bluuebunny/crossref_metadata_2025_split huggingface dataset:
PDGF α-receptor is unresponsive to PDGF-AA in aortic smooth muscle cells from the NG2 knockout mouse
Authors: Grako Kathryn A. , Ochiya Takahiro , Barritt Diana , Nishiyama Akiko , Stallcup William B. 
Published: April 2021

ABSTRACT
A line of null mice has been produced which fails to express the transmembrane chondroitin sulfate proteoglycan NG2. Homozygous NG2 null mice do not exhibit gross phenotypic differences from wild-type mice, suggesting that detailed analyses are required to detect subtle alterations caused by the absence of NG2. Accordingly, dissociated cultures of aortic smooth muscle cells from null mice were compared to parallel cultures from wild-type mice for their ability to proliferate and migrate in response to specific growth factors. Both null and wild-type smooth muscle cells exhibited identical abilities to proliferate and migrate in response to PDGF-BB. In contrast, only the wild-type cells responded to PDGF-AA in both types of assays. NG2 null cells failed to proliferate or migrate in response to PDGF-AA, implying a defect in the signaling cascade normally initiated by activation of the PDGF α-receptor. In agreement with this idea, activation of the extracellular signal-regulated kinase (ERK) in response to PDGF-AA treatment occured only in wild-type cells. Failure to observe autophosphorylation of the PDGF α-receptor in PDGF-AA-treated null cells indicates that the absence of NG2 causes a defect in signal transduction at the level of α-receptor activation.
Caffeine can override the S-M checkpoint in fission yeast
Authors: Wang Shao-Win , Norbury Chris , Harris Adrian L , Toda Takashi 
Published: April 2021

ABSTRACT
The replication checkpoint (or ‘S-M checkpoint’) control prevents progression into mitosis when DNA replication is incomplete. Caffeine has been known for some time to have the capacity to override the S-M checkpoint in animal cells. We show here that caffeine also disrupts the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. By contrast, no comparable effects of caffeine on the S. pombe DNA damage checkpoint were seen. S. pombe cells arrested in early S phase and then exposed to caffeine lost viability rapidly as they attempted to enter mitosis, which was accompanied by tyrosine dephosphorylation of Cdc2. Despite this, the caffeine-induced loss of viability was not blocked in a temperature-sensitive cdc2 mutant incubated at the restrictive temperature, although catastrophic mitosis was prevented under these conditions. This suggests that, in addition to S-M checkpoint control, a caffeine-sensitive function may be important for maintenance of cell viability during S phase arrest. The lethality of a combination of caffeine with the DNA replication inhibitor hydroxyurea was suppressed by overexpression of Cds1 or Chk1, protein kinases previously implicated in S-M checkpoint control and recovery from S phase arrest. In addition, the same combination of drugs was specifically tolerated in cells overexpressing either of two novel S. pombe genes isolated in a cDNA library screen. These findings should allow further molecular investigation of the regulation of S phase arrest, and may provide a useful system with which to identify novel drugs that specifically abrogate the checkpoint control.
Mitotic control in the absence of cdc25 mitotic inducer in fission yeast
Authors: A. Sveiczer , Novak B. , Mitchison J. M. 
Published: April 2021

ABSTRACT
Fission yeast cells tolerate the total absence of the cdc25 mitotic inducer in two cases, either in cdc2-3w or in wee1 genetic backgrounds. In the cdc2-3w cdc25Δ double mutant, the rate-limiting step leading to mitosis is reaching a critical size. However, the size control of this mutant operates in late G2, which is different from wild-type (WT) cells. This fact suggests that in WT the rate-limiting molecular process during the G2 timer is the Tyr15 dephosphorylation of cdc2, for which the cdc25 phosphatase (together with its back-up, pyp3) is dependent. In the wee1-50 cdc25Δ mutant, the population splits into different clusters, all lacking mitotic size control. This strain maintains size homeostasis by a novel method, which is random movement of the cells from one cluster to another in the successive generations. These cells should normally have a ‘minimal cycle’, a ‘timer’ with short G1 and G2 phases. However, very often the cells abort mitosis, possibly at an early event and return back to early G2, thus lengthening their cycles. The inability of these cells to start anaphase might be caused by the absence of the main mitotic regulators (wee1 and cdc25) and the improper regulation of their back-up copies (mik1 and pyp3, respectively).
Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues Application of a novel fixation protocol using trichloroacetic acid (TCA) as a fixative
Authors: Hayashi Ken , Yonemura Shigenobu , Matsui Takeshi , Tsukita Sachiko , Tsukita Shoichiro 
Published: April 2021

ABSTRACT
Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.
Regulation of actin organisation by TGF-β in H-ras-transformed fibroblasts
Authors: Moustakas Aristidis , Stournaras Christos 
Published: April 2021

ABSTRACT
The actin cytoskeleton undergoes architectural changes during the processes of cell transformation and tumourigenesis. Transforming growth factors β arrest cell cycle progression, regulate differentiation and modulate the onset of oncogenesis and tumourigenesis. Here, we investigated the direct role of transforming growth factor β-1 in altering the transformed phenotype and regulating the actin organisation of oncogenic fibroblasts that constitutively or inducibly express the H-ras oncogene. Following transforming growth factor β-1 treatment, these transformed fibroblasts undergo a dramatic morphological alteration that includes a discrete reorganisation of their actin cytoskeleton and focal adhesions. Quantitative biochemical analysis demonstrated that transforming growth factor β-1 potently induced polymerisation of globular to filamentous actin, thus corroborating the morphological analysis. The effect of transforming growth factor β-1 on the cytoskeleton correlates with the ability of this cytokine to suppress anchorage-independent growth of the transformed fibroblasts. Furthermore, transforming growth factor β-1 upregulates considerably the levels of the RhoB small GTPase and less the RhoA levels. Finally, The Rho GTPase inhibitor, C3 exotransferase, blocks the ability of TGF-β1 to induce cytoskeletal reorganisation. These findings indicate that transforming growth factor β can regulate cell morphology and growth in a concerted manner possibly via mechanisms that control the actin cytoskeleton.
Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1α
Authors: Chilov Dmitri , Camenisch Gieri , Kvietikova Ivica , Ziegler Urs , Gassmann Max , Wenger Roland H. 
Published: April 2021

ABSTRACT
Hypoxia-inducible factor-1 (HIF-1) is a master regulator of mammalian oxygen homeostasis. HIF-1 consists of two subunits, HIF-1α and the aryl hydrocarbon receptor nuclear translocator (ARNT). Whereas hypoxia prevents proteasomal degradation of HIF-1α, ARNT expression is thought to be oxygen-independent. We and others previously showed that ARNT is indispensable for HIF-1 DNA-binding and transactivation function. Here, we have used ARNT-mutant mouse hepatoma and embryonic stem cells to examine the requirement of ARNT for accumulation and nuclear translocation of HIF-1α in hypoxia. As shown by immunofluorescence, HIF-1α accumulation in the nucleus of hypoxic cells was independent of the presence of ARNT, suggesting that nuclear translocation is intrinsic to HIF-1α? Co-immunoprecipitation of HIF-1α together with ARNT could be performed in nuclear extracts but not in cytosolic fractions, implying that formation of the HIF-1 complex occurs in the nucleus. A proteasome inhibitor and a thiol-reducing agent could mimic hypoxia by inducing HIF-1α in the nucleus, indicating that escape from proteolytic degradation is sufficient for accumulation and nuclear translocation of HIF-1α. During biochemical separation, both HIF-1α and ARNT tend to leak from the nuclei in the absence of either subunit, suggesting that heterodimerization is required for stable association within the nuclear compartment. Nuclear stabilization of the heterodimer might also explain the hypoxically increased total cellular ARNT levels observed in some of the cell lines examined.
Examining the relationship between the gelatinolytic balance and the invasive capacity of endothelial cells
Authors: Puyraimond Alain , Weitzman Jonathan B. , Babiole Emeline , Menashi Suzanne 
Published: April 2021

ABSTRACT
Angiogenesis and the formation of new blood vessels requires coordinated regulation of matrix proteolysis and endothelial cell migration. Cellular proteolytic capacity is the balance between secreted matrix metalloproteinases (MMP) and their inhibitors (TIMPs). We have examined the regulation of the gelatinase/TIMP balance by transforming growth factor-β1 (TGF-β1) and phorbol myristate acetate (PMA) in bovine endothelial cells. The low constitutive expression of gelatinase A/MMP-2 was upregulated by TGF-β1 in a dose-dependent manner. Gelatinase B/MMP-9 was only detected upon treatment with either PMA or TGF-β1. However, addition of both factors together revealed a striking synergistic effect causing upregulation of MMP-9 and downregulation of TIMPs, thereby increasing the net MMP-9/TIMP balance and the gelatinolytic capacity. These effects were observed at both the protein and mRNA levels. We demonstrate that changes in different members of the Jun oncogene family with distinct transactivation properties may account for this synergistic effect. We investigated the contribution of these changes in gelatinolytic balance to endothelial cell migration and invasion. The endothelial cells showed increased cell motility in response to PMA, but the addition of TGF-β1 had an inhibitory effect. Hence, regulation of the MMP-9/TIMP balance failed to correlate with the migratory or invasive capacity. These results question a direct role for MMP-9 in endothelial cell motility and suggest that gelatinases may contribute in alternative ways to the angiogenic process.
New actin mutants allow further characterization of the nucleotide binding cleft and drug binding sites
Authors: Belmont Lisa D. , Patterson Gregory M. L. , Drubin David G. 
Published: April 2021

ABSTRACT
We have generated 9 site-specific mutations in Saccharomyces cerevisiae actin. These mutants display a variety of phenotypes when expressed in vivo, including slow actin filament turnover, slow fluid-phase endocytosis, and defects in actin organization. Actin mutation D157E confers resistance to the actin-sequestering drug, latrunculin A. Latrunculin A inhibits nucleotide exchange on wild-type yeast actin but not on D157E actin, suggesting that this residue is part of the latrunculin A binding site. We have refined our earlier map of the phalloidin binding site on actin, demonstrating a requirement for residue G158 in addition to D179 and R177. The nine new actin mutants as well as a large collection of existing actin mutants were also used to identify the putative binding site of another actin binding drug, tolytoxin, on actin. The actin alleles that result in decreased sensitivity to this drug cluster at a site near the nucleotide-binding pocket. Actin purified from one of these mutants has a reduced affinity for tolytoxin. In addition, tolytoxin causes a 2.4-fold increase in the t1/2 of ATP exchange, further suggesting that this drug binds near the nucleotide-binding pocket of actin. We note that the binding sites for latrunculin A, phalloidin, and tolytoxin all map close to the actin nucleotide binding pocket.
Reprogramming nuclei: insights from cloning, nuclear transfer and heterokaryons
Authors: Kikyo Nobuaki , Wolffe Alan P. 
Published: April 2021

ABSTRACT
Mammals and amphibians can be cloned following the transfer of embryonic nuclei into enucleated eggs or oocytes. As nuclear functions become more specialized in the differentiated cells of an adult, successful cloning using these nuclei as donors becomes more difficult. Differentiation involves the assembly of specialized forms of repressive chromatin including linker histones, Polycomb group proteins and methyl-CpG-binding proteins. These structures compartmentalize chromatin into functional domains and maintain the stability of the differentiated state through successive cell divisions. Efficient cloning requires the erasure of these structures.
The erasure can be accomplished through use of molecular chaperones and enzymatic activities present in the oocyte, egg or zygote. We discuss the mechanisms involved in reprogramming nuclei after nuclear transfer and compare them with those that occur during remodeling of somatic nuclei after heterokaryon formation. Finally we discuss how one might alter the properties of adult nuclei to improve the efficiency of cloning.
Source, catabolism and role of the tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro within the testis
Authors: Stéphan Jean-Philippe , Melaine Nathalie , Ézan Eric , Hakovirta Harri , Maddocks Simon , Toppari Jorma , Garnier Danielle-Hélène , Wdzieczak-Bakala Joanna , Jégou Bernard 
Published: April 2021

ABSTRACT
The tetrapeptide N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (AcSDKP) is a natural regulator of hematopoietic stem cell proliferation. The present study was aimed at investigating the presence and the role of AcSDKP in rat testis. Specific immunoreactivity was always observed in the interstitial tissue at all stages of testicular development and in elongated spermatids at 45 days of age and in adults. In accordance with the interstitial labeling, high AcSDKP levels were detected in Leydig cell and testicular macrophage culture media and cell extracts, as well as in the testicular interstitial fluid (TIF). Much lower concentrations were found in peritubular cells and Sertoli cells cultures, whereas very low concentrations were present in cultured spermatocytes and spermatids. In contrast to the slight degradation rate of AcSDKP observed in the spermatocyte and spermatid culture media, no catabolism of the peptide was seen in testicular somatic cell culture medium. Furthermore, the degradation rate of AcSDKP was much lower in TIF than in peripheral blood plasma. Despite the very strong evidence indicating that Leydig cells and testicular macrophages produce AcSDKP, the selective destruction of these cells did not result in any change in AcSDKP levels in TIF or in plasma. This suggests a compensatory mechanism ensuring constant levels of the peptide in TIF when interstitial cells are absent. Finally, in vitro, in the presence of AcSDKP, significantly more [3H]thymidine incorporation was found in A spermatogonia. In conclusion, this study establishes the presence of very high concentrations of AcSDKP in rat testis and demonstrates its Leydig cell and testicular macrophage origin. The presence of AcSDKP in the TIF and its stimulatory effect on thymidine incorporation in spermatogonia very strongly suggest its implication in the paracrine control of spermatogenesis.
Stem cell factor protects germ cells from apoptosis in vitro
Authors: Yan Wei , Suominen Janne , Toppari Jorma 
Published: April 2021

ABSTRACT
Stem cell factor (SCF) plays an important role in migration, adhesion, proliferation, and survival of primordial germ cells and spermatogonia during testicular development. However, the function of SCF in the adult testis is poorly described. We have previously shown that, in the presence of SCF, there were more type A spermatogonia incorporating thymidine at stage XII of rat seminiferous tubules cultured in vitro than in the absence of SCF, implying that the increased DNA synthesis might result from enhanced survival of spermatogonia. To explore the potential pro-survival function of SCF during spermatogenesis, the seminiferous tubules from stage XII were cultured in the presence or absence of SCF (100 ng/ml) for 8, 24, 48, and 72 hours, respectively, and apoptosis was analyzed by DNA laddering and in situ 3′-end labeling (ISEL) staining. Surprisingly, not only spermatogonia, but also spermatocytes and spermatids, were protected from apoptosis in the presence of SCF.
Apoptosis took place much later and was less severe in the SCF-treated tubules than in the controls. Based on previous studies showing that FSH prevents germ cells from undergoing apoptosis in vitro, and that SCF level is increased dramatically in response to FSH stimulation, we also tested if the pro-survival effect of FSH is mediated through SCF by using a function-blocking monoclonal antibody, ACK-2, to block SCF/c-kit interaction. After 24 hours of blockade, the protective effect of FSH was partially abolished, as manifested by DNA laddering and ISEL analyses. The present study demonstrates that SCF acts as an important survival factor for germ cells in the adult rat testis and FSH pro-survival effect on germ cells is mediated partially through the SCF/c-kit pathway.
Human embryonic stem cells
Authors: Pera Martin F. , Reubinoff Benjamin , Trounson Alan 
Published: April 2021

ABSTRACT
Embryonic stem (ES) cells are cells derived from the early embryo that can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent; they share these properties with embryonic germ (EG) cells. Candidate ES and EG cell lines from the human blastocyst and embryonic gonad can differentiate into multiple types of somatic cell. The phenotype of the blastocyst-derived cell lines is very similar to that of monkey ES cells and pluripotent human embryonal carcinoma cells, but differs from that of mouse ES cells or the human germ-cell-derived stem cells. Although our understanding of the control of growth and differentiation of human ES cells is quite limited, it is clear that the development of these cell lines will have a widespread impact on biomedical research.
Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation
Authors: Carlevaro Mariella F. , Cermelli Silvia , Cancedda Ranieri , Cancedda Fiorella Descalzi 
Published: April 2021

ABSTRACT
Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces endothelial cell migration and proliferation in culture and is strongly angiogenic in vivo. VEGF synthesis has been shown to occur in both normal and transformed cells. The receptors for the factor have been shown to be localized mainly in endothelial cells, however, the presence of VEGF synthesis and the VEGF receptor in cells other than endothelial cells has been demonstrated. Neoangiogenesis in cartilage growth plate plays a fundamental role in endochondral ossification. We have shown that, in an avian in vitro system for chondrocyte differentiation, VEGF was produced and localized in cell clusters totally resembling in vivo cartilage. The factor was synthesized by hypertrophic chondrocytes and was released into their conditioned medium, which is highly chemotactic for endothelial cells. Antibodies against VEGF inhibited endothelial cell migration induced by chondrocyte conditioned media. Similarly, endothelial cell migration was inhibited also by antibodies directed against the VEGF receptor 2/Flk1 (VEGFR2). In avian and mammalian embryo long bones, immediately before vascular invasion, VEGF was distinctly localized in growth plate hypertrophic chondrocytes. In contrast, VEGF was not observed in quiescent and proliferating chondrocytes earlier in development.
VEGF receptor 2 colocalized with the factor both in hypertrophic cartilage in vivo and hypertrophic cartilage engineered in vitro, suggesting an autocrine loop in chondrocytes at the time of their maturation to hypertrophic cells and of cartilage erosion. Regardless of cell exposure to exogenous VEGF, VEGFR-2 phosphorylation was recognized in cultured hypertrophic chondrocytes, supporting the idea of an autocrine functional activation of signal transduction in this non-endothelial cell type as a consequence of the endogenous VEGF production.
In summary we propose that VEGF is actively responsible for hypertrophic cartilage neovascularization through a paracrine release by chondrocytes, with invading endothelial cells as a target. Furthermore, VEGF receptor localization and signal transduction in chondrocytes strongly support the hypothesis of a VEGF autocrine activity also in morphogenesis and differentiation of a mesoderm derived cell.
The p53/p63/p73 family of transcription factors: overlapping and distinct functions
Authors: Levrero M. , Laurenzi V. De , Costanzo A. , Sabatini S. , Gong J. , Wang J. Y. J. , Melino G. 
Published: April 2021

ABSTRACT
The p53 gene is the most frequently mutated gene in human cancer. The identification of two homologues, p63 and p73, revealed that p53 is a member of a family of related transcription factors. Given that they share amino acid sequence identity reaching 63% in the DNA-binding domain, p53, p63 and p73 should have redundant functions in the regulation of gene expression. Indeed, p73 can activate p53-regulated genes and suppress growth or induce apoptosis. Moreover, p53 and p73 are both induced by DNA damage – albeit through distinct mechanisms. Other evidence, however, suggests that p63 and p73 are important for regulation of normal development. An extended C-terminal region, not found in p53, is alternatively spliced in p63 and p73. Within this C-terminal extension is a sterile alpha motif (SAM) previously found in other proteins that regulate development. The p63-deficient mice showed developmental abnormalities. Interestingly, the human p63 gene is mutated in children who have the disease Ectrodactyly, Ectodermal dysplasia and facial Clefts (EEC) syndrome, and the disease phenotype is similar to the one of p63-deficient mice. The p63 and p73 genes are rarely mutated in human cancer, although p73 loss is observed in neuroblastoma and a subtype of T-cell lymphoma. p53, p63 and p73 appear to have overlapping and distinct functions: p53 regulates the stress response to suppress tumors; p63 is essential for ectoderm development; and p73 might regulate both the stress response and development. Because p53 and p73 are linked to different upstream pathways, this family of transcription factors might regulate a common set of genes in response to different extracellular signals and developmental cues.