in focal adhesions. Our results indicate that two forms of the β6β1 laminin receptor coexpressed in the same cells show distinctive distributions, and suggest that receptor occupancy by laminin is not essential for the accumulation of the α6Aβ1 integrin in adhesion plaques.
Purification of bovine lens cell-to-cell channels composed of connexin44 and connexin50
Authors: Konig Nicola , Zampighi Guido A.
Published: April 2021

Cell-to-cell channels composed of connexin44 and connexin50 were purified from plasma membranes of calf and fetal bovine lenses. The channels were treated with the nonionic detergents octyl-β-P-glucopyranoside and decyl-β-D-maltopyranoside, and the channel/detergent complexes purified by ion and gel filtration column chromatography. In negative staining, the channels appeared as annuli 11±0.6 nm (s.d., n=105) in diameter and as 16±0.8 nm (s.d., n=96) long particles which corresponded to top and side views of ‘complete’ cell-to-cell channels. The purified cell-to-cell channels were composed principally of a protein, called MP70, that appeared as a diffuse 55–75 kDa band in SDS-PAGE. Dephosphorylation with alkaline phosphatase transformed the diffuse 55–75 kDa band into two distinct bands of almost equal intensity. Immunoblotting showed the bands to be connexin44 and connexin50, respectively. The antibodies also recognized weaker bands composed of the unphosphorylated form of both connexins. The connexins appear to be processed independently ‘in vivo’. The unphosphorylated form of connexin50 was present in channels and membranes from fetal, calf and adult bovine lenses, while unphosphorylated connexin44 only in channels purified from fetal lenses. Therefore, lens cell-to-cell channels are composed principally of equal amounts of phosphorylated connexins 44 and 50 that appear to be assembled in the same channel (‘hybrid’).