bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a molecular chaperone in the eukaryotic flagellum and its localization to the site of microtubule assembly
Authors: Bloch Melissa A. , Johnson Karl A. 
Published: April 2021

ABSTRACT
Monoclonal antibodies that recognize HSP70 family members from evolutionarily divergent organisms were used to identify both constitutively-expressed and stress-inducible HSP70 proteins in the green alga Chlamy-domonas. These monoclonal antibodies also cross-reacted with a 70 kDa flagellar protein that comigrated with the constitutively-expressed HSP70 isoform(s) present in the cell body; this is the first identification of a molecular chaperone within the eukaryotic flagellum. Fractionation experiments demonstrated that much of the flagellar HSP70 was bound to the ‘9+2’ microtubule axoneme. Incubation of isolated axonemes in ATP, but not AMP or AMP-PNP, caused significant release of the previously bound HSP70 as is characteristic of complexed HSP70s. Immuno-fluorescent localization in whole flagella showed that flagellar HSP70 was concentrated at the distal ends of flagella, sites of axonemal assembly in vivo. Extraction of axonemes under ionic conditions known to cause the release of capping structures that link the distal ends of the axonemal microtubules to the flagellar membrane also caused the release of axonemal-bound HSP70. Taken together, these results suggest a model in which an HSP70 chaperone may assist in targeting tubulin and other unassembled axonemal components to the flagellar tip where the chaperone may also participate in the assembly of the ‘9+2’ flagellar axoneme.
A survey of gtp-binding proteins and other potential key regulators of exocytotic secretion in eosinophils : Apparent absence of rab3 and vesicle fusion protein homologues
Authors: Lacy Paige , Thompson Nicki , Tian Min , Solari Roberto , Hide Izumi , Newman Terence M. , Gomperts Bastien D. 
Published: April 2021

ABSTRACT
We set out to identify potential key regulators of exocytotic fusion in the eosinophil, in the knowledge that granule exocytosis can be stimulated in these cells by intracellular application of nonhydrolyzable analogues of guanosine triphosphate, with Ca2+ acting as a modulator of guanine nucleotide-dependent secretion. To screen for GTP-binding proteins, guinea pig eosinophils were purified from peritoneal washings and subjected to western blotting analysis using specific immune sera raised against recombinant proteins or consensus peptide sequences within proteins of interest. We found a number of heterotrimeric G proteins (Gαi3, Gαo, Gαq11, Gαs and Gβ subunits) and members of the small GTP-binding proteins expressed in eosinophils. Two subtypes of G-protein alpha subunits (Gαi1 and Gαz) could not be detected. Separation of sub-cellular organelles from homogenized eosinophils by density gradient centrifugation revealed that all of the detected GTP-binding proteins were mainly expressed in fractions containing peak plasma membrane and Golgi marker enzyme activities, while Gβ subunits were also detected in secretory granule fractions. However, isoforms of Rab3, a putative GTP-binding regulator of exocytotic fusion, were undetectable in eosinophils. Neither, with the exception of syntaxin-3, could we detect any of the proteins belonging to the proposed synaptic vesicle fusion complex (SNAP-25; synaptobrevin (VAMP) and its non-neuronal homologue, cellubrevin; synaptophysin; synaptotagmin). The results from this study, based on western blotting, suggest that eosinophils express a different class of exocytotic fusion complex proteins from those found in neuronal tissues, although a number of potential candidates fulfilling the role of GE were identified in this important inflammatory cell.
Lung surfactant protein A (SP-A) activates a phosphoinositide/calcium signaling pathway in alveolar macrophages
Authors: Ohmer-Schröck Daniela , Schlatterer Christina , Plattner Helmut , Schlepper-Schäfer Jutta 
Published: April 2021

ABSTRACT
Lung surfactant protein A (SP-A), the main protein component of lung surfactant which lines the alveoli, strongly enhances serum-independent phagocytosis of bacteria by rat alveolar macrophages. We tested if the effect of SP-A is due to interaction with the macrophages or to opsonization of the bacteria. In phagocytosis assays with fluorescein isothiocyanate labeled bacteria, SP-A had no opsonic effect on Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, but enhanced phagocytosis by acting only on the macrophages. We characterized this activation mechanism. With single cell measurements of fura-2 loaded cells we demonstrate that SP-A raises the intracellular free calcium ion concentration 6 to 8 seconds after addition. This calcium mobilization is dosedependent in that increased SP-A concentrations lead to a higher percentage of responding cells. Additionally, SP-A leads to a dosedependent and transient generation of inositol 1,4,5-trisphosphate. Release of intracellular stored calcium by SP-A is a prerequisite for its stimulatory effect on phagocytosis, since SP-A-induced enhancement of phagocytosis can be impaired by prior addition of thapsigargin, a Ca2+-ATPase inhibitor that leads to depletion of intracellular calcium stores.
We conclude that SP-A activates a phosphoinositide/calcium signaling pathway in alveolar macrophages leading to enhanced serumindependent phagocytosis of bacteria.
The role of macrophage receptors in adhesion and uptake of Leishmania mexicana amastigotes
Authors: Peters Christopher , Aebischer Toni , Stierhof York-Dieter , Fuchs Manuela , Overath Peter 
Published: April 2021

ABSTRACT
Amastigotes of the protozoan parasite Leishmania proliferate in phagolysosomes of mammalian macrophages. Propagation of the infection is considered to occur by hostcell rupture and uptake of released parasites by uninfected macrophages. In this study, the kinetics of binding of L. mexicana mexicana amastigotes to COS cells and to COS cells transfected with three different macrophage receptors (FcRII-B2, receptor for the Fc-domain of immunoglobulins; CR3, complement type 3 receptor and the mannose receptor) is compared to the rate of adhesion to peritoneal macrophages. Amastigotes isolated from macrophages cultivated in vitro bind with slow, sigmoid kinetics to COS cells expressing either of the three receptors, or to peritoneal macrophages. In contrast, amastigotes isolated from mouse lesions bind with rapid, hyperbolic kinetics to COS cells expressing the Fc receptor or to peritoneal macrophages but with slow, sigmoid kinetics to COS cells expressing the CR3 or the mannose receptor. As shown by immunofluorescence experiments, lesion-derived amastigotes contain host-derived immunoglobulins (Ig) but no complement component 3 at their surface. It is concluded that amastigotes contain no intrinsic ligand at their surface, which enables high-affinity interactions with macrophages. Opsonization by specific Ig may be of relevance in vivo because firstly, in cryosections of mouse lesions extracellular amastigotes containing surface Ig can be detected and, secondly, B cell-deficient mice reconstituted with parasite-specific Ig show a modest increase in the rate of lesion development. In addition, it is shown that amastigotes are internalized by COS cells and grow in large para-sitophorous vacuoles similar to those observed in macrophages.
Synthesis and assembly of human β1 gap junctions in BHK cells by DNA transfection with the human β1 cDNA
Authors: Kumar Nalin M. , Friend Daniel S. , Gilula Norton B. 
Published: April 2021

ABSTRACT
Gap junctional communication is important in many phys-iological processes, including growth control, patterning, and the synchronization of cell-to-cell activities. It has been difficult to study the synthesis and assembly of gap junctions due to their low abundance. To overcome this limitation, baby hamster kidney cells (BHK) have been transfected with a human β1 (Cx32) connexin cDNA construct. Expression was placed under the control of the mouse metallothionein promoter that can be induced by heavy metals. The transfected cells were characterized by DNA, RNA and protein analysis, as well as by scrape loading to detect functional channels. Functional β1 connexin was detected only in cells transfected with β1 connexin cDNA in the correct orientation (β1-BHK). Analysis of the cells by light microscopic immunocyto-chemistry indicated that β1 connexin antigen was localized to the plasma membrane and to several intracellular compartments. Characterization with thin section electron microscopy revealed extensive areas of assembled double membrane gap junctions between cells (on the cell surface), in the endoplasmic reticulum (ER), and the nuclear envelope. This unusual intracellular distribution for assembled gap junction protein was confirmed by freeze fracture analysis, which revealed large particle aggregates, characteristic of gap junction plaques, on the fracture faces of all these membranes. The presence of gap junction particle aggregates in the ER suggests that the oligomer-ization of connexin can occur at its site of synthesis. Further, the process of assembly into double membrane junction structures in intracellular membranes may be driven by connexin protein concentration.
A tumor-associated glycosylation change in the glucose transporter GLUT1 controlled by tumor suppressor function in human cell hybrids
Authors: Kitagawa Takayuki , Tsuruhara Yasuko , Hayashi Masahiro , Endo Tamao , Stanbridge Eric J. 
Published: April 2021

ABSTRACT
Studies of human cell hybrids have provided evidence that the tumorigenicity of a cervical carcinoma (HeLa) is under the control of a putative tumor suppressor on chromosome 11. Using these human cell hybrids, we found a tumor-associated glycosylation change in the glucose transporter GLUT1, which is an N-linked glycoprotein at the plasma membrane. The non-tumorigenic HeLa × fibroblast cell hybrid CGL1 and the normal diploid fibroblast WI38 expressed the 50-55 kDa GLUT1, whereas in a tumorigenic segregant hybrid, CGL4, as well as in parental HeLa cells, GLUT1 glycosylation was altered and its molecular mass was about 70 kDa. However, the altered GLUT1 glycosylation was not observed in SV40-transformed WI38 cells, suggesting a correlation between this glycosylation change and a putative tumor suppressor function. Further investigations using glycosidases, glycosylation inhibitors and lectin-affinity chromatography demonstrated that the tumor-associated glycosylation change in GLUT1 was mainly due to the increase in N-acetyl-lactosamine repeats in the N-linked oligosaccharides. In accordance with the altered glycosylation, affinity for 2-deoxyglucose in the tumorigenic CGL4 cells increased 2-fold, but there was little change in the Vmax. These results suggest there may be a functional role for the modulation by glycosylation of GLUT1 in the tumorigenic behavior of CGL4 and HeLa cells.
Cell spreading and the regulation of ornithine decarboxylase
Authors: Morrison Ron F. , Seidel Edward R. 
Published: April 2021

ABSTRACT
The aim of this study was to investigate the effect of cell spreading on the induction of ornithine decarboxylase and the rate of putrescine uptake in anchorage-dependent and anchorage-independent cells. Plating non-transformed IEC-6 epithelial cells at high versus low cell density restricted cell spreading from 900 μm2 to approximately 140 μm2, blunted the transient induction of ornithine decarboxylase activity from 202 to 32 pmol 14CO2/mg protein per hour and reduced the rate of [14C]putrescine uptake from 46 to 23 pmol/105 cells per hour. The mean spreading area of the cell population was controlled by coating tissue culture dishes with the nonadhesive polymer, polyHEMA. Ornithine decarboxylase activity and putrescine uptake correlated with cell spreading with minimal spreading (263 μm2) corresponding to an 83% decrease in ornithine decarboxylase activity and 51% decrease in the rate of putrescine uptake. Adding the RGD peptide, Gly-Arg-Gly-Glu-Ser-Pro to the medium of sparsely plated cells resulted in rapid reductions in cell spreading concomitant with dose-dependent decreases in ornithine decarboxylase activity and putrescine uptake. Finally, minimizing cell spreading by depriving cells of sub-stratum contact completely abolished serum-induced increases in ornithine decarboxylase and reduced the rate of putrescine uptake by 47%. In contrast to IEC-6 cells, ornithine decarboxylase of neoplastic HTC-116 cells was constitutively expressed with basal and stimulated activity (193 and 982 pmol 14CO2/mg protein per hour, respec-tively) completely independent of cell adhesion. Putrescine uptake, however, was abolished in the absence of cell adhesion. These data suggest that the induction of ornithine decarboxylase activity and the rate of putrescine uptake correlate with spreading of anchorage-dependent IEC-6 cells and that ornithine decarboxylase activity, but not putrescine uptake, appears to be independent of spreading of neoplastic HTC-116 cells.
Microtubule organization and the distribution of γ-tubulin in spermatogenesis of a beetle, Tenebrio molitor (Tenebrionidae, Coleoptera, Insecta)
Authors: Wolf Klaus Werner , Joshi Harish C. 
Published: April 2021

ABSTRACT
The present study focuses on the restructuring of the microtubule (MT) cytoskeleton and microtubule-organizing centres (MTOCs) throughout spermatogenesis of a darkling beetle, Tenebrio molitor (Tenebrionidae, Coleoptera, Insecta). To this end, serial ultrathin sections through male germ cells were studied using transmission electron microscopy. Additionaly, spindles and young sper-matids were isolated from testes under MT-stabilizing con-ditions and doubly labeled with antibodies against β- and γ-tubulin. The latter is a tubulin isoform detected in MTOCs of a wide variety of species. The observations suggest that microtubules may be nucleated from sites with and without high γ-tubulin content and that these sites do not necessarily possess canonical centrosomes. In a prominent cytoplasmic MT system of primary spermato-cytes in prophase, microtubule nucleation apparently occurs in the absence of immunologically detectable γ-tubulin. At the poles of the meiotic spindles, MTs are directly inserted into γ-tubulin-containing material and this connection is considered responsible for their nucleation. The interzone spindle MTs of telophase cells contain γ-tubulin and this may confer stability to them. Finally, manchette MTs of spermatids originate in the vicinity of the acrosome precursor but are not inserted into this body. The acrosome precursor is surrounded by a membrane and is clearly detected by the antibody against γ-tubulin.
Coupling of [ca2+]i and ciliary beating in cultured tracheal epithelial cells
Authors: Salathe Matthias , Bookman Richard J. 
Published: April 2021

ABSTRACT
The molecular mechanisms responsible for the regulation of ciliary beating frequency (CBF) are only partially characterized. To determine whether elevation of intracellular Ca2+ ([Ca2+]i) can cause an increase in CBF, we measured CBF and Ca2+ in single cells. Ovine tracheal epithelial cells, obtained by dissociation with protease, were grown in primary culture for 1 to 28 days in a mucus-free system. CBF of a single cilium was measured by digital video phase-contrast microscopy and on-line Fourier-transform analysis. Changes in [Ca2+]i from single cells were determined with fura-2, using ratio imaging video microscopy. Activation of a muscarinic pathway with 10 μM ACh (acetylcholine) increased [Ca2+]i from 53±9 nM (mean ± s.e.m.) to 146±12 nM or to 264±51% above initial baseline. In the same cells, ACh increased CBF from a baseline of 7±0.5 Hz to 9±0.2 Hz or to 31±2.8% above baseline (n=14). The elevations of both [Ca2+]i and CBF were transient and relaxed back to an elevated plateau (10/14 cells) as long as ACh was present. To elevate [Ca2+]i by mechanisms independent of a G-protein-coupled receptor, we measured [Ca2+]i and CBF of the same cells in extracellular solutions with either 0 Ca2+ (+ 1 mM EGTA) or 10 mM Ca2+. Both signals rose and fell with similar kinetics in response to changing [Ca2+]o, suggesting that changes in [Ca2+]i alone can modulate CBF. In a second independent manipulation, cells were treated with 1 μM thapsigargin, an irreversible inhibitor of the endoplasmic reticulum Ca2+-ATPase. Upon thapsigargin addition, 37 of 42 cells showed a transient [Ca2+]i increase and, as measured in different experiments, 8 of 9 cells showed a transient increase in CBF. Interestingly, application of ACh after cells were treated with thapsigargin produced decreases in both [Ca2+]i and CBF in 8/8 cells. Lastly, after 1-3 days in culture, addition of 10 μM ACh often produced [Ca2+]i oscillations rather than transients in [Ca2+]i. Measurements of CBF in these cells showed frequency modulation of CBF with the same peak-to-peak time interval as the Ca2+ oscillation. These results show that: (1) CBF can be measured from a single cilium and monitored on-line to track changes; (2) CBF and [Ca2+]i can be measured in the same single cell; (3) transient changes in [Ca2+]i (induced by 4 different manipulations) are associated with kinetically similar changes in CBF; and (4) [Ca2+]i oscillations are coupled to frequency modulation of ciliary beating. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of CBF in mammalian tracheal epithelial cells.
Mg-160, a membrane sialoglycoprotein of the medial cisternae of the rat golgi apparatus, binds basic fibroblast growth factor and exhibits a high level of sequence identity to a chicken fibroblast growth factor receptor
Authors: Gonatas Jacqueline O. , Mourelatos Zissimos , Stieber Anna , Lane William S. , Brosius Jurgen , Gonatas Nicholas K. 
Published: April 2021

ABSTRACT
We report the primary structure of MG-160, a 160 kDa membrane sialoglycoprotein residing in the medial cisternae of the Golgi apparatus of rat neurons, pheochromocytoma (PC-12), and several other cells. The cDNA encodes a polypeptide of 1,171 amino acids with an Mr of 133,403. An intralumenal cleavable signal peptide is followed by a Pro-Gln-rich segment and 16 contiguous, approx. 60-residue-long, regularly spaced cysteine-rich segments showing sequence identities ranging from 15 to 35%. The lumenal domain is followed by a single membrane spanning domain and a short carboxy-terminal cytoplasmic tail. The protein contains 5 potential NXT glycosylation sites.
The sequence of MG-160 shows no homologies with enzymes and other membrane proteins of the Golgi apparatus. MG-160 displays a so far unique feature for a membrane protein of the Golgi apparatus: namely, an upstream open reading frame (uORF), encoding 58 amino acids, located in front of the major open reading frame (ORF). Most vertebrate mRNAs containing uORF or AUG codons in front of the major ORF encode growth factors and cell surface receptors (Geballe and Morris 1994). In that regard a 90% identity between the primary structure of MG-160 and a receptor for acidic and basic fibroblast growth factors (CFR), isolated from chicken embryos (Burrus et. al., 1992), may be relevant.
Immunoreactivity for MG-160 has been detected in the Golgi apparatus of neural and other cells of 2-day-old chicken embryos and adult chicken; furthermore, recombinant human basic fibroblast growth factor (bFGF) binds MG-160 purified from rat brain. MG-160 shows no sequence similarity with members of the family of fibroblast growth factor receptors (FGFR) involved in signal transduction. These findings are consistent with the hypothesis that MG-160 is involved in the traffic and processing of endogenous or autocrine FGFs. This is the first example of an intrinsic membrane protein of the Golgi apparatus which binds a growth factor and may be involved in its regulation.
The Schizosaccharomyces Pombe Hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis
Authors: Al-Khodairy Fahad , Enoch Tamar , Hagan Iain M. , Carr Antony M. 
Published: April 2021

SUMMARY
Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called ‘check-points’, mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs).
To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.
Sequence and tissue distribution of the human integrin α8 subunit: a β1-associated α subunit expressed in smooth muscle cells
Authors: Schnapp Lynn M. , Breuss Johannes M. , Ramos Daniel M. , Sheppard Dean , Pytela Robert 
Published: April 2021

ABSTRACT
Integrins are a major family of cell adhesion molecules involved in cell-cell and cell-extracellular matrix interactions. Each integrin is a heterodimeric glycoprotein composed of an α and a β subunit. We now report the cDNA sequence and distribution of a new human integrin α subunit. This sequence is 78% identical to the previously reported chicken α8 integrin sequence. Thus, we have designated this subunit as human α8. By northern blot analysis, an α8 probe detects two mRNA species of approximately 6.5 and 4.0 kb in neuroglioma H4 cells. An anti-α8 polyclonal antibody precipitates a protein complex containing the β1 subunit associated with the putative α8 subunit, which has an apparent molecular mass of 180 kDa (non-reduced) or 155 kDa and 25 kDa (reduced). Immunohistochemistry with anti-α8 polyclonal antibody in adult rat tissues shows prominent staining in vascular and visceral smooth muscle. In addition, the antibody strongly stained kidney mesangial cells and a cell type in the alveolar wall of the lungs, most likely corresponding to alveolar myofibroblasts. These results suggest that in adult mammalian tissues, α8 is predominantly expressed in smooth muscle and smooth muscle-like contractile cells.