bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of the CYP1A1 promoter in transgenic mice: an exquisitely sensitive on-off system for cell specific gene regulation
Authors: Campbell Sandra J. , Carlotti Franco , Hall Peter A. , Clark A. John , Wolf C. Roland 
Published: April 2021

ABSTRACT
Mammalian cytochrome P-450s in the CYP1A gene family catalyse the oxidation of a wide range of drugs and foreign compounds resulting in their excretion. These enzymes are highly inducible by a range of compounds, including polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (3-MC) and dioxins. Analysis of the CYP1A1 promoter has identified dioxin responsive enhancer elements which mediate the induction response. In order to evaluate this promoter as an in vivo regulatable expression system and to gain further insights into the tissue specific regulation of this gene, an 8.5 kb genomic fragment of the rat CYP1A1 promoter was cloned upstream of the lacZ reporter gene. This construct was used to generate transgenic mice and three independent lines were expanded for further study. The regulation of β-galactosidase expression was determined in mock and 3-MC-treated mice in an extensive range of tissues. In untreated animals no transgene expression was detectable over non-transgenic controls. Treatment with 3-MC caused a profound increase in transgene expression (&gt;1,000-fold) in many tissues including liver, adrenal, kidney and intestine. Inducible transgene expression was also detectable in many of the other tissues including the spleen, lung, pancreas and the reproductive organs. Although the absolute levels of induction varied, no significant differences in the pattern of transgene expression were observed between the three different transgenic mouse lines. In addition, the pattern of transgene expression correlated closely with the reported regulation of CYP1A1 protein. These results indicate that the CYP1A1 promoter can drive expression of heterologous genes in a truly on/off manner in a variety of tissues and cell types which will allow the expression of other proteins to be controlled in vivo. This reporter system also provides a model for establishing the environmental and hormonal factors regulating the expression of the CYP1A1 gene.
MT-MMP, the cell surface activator of proMMP-2 (progelatinase A), is expressed with its substrate in mouse tissue during embryogenesis
Authors: Kinoh Hiroaki , Sato Hiroshi , Tsunezuka Yoshio , Takino Takahisa , Kawashima Atsuhiro , Okada Yasunori , Seiki Motoharu 
Published: April 2021

ABSTRACT
Matrix metalloproteinases (MMPs), which degrade the components of the extracellular matrix, are key enzymes involved in the tissue remodeling of multicellular organisms. Since MMPs are secreted as inactive zymogens (pro-MMPs), they have to be activated to function. We identified a membranetype MMP (MT-MMP) that activated proMMP-2 (progelatinase A = 72 kDa type IV procollagenase) and described its expression on the invasive tumor cell surface. In this study we further examined the expression and role of MT-MMP in the activation of proMMP-2 during mouse embryogenesis. Northern blotting demonstrated that MT-MMP expression was increased together with that of MMP-2 and its inhibitor gene, TIMP-2, in embryos depending upon the number of days after gestation, and decreased with maturation after birth. In situ hybridization and immunohistochemistry localized MT-MMP mRNA and protein in the cells of ossifying tissues where both MMP-2 and TIMP-2 were expressed. Activated MMP-2 was detected by gelatin zymography in the lysates prepared from the micro dissected tissues that expressed the three genes. The activation rate of proMMP-2 was proportional to the expression of MMP-2 and MT-MMP. These results indicated that proMMP-2 activation through its activator, MT-MMP, is a physiological system used by organisms to initiate tissue remodeling on the cell surface.
Xenopus cyclin E, a nuclear phosphoprotein, accumulates when oocytes gain the ability to initiate DNA replication
Authors: Chevalier S. , Couturier A. , Chartrain I. , Guellec R. Le , Beckhelling C. , Guellec K. Le , Philippe M. , Ford C. C. 
Published: April 2021

ABSTRACT
The capacity to initiate DNA replication appears during oocyte maturation in Xenopus. Initiation of S phase is driven by several components which include active cyclin/cdk complexes. We have identified three Xenopus cyclin E clones showing 59% amino acid identity with human cyclin E. The recruitment of cyclin E mRNA, like cdk2 mRNA, into the polysomal fraction during oocyte maturation, results in the accumulation of the corresponding proteins in unfertilized eggs. Cyclin E mRNA remains polyadenylated during cleavage and anti-cyclin E anti-bodies detect Xlcyclin E in embryonic nuclei at this time. Cdk2 protein is necessary for the phosphorylation of radio-labelled cyclin E added to egg extracts. Radiolabelled Xlcyclin E enters interphase nuclei and, though stable through interphase and mitosis, is not associated with condensed mitotic chromatin. In egg extracts, endogenous Xlcyclin E rapidly associates with nuclei before S phase and remains nuclear throughout interphase, becoming nucleo-plasmic in G2/prophase. Under conditions where initiation of replication is limiting in extracts, Xlcyclin E associates only with those nuclei that undergo S phase. These features are entirely consistent with the view that Xlcyclin E is required for initiation of S phase.
The transcytotic pathway of an apical plasma membrane protein (B10) in hepatocytes is similar to that of IgA and occurs via a tubular pericentriolar compartment
Authors: Hemery Isabelle , Durand-Schneider Anne-Marie , Feldmann Gérard , Vaerman Jean-Pierre , Maurice Michèle 
Published: April 2021

ABSTRACT
In hepatocytes, newly synthesized apical plasma membrane proteins are first delivered to the basolateral surface and are supposed to reach the apical surface by transcytosis. The transcytotic pathway of apical membrane proteins and its relationship with other endosomal pathways has not been demonstrated morphologically. We compared the intracellular route of an apical plasma membrane protein, B10, with that of polymeric IgA (pIgA), which is transcy-tosed, transferrin (Tf) which is recycled, and asialooroso-mucoid (ASOR) which is delivered to lysosomes. Ligands and anti-B10 monoclonal IgG were linked to fluorochromes or with peroxidase. The fate of each ligand was followed by confocal and electron microscopy in polarized primary monolayers of rat hepatocytes. When fluorescent anti-B10 IgG and fluorescent pIgA were simultaneously endocytosed for 15-30 minutes, they both uniformly labelled a juxtanuclear compartment. By 30-60 minutes, they reached the bile canaliculi. Tf and ASOR were also routed to the juxtanu-clear area, but their fluorescence patterns were more punctate. Microtubule disruption prevented all ligands from reaching the juxtanuclear area. This area corresponded, at least partially, to the localization of the mannose 6-phosphate receptor, an endosomal marker. By electron microscopy, the juxtanuclear compartment was made up of anastomosing tubules connected to vacuoles, and was organized around the centrioles. B10 and pIgA were mainly found in the tubules, whereas ASOR was segregated inside the vacuolar elements and Tf within thinner, recycling tubules. In conclusion, transcytosis of the apical membrane protein B10 occurs inside tubules similar to those carrying pIgA, and involves passage via the pericentriolar area. In the pericentriolar area, the transcytotic tubules appear to maintain connections with other endosomal elements where sorting between recycled and degraded ligands occurs.
Identification and characterization of espin, an actin-binding protein localized to the F-actin-rich junctional plaques of Sertoli cell ectoplasmic specializations
Authors: Bartles James R. , Wierda Allison , Zheng Lili 
Published: April 2021

ABSTRACT
Ectoplasmic specializations are membrane-cytoskeletal assemblages found in Sertoli cells at sites of attachment to elongate spermatids or neighboring Sertoli cells. They are characterized in part by the presence of a unique junctional plaque which contains a narrow layer of parallel actin bundles sandwiched between the Sertoli cell plasma membrane and an affiliated cistern of endoplasmic reticulum. Using a monoclonal antibody, we have identified ‘espin,’ a novel actin-binding protein localized to ectoplas-mic specializations. By immunogold electron microscopy, espin was localized to the parallel actin bundles of ecto-plasmic specializations at sites where Sertoli cells contacted the heads of elongate spermatids. The protein was also detected at the sites of ectoplasmic specializations between neighboring Sertoli cells. Espin exhibits an apparent molecular mass of ∼110 kDa in SDS gels. It is encoded by an ∼2.9 kb mRNA, which was found to be specific to testis among the 11 rat organs and tissues examined. On the basis of cDNA sequence, espin is predicted to be an 836 amino acid protein which contains 8 ankyrin-like repeats in its N-terminal third, a potential P-loop, two proline-rich peptides and two peptides which contain clusters of multiple gluta-mates bracketed by arginines, lysines and glutamines in a pattern reminiscent of the repetitive motif found in the protein trichohyalin. The ankyrin-like repeats and a 66 amino acid peptide in the C terminus show significant sequence similarity to proteins encoded by the forked gene of Drosophila. A fusion protein containing the C-terminal 378 amino acids of espin was found to bind with high affinity (Kd=∼10 nM) to F-actin in vitro with a stoichiome-try of ∼1 espin per 6 actin monomers. When expressed by transfected NRK fibroblasts, the same C-terminal fragment of espin was observed to decorate actin fibers or cables. On the basis of its structure, localization and prop-erties, we hypothesize that espin is involved in linking actin filaments to each other or to membranes, thereby poten-tially playing a key role in the organization and function of the ectoplasmic specialization.
The organization of ribosomal RNA processing correlates with the distribution of nucleolar snRNAs
Authors: Beven A. F. , Lee R. , Razaz M. , Leader D. J. , Brown J. W. S. , Shaw P. J. 
Published: April 2021

ABSTRACT
We have analyzed the organization of pre-rRNA processing by confocal microscopy in pea root cell nucleoli using a variety of probes for fluorescence in situ hybridization and immunofluorescence. Our results show that transcript processing within the nucleolus is spatially highly organized. Probes to the 5′ external transcribed spacer (ETS) and first internal transcribed spacer (ITS1) showed that the excision of the ETS occurred in a sub-region of the dense fibrillar component (DFC), whereas the excision of ITS1 occurred in the surrounding region, broadly corresponding to the granular component. In situ labelling with probes to the snoRNAs U3 and U14, and immunofluorescence labelling with antibodies to fibrillarin and SSB1 showed a high degree of coincidence with the ETS pattern, confirming that ETS cleavage and 18 S rRNA production occur in the DFC. ETS, U14, fibrillarin and SSB1 showed a fine substructure within the DFC comprising closely packed small foci, whereas U3 appeared more diffuse throughout the DFC. A third snoRNA, 7-2/MRP, was localised to the region surrounding the ETS, in agreement with its suggested role in ITS1 cleavage. All three snoRNAs were also frequently observed in numerous small foci in the nucleolar vacuoles, but none was detectable in coiled bodies. Antibodies to fibrillarin and SSB1 labelled coiled bodies strongly, though neither protein was detected in the nucleolar vacuoles. During mitosis, all the components analyzed, including pre-rRNA, were dispersed through the cell at metaphase, then became concentrated around the periphery of all the chromosomes at anaphase, before being localized to the developing nucleoli at late telophase. Pre-rRNA (ETS and ITS1 probes), U3 and U14 were also concentrated into small bodies, presumed to be pre-nucleolar bodies at anaphase.
Identification of a novel member of the Rab8 family from the rat basophilic leukaemia cell line, RBL.2H3
Authors: Armstrong John , Thompson Nicola , Squire Jane H. , Smith Janet , Hayes Brian , Solari Roberto 
Published: April 2021

ABSTRACT
We describe the cloning of a cDNA from the rat basophilic leukaemia cell line (RBL.2H3) encoding a novel member of the Rab family of small GTP binding proteins. The novel clone, which we call Rab8b, is most highly related to the Rab8 family with substantial divergence in the variable C-terminal domain. Northern blot analysis reveals highest levels of expression of Rab8b in the spleen, testis and brain, which is in marked contrast to the tissue distribution of Rab8. The Rab8b cDNA was modified to introduce a c-myc epitope tag at the extreme N terminus of the protein, and transient transfection studies were performed to analyse the intracellular localization of Rab8b by confocal microscopy. Transient expression of the c-myc/Rab8b fusion protein in both PC12 and RBL.2H3 cells shows staining of both the plasma membrane and ill-defined vesicular structures, and in the case of RBL.2H3 cells appears to induce striking outgrowths of the plasma membrane.
Distinct egg membrane vesicles differing in binding and fusion properties contribute to sea urchin male pronuclear envelopes formed in vitro
Authors: Collas Philippe , Poccia Dominic 
Published: April 2021

ABSTRACT
We have identified three distinct membrane vesicle populations from sea urchin egg cytoplasm that cooperate in assembling the male pronuclear envelope in vitro. Membranes from sea urchin egg homogenates were separated by buoyant density into five vesicle fractions, three of which bind to demembranated sperm nuclei. Each requires a membranous element (lipophilic structure) derived from the sperm nuclear envelope at the tip and base (poles) of the nucleus in order to bind. Binding is differentially sensitive to protease, high salt and N-ethyl maleimide treatment of the membrane vesicles. MV1 binds at the poles and is required for fusion of the membrane vesicle fractions to each other and to the lipophilic structures. MV2β binds over the entire chromatin surface and is enriched in an endoplasmic reticulum marker enzyme. MV2α binds at the nuclear poles, is enriched in a Golgi enzyme marker and is required for fusion of MV2β. All three fractions are required for nuclear envelope formation in vitro. The results suggest a multistep process for nuclear envelope formation involving contributions from both sperm and egg, roles for both endoplasmic reticulum and non-endoplasmic reticulum-derived vesicles, and the localization of a critical element of the fusion machinery in MV1.
Apical recruitment of CFTR in T-84 cells is dependent on cAMP and microtubules but not Ca2+ or microfilaments
Authors: Tousson Albert , Fuller Catherine M. , Benos Dale J. 
Published: April 2021

ABSTRACT
Previous studies from this laboratory have demonstrated that chloride transport induced by forskolin, but not ionomycin, in T84 cells is highly dependent on an intact microtubular network. Using an antibody raised against a region of the R domain of CFTR, we now show by indirect immunofluorescence that forskolin causes relocation of CFTR to the apical domain of T84 cells. T84 cells grown on transparent filters were incubated with agonists and/or cytoskeletal inhibitors prior to fixation, permeabilization, and staining with the antibody. A 30 second stimulation with forskolin (10 μM) caused a twofold increase in relative fluorescence intensity at the apical surface. In contrast, a 30 second exposure to ionomycin (2 μM), had no effect on the distribution of CFTR-related fluorescence. Incubation of the cells with nocodazole (33 μM), a microtubule disrupting agent, prevented the forskolin-induced rise in CFTR fluorescence at the apical surface. However, incubation of the cells with cytochalasin D, an actin inhibitor, was without effect on forskolin-related re-distribution of CFTR-associated fluorescence. In double label experiments using antibodies against both β-tubulin and actin, CFTR-related fluorescence was found to co-localize with the microtubule network, but not with actin filaments. These observations are consistent with the microtubule-dependent acute recruitment of CFTR to the apical plasma membrane of T84 cells in response to elevations in intracellular cAMP.
A nitrogen starvation-induced dormant G0 state in fission yeast: the establishment from uncommitted G1 state and its delay for return to proliferation
Authors: Su Sophia S. Y. , Tanaka Yusuke , Samejima Itaru , Tanaka Kenji , Yanagida Mitsuhiro 
Published: April 2021

ABSTRACT
Fission yeast cells either remain in the mitotic cell cycle or exit to meiotic sporulation from an uncommitted G1 state dependent on the presence or absence of nitrogen source in the medium (Nurse and Bissett, 1981). We examined how heterothallic haploid cells, which cannot sporulate, behave under nitrogen-starvation for longer than 25 days at 26°C. These cells were shown to enter a stable state (designated the dormant G0) with nearly full viability. Maintaining the dormant cells required glucose, suggesting that the cells remained metabolically active although cell division had ceased. They differed dramatically from mitotic and uncommitted G1 cells in heat resistance, and also in cytoplasmic and nuclear morphologies. After nitrogen replenishment, the initial responses of dormant G0 cells were investigated. The kinetics for reentry into the proliferative state were delayed considerably, and the changes in cell shape were enhanced particularly for those recovering from extended nitrogen starvation. A part of the delay could be accounted for by the duration of nuclear decondensation and cell elongation for the first cell division.
Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn
Authors: Cary Leslie A. , Chang James F. , Guan Jun-Lin 
Published: April 2021

ABSTRACT
Cellular interactions with the extracellular matrix proteins play important roles in a variety of biological processes. Recent studies suggest that integrin-mediated cell-matrix interaction can transduce biochemical signals across the plasma membrane to regulate cellular functions such as proliferation, differentiation and migration. These studies have implicated a critical role of focal adhesion kinase (FAK) in integrin-mediated signal transduction pathways. We report here that overexpression of FAK in CHO cells increased their migration on fibronectin. A mutation of the major autophosphorylation site Y397 in FAK abolished its ability to stimulate cell migration, while phosphorylation of Y397 in a kinase-defective FAK by endogenous FAK led to increased migration. We also find that the wild-type and the kinase-defective FAK were associated with Src and Fyn in CHO cells whereas the F397 mutant was not. These results directly demonstrate a functional role for FAK in integrin signaling leading to cell migration. They also provide evidence for the functional significance of FAK/Src complex formation in vivo.
Sequence and expression of Drosophila myristoyl-CoA:protein N-myristoyl transferase: evidence for proteolytic processing and membrane localisation
Authors: Ntwasa Monde , Egerton Mark , Gay Nicholas J. 
Published: April 2021

ABSTRACT
The enzyme N-myristoyl transferase transfers the 14 carbon fatty acid myristate to an N-terminal glycine residue in a small subset of cytoplasmic proteins. Many myristoyl proteins are components of cellular signalling pathways, some of which play important roles during embryonic development, for example protein kinase A. Thus, the function of N-myristoyl transferase is probably essential for embryogenesis and it is of some interest to study the enzyme in an organism with well understood developmental biology. In this paper we report the purification of a processed form of the Drosophila enzyme from peripheral membrane fractions of embryos by affinity chromatography to a protein containing leucine rich repeats. We have also isolated the Drosophila N-myristoyl transferase gene and determined its nucleotide sequence. The predicted amino acid sequence of the Drosophila enzyme is closely related to that of mammalian and fungal N-myristoyl transferases and residues essential for enzyme function are conserved. Our findings indicate that a fraction of Drosphila NMT is bound to the membrane and they are consistent with recent results for the human enzyme. We suggest that N-myristoyl transferase may be recruited to the membrane as part of a translational complex, perhaps by binding to p34 ribosome binding protein, a leucine rich repeat receptor of the microsomal membranes. We have also studied the expression pattern of the gene in the embryo by northern blot analysis and in situ hybridization. The transcripts appear to be uniformly distributed in the pre-cellular embryo but at later stages the RNA is barely detectable with these methods. However, from about stage 14, high levels of transcript are detected in a small number of randomly distributed cells of the central and peripheral nervous system.
Monastral bipolar spindles: implications for dynamic centrosome organization
Authors: Wilson P. G. , Fuller M. T. , Borisy G. G. 
Published: April 2021

ABSTRACT
Implicit to all models for mitotic spindle assembly is the view that centrosomes are essentially permanent structures. Yet, immunofluorescence revealed that spindles in larval brains of urchin mutants in Drosophila were frequently monastral but bipolar; the astral pole contained a centrosome while the opposing anastral pole showed neither gamma tubulin nor a radial array of astral microtubules. Thus, mutations in the urchin gene seem to uncouple centrosome organization and spindle bipolarity in mitotic cells. Hypomorphic mutants showed a high frequency of monastral bipolar spindles but low frequencies of polyploidy, suggesting that monastral bipolar spindles might be functional. To test this hypothesis, we performed pedigree analysis of centrosome distribution and spindle structure in the four mitotic divisions of gonial cells. Prophase gonial cells showed two centrosomes, suggesting cells entered mitosis with the normal number of centrosomes and that centrosomes separated during prophase. Despite a high frequency of monastral bipolar spindles, the end products of the four mitotic divisions were equivalent in size and chromatin content. These results indicate that monastral bipolar spindles are functional and that the daughter cell derived from the anastral pole can assemble a functional bipolar spindle in the subsequent cell cycle. Cell proliferation despite high frequencies of monastral bipolar spindles can be explained if centrosome structure in mitotic cells is dynamic, allowing transient and benign disorganization of pericentriolar components. Since urchin proved to be allelic to KLP61F which encodes a kinesin related motor protein (Heck et al. (1993)J. Cell Biol. 123, 665-671), our results suggest that motors influence the dynamic organization of centrosomes.