bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of differentiation on platelet-activating factor metabolism in HL-60 cells
Authors: Stoll Lynn L. , Yerram Nagender R. , Spector Arthur A. 
Published: April 2021

ABSTRACT
The formation and metabolism of l-O-alkyl-2-acetyl-sn-glycerol (AAG), a protein kinase C (PKC) activator formed from platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF), was studied in HL-60 cells to determine whether differentiation may influence this process. HL-60 cells differentiated to macrophages (HL-60/Mø) with a phorbol ester convert added [3 H]PAF to AAG; 22% of the incorporated radioactivity is converted to AAG within 15 s. By contrast, neither undifferentiated HL-60 cells (HL-60/U) nor HL-60 cells differentiated to granulocytes (HL-60/GN) with retinoic acid produce AAG from PAF. The HL-60/Mϕ rapidly convert radiolabeled AAG to l-O-alkyl-sn-glycerol and, subsequently, to two other unidentified metabolites. However, some apparently unmodified AAG persists in the cell lipids for at least 6h. The HL-60 subtypes which do not convert PAF to AAG can nevertheless catabolize AAG; HL-60/U and HL-60/GN produce alkylglycerol and the other AAG metabolites. These findings demonstrate that differentiation can alter the processing of PAF in a human leukocyte cell line. Furthermore, they suggest that PAF may produce at least some of its biological effects in macrophages by conversion to AAG.
Changes in the abundance and distribution of actin and associated proteins during terminal differentiation of human epidermal kératinocytes
Authors: Kubler M.-Dominique , Jordan Peter W. , O’neill Charles H. , Watt Fiona M. 
Published: April 2021

ABSTRACT
We have examined the abundance and distribution of actin and several actin-associated proteins in human epidermal keratinocytes before and after initiation of terminal differentiation. Keratinocytes were placed in suspension in methylcellulose for lh or 24 h and then extracted for immunoblotting. At 24 h, when the proportion of cells expressing the terminal differentiation marker, involucrin, had increased approximately 3-fold, there were marked decreases in the levels of vinculin, talin, filamin and gelsolin. The level of actin was unchanged and the level of a-actinin decreased only slightly. To complement the immunoblot analysis, we also examined the distribution of each protein in basal (involucrin-negative) and suprabasal (involucrin-positive) cells in stratified colonies, using confocal microscopy. Gelsolin, filamin, vinculin, talin, a-actinin and filamentous actin were all less abundant in suprabasal cells than in basal cells. There were also differences in the distribution of all the proteins in the basal compared to the suprabasal layers. In addition to the changes associated with terminal differentiation, there was variation in the distribution of focal contacts and stress fibres and in gelsolin levels between basal cells at the periphery of colonies and those in the centre. These results are discussed in the context of the known association of the actin cytoskeleton with receptors of the integrin family, the loss of integrine that occurs during kératinocyte terminal differentiation, and the possible role of the cytoskeleton in signalling between integrine and the nucleus.
Insulin receptors on Xenopus laevis oocytes: Effects of injection of ob/ob mouse liver mRNA
Authors: Diss David A. G. , Greenstein Benjamin D. 
Published: April 2021

ABSTRACT
We describe here conditions for the detection of insulin binding sites on Xenopus laevis oocytes. The binding of 125 I-labelled insulin displayed sigmoidal behaviour, which is characteristic of the binding relationship between insulin and its receptor. Resolution of the resulting curvilinear Scatchard plot into two components revealed KD values of 8.86×10−10 ±1.9× 10−10 and 5.32× 10−9 ±2.4× 10−9 M and n values of 9.7×107 ±0.4×107 and 3.3×108 ±0.5×108 binding sites per oocyte, respectively. The possibility cannot be excluded, however, that receptors for IGF-1 were also being detected. Also described are conditions for the rapid and efficient removal of all tissues surrounding the oocyte, including the vitelline membrane. We could not detect any specific 125 I-labelled insulin binding to oocytes that had their follicle cells or vitelline membrane removed and this was not due to the enzymic treatment used in the process. Microinjection of oocytes without follicular layers did not result in the appearance of any detectable insulin binding sites, which were, however, observed if oocytes were first stripped of the vitelline membrane. We suggest that oocytes may possess endogenous insulin receptors on their surface in numbers of the same order of magnitude as those present on somatic cells. The removal of tissues surrounding the oocyte should facilitate studies aimed at determining functional interactions of the various cell types during oocyte development and for studying insulin receptors on the oocyte-follicular cell complex.
A comparison of spreading and motility behaviour of 8701 -BC breast carcinoma cells on type I, I-trimer and type V collagen substrata evidence for a permissive effect of type I-trimer collagen on cell locomotion
Authors: Luparello Claudio , Sheterline Peter , Pucci-Minafra Ida , Minmra Salvatore 
Published: April 2021

ABSTRACT
Ductal infiltrating carcinoma (d.i.c.) of human breast is a highly invasive neoplasm characterized by enhanced deposition of collagen. Paradoxically, enhanced collagen deposition is not correlated with inhibition of the migration of tumour cells into the host tissue, d.i.c. is characterized by the reappearance of ‘embryonic’ type l-trimer collagen and an increase in type V collagen content in the matrix. The effects of these two collagen types were compared with type I collagen as culture substrata on the spreading pattern, cytoskeletal organization and motile behaviour of 8701-BC breast carcinoma cells using rhodamine-phalloidin staining, a DNAase I-competition assay, scanning electron microscopy and time-lapse video-microscopy. Cells grown on type I collagen were stationary, showing a wellspread morphology and an extensive stress fibre pattern. Cells grown on type V collagen were also stationary, but displayed a poorly spread and elongated morphology. In contrast, cells grown on trimer collagen were motile and displayed a compact morphology and a reduced content of stress fibres. Both single-cell and group motility were detectable on trimer collagen substratum. These data are consistent with the existence of two opposite local signals, type l-trimer and type V collagens, which may confer a more or a less metastatic phenotype on breast carcinoma cells. Moreover, the synthesis of trimer collagen in d.i.c. is conceivably instrumental in providing new stromal pathways permitting tumour cells to infiltrate the host tissue.
Pole-to-chromosome movements induced at metaphase: Sites of microtubule disassembly
Authors: Centonze Victoria E. , Borisy Gary G. 
Published: April 2021

ABSTRACT
Metaphase spindles can be induced to shrink by treating cells with microtubule-depolymerizing agents. During treatment, the paired sister chromatids remain at the metaphase plate and the poles move toward them. The question we asked is whether this pole-to-chromosome movement was accompanied by a loss of subunits from the kineto-chore ends of the microtubules, the polar ends, or both ends. LLC-PK cells were injected at late prometaphase with Xrhodamine tubulin and at metaphase the fluorescent spindles were marked by photobleaching a bar between one pole and the chromosomes. Nocodazole at low concentrations was briefly applied to the cells to induce the shortening of the spindle and movement of the poles inward toward the chromosomes. In the induced shortening, the distance between the photobleached bar and the chromosomes decreased substantially while the distance between the bar and the pole showed a smaller change. Upon reversal from nocodazole, new polymer was added to the spindle as determined by recovery of fluorescence, and the cells progressed through mitosis and cytokinesis. We conclude that the movement of the poles to the chromosomes induced by nocodazole treatment during metaphase is similar to the chromosome-to-pole movement occurring during anaphase in that under both conditions the primary site for kinetochore microtubule disassembly is at the kinetochore.
Localization of centromeric satellite and telomeric dna sequences in dorsal root ganglion neurons, in vitro
Authors: Billia Filio , Boni Umberto De 
Published: April 2021

ABSTRACT
Chromatin domains of interphase nuclei are organized in a tissue-specific, non-random manner. In the present work, the spatial arrangement of satellite (sDNA) and telomeric (tDNA) DNA was examined in nuclei of murine Dorsal Root Ganglion (DRG) cells, maintained in vitro. In situ hybridization in conjunction with three-dimensional reconstruction was employed. A mean number of 8.02 ±0.40 sDNA signals/nucleus was detected, of which 41.65±0.59% were associated with the nucleolus. The remaining fraction of signals was localized between the nucleolus and the nuclear membrane. sDNA signals were reproducibly localized at a mean distance of 3.15±0.06gm from the nuclear center and measured 1–2 μm in diameter. Given a centromere complement of 40 per murine nucleus, the relatively low number of signals detected and their large signal volumes were interpreted to reflect clustering of centromeres, a phenomenon common in mammalian cells. An average of 37.00±1.52 tDNA signals was detected per nucleus. Of these, and in contrast to sDNA signals, only 18.45±0.41% of these signals were associated with the nucleolus while the remainder was distributed between the nucleolus and the nuclear membrane. Both centromeric and telomeric signals often occurred in pairs and were distributed throughout the nucleoplasm. No evidence for a classical Rabi configuration was found.
Intracellular localization of types i and ii collagen mrna and endoplasmic reticulum in embryonic corneal epithelia
Authors: Svoboda Kathy Kay Hartford 
Published: April 2021

ABSTRACT
The intracellular distribution of endoplasmic reticulum (ER) and types I and II collagen mRNA was analyzed in whole-mount preparations of freshly isolated corneal epithelia using in situ hybridization combined with confocal laser scanning analysis. The ER stained with DiOC6(3) was prominent in both the periderm and basal cells. The basal cell ER distribution was perinuclear in the center of the cells, but below the nucleus the ER occupied nearly all of the cytoplasm in a reticular pattern similar to that seen with TEM cross-sections. Initial single label in situ hybridization studies showed that both the periderm and basal cells were positive for both types I and II collagen mRNA. The collagen cDNA probes appeared perinuclear in the center of the basal cells, similar to the DiOC8(3) staining pattern. In doublelabeling experiments, the two mRNAs that translate chains of type I collagen, αl and α2, colocalized within the same cell. However, the hybridization of probes specific for type I and II collagen mRNAs had separate, but overlapping, distributions within the same cell.
Lepidopteran cell variants resistant to 5-bromodeoxyuridine and their use for transfection of the hsv-tk gene
Authors: Xie Weidong , He Daifen , Wang Xunzhang , Long Qingxin , Pu Zhelong 
Published: April 2021

ABSTRACT
Following mutagenesis of cultured lepidopteran cells (Spodoptera frugiperda) by ethylmethanesulfonate, three variants resistant to 5-bromodeoxyuridine (BrdUrd) were isolated. These clones were 100-to 200-fold more resistant to BrdUrd than the parental cells and were shown to be deficient in thymidine kinase (TK). The drug-resistant phenotype was stable for up to three years of culture under nonselective conditions. It was also found that the S. frugiperda cell line was highly resistant to aminopterin. A selective medium was formulated and used to select herpes simplex virus thymidine kinase (HSV-TK) transfectants from the TK-deficient cells.
Sea urchin egg villin: Identification of villin in a non-epithelial cell from an invertebrate species
Authors: Wang Feng-Song , Bonder Edward M. 
Published: April 2021

ABSTRACT
Fertilization of sea urchin eggs results in the rapid polymerization of actin filaments and subsequent formation of a brush border-like cortical cytoskeleton. A 110×103Mr (110K) actin binding protein has been purified from extracts of unfertilized Strongylo-centrotus purpuratus eggs. Analysis of polymerization kinetics using fluorescence and viscometry assays demonstrated that 110K accelerated the nucleation phase of actin assembly only in the presence of elevated Ca2+. The Ca2+-mediated effects were correlated with a decrease in sedimentable polymer and a decrease in average filament length. Addition of Ca2+ to solutions of 110K and F-actin, polymerized in the presence of EGTA, resulted in a precipitous drop in viscosity and the decreased viscosity was fully reversible upon chelation of Ca2+. The Ca2+ threshold for 110K activation was in the 10 −6 to 10 −7M range. Nucleated assembly experiments using Limulus sperm acrosomal processes demonstrated that egg 110K capped the barbed ends of actin filaments. In the absence of Ca2+, 110K organized actin filaments into bundles at pH values less than 7.4. Anti-egg 110K antibody crossreacted with chicken intestinal epithelial cell villin and antiporcine villin headpiece monoclonal antibody crossreacted with 110K. Further, 110K possesses an ∼10×10’3Mr terminal polypeptide segment that is immunologically related to villin headpiece. These studies demonstrate that sea urchin egg 110K is functionally, immunologically and structurally related to villin, an actin binding protein expressed in specific epithelial tissues in vertebrates. Consequently, this finding provides insight into the potential mechanisms that might determine the genesis of the cortical brush border cytoarchitecture in sea urchin eggs and further sheds light on the evolution of the villin protein family.
Mass isolation of cleavage furrows from dividing sea urchin eggs
Authors: Yonemura Shigenobu , Mabuchi Issei , Tsuktta Shoichiro 
Published: April 2021

ABSTRACT
To develop a mass isolation procedure for the cleavage furrow from synchronized sea urchin eggs, we compared the stability of the cleavage furrow with that of the rest of the cortex (polar-region cortex) and the inner cytoplasm under various conditions using the rhodamine-phalloidin staining method. As a result, to remove the polar-region cortex and leave the cleavage furrow intact, it became clear that the type and concentration of detergent, the pH and Ca concentration of the isolation solution and the temperature were of critical importance, and that 0.04–0.1% Nonidet P-40, pH 7.0–7.5, low calcium ion concentration and room temperature were optimal conditions. To solubilize the inner cytoplasm to release intact cleavage furrows, two factors, osmotic pressure and sea urchin species, were found to be important: 0.16 M glucose (or sucrose) was optimal, and we found Clypeaster japonicus to be the most appropriate. A shearing force, by gentle pipetting, was also required for furrow isolation. Taking these results into consideration, we have succeeded in developing a mass isolation procedure for cleavage furrow from C. japonicus. A total of 20–50 μg of protein of isolated cleavage furrow was recovered from 1 ml of packed dividing eggs. The structural integrity of the isolated cleavage furrow was well maintained and it was characterized by remnants of plasma membranes, actin filament meshwork including a contractile ring, and cytoplasmic vacuoles. Although the isolated furrow contained myosin II molecules, it showed no capability of in vitro reactivation.
Kinetochore function can be altered by ultraviolet microbeam irradiation without loss of the associated birefringent spindle fibre
Authors: Swedak Julia A. M. , Forer Arthur 
Published: April 2021

ABSTRACT
We have irradiated kinetochores of chromosomes in spermatocytes of crane flies (Nephrotoma abbreviata (Loew) and Nephrotoma suturalis (Loew)), while observing the cells using polarization microscopy. Irradiation of a kinetochore of one sex chromosome with 0.106 ergsμm−2, the minimum dose needed to stop movement, had no effect on the birefringence of the irradiated kinetochore’s spindle fibre. Irradiation of the kinetochore of an autosomal halfbivalent in anaphase, with the same dose, had no effect on the birefringence of the irradiated kinetochore’s spindle fibre, but nonetheless the anaphase movements of all six autosomal half-bivalents were stopped, temporarily, for up to 20 min. Irradiations of the kinetochores of an autosomal half-bivalent with higher doses (0.301 ergs μm-2) caused loss of birefringence of the irradiated kinetochore’s spindle fibre, and the movements of all six autosomal half-bivalents were stopped permanently. We argue that the ultraviolet microbeam differentially affects two functions of the kinetochore: (1) a ‘signalling’ function, and (2) microtubule attachment, with the signalling function being altered at doses lower than that of microtubule attachment.
Electron-microscopic and immunochemical analysis of kinetochore microtubules after ultraviolet microbeam irradiation of kinetochores
Authors: Swedak Julia A. M. , Leggiadro Cynthia , Forer Arthur 
Published: April 2021

ABSTRACT
We used an ultraviolet microbeam to irradiate kinetochores of chromosomes in crane-fly spermatocytes. We used one of two doses, low (0.106 ergμm−2) or high (0.301 ergμm−2), and then studied the microtubules in those spindles using electron microscopy or immunofluorescence microscopy. After irradiation with low doses microtubules are present as usual, with normal fluorescence and in normal numbers. After irradiation with high doses microtubules are no longer associated with the irradiated kinetochore. After irradiation with either dose, nonkinetochore microtubules are in smaller numbers in the irradiated half-spindle than in the non-irradiated half-spindle or in non-irradiated cells. Since irradiation with low doses alters interchromosomal ‘signals’, but microtubules remain attached to the kinetochore, we argue that low doses of ultraviolet light damage a signal-related function of kinetochores without altering the ability of the kinetochores to bind microtubules.
Direct experimental evidence for the existence, structural basis and function of astral forces during anaphase B in vivo
Authors: Aist James R. , Bayles Carol J. , Tao Wen , Berns Michael W. 
Published: April 2021

ABSTRACT
The existence, structural basis and function of astral forces that are active during anaphase B in the fungus, Nectria haematococca, were revealed by experiments performed on living cells. When one of the two asters of a mitotic apparatus was damaged, the entire mitotic apparatus migrated rapidly in the direction of the opposing astral forces, showing that the force that accelerated spindle pole body separation in earlier experiments is located in the asters. When a strong solution of the antimicrotubule drug, MBC, was applied at anaphase A, tubulin immunocytochemistry showed that both astral and spindle microtubules were destroyed completely in less than a minute. As a result, separation of the spindle pole bodies during anaphase B almost stopped. By contrast, disrupting only the spindle microtubules with a laser microbeam increased the rate of spindle pole body separation more than fourfold. Taken together, these two experiments show that the astral forces are microtubule-dependent. When only one of the two or three bundles of spindle microtubules was broken at very early anaphase B, most such diminished spindles elongated at a normal rate, whereas others elongated at an increased rate. This result suggests that only a critical mass or number of spindle microtubules needs be present for the rate of spindle elongation to be fully governed, and that astral forces can accelerate the elongation of a weakened or diminished spindle.
Calcium/calmodulin affects microtubule stability in lysed protoplasts
Authors: Cyr Richard J. 
Published: April 2021

ABSTRACT
Microtubules (Mts) are found in four distinct arrays appearing sequentially in a cell-cycle-dependent fashion within the cells of higher plants. Additionally, the cortical Mts of non-cycling cells are spatially altered in a variety of differentiated states. Information regarding the molecular details underlying these Mt-reorientation events in plant cells is scarce. Moreover, it is unclear how cytoskeletal behavior integrates with the myriad of other cellular activities that are altered concomitantly in both differentiating and cycling cells. Data are presented herein to indicate that calcium, in the form of a Ca2+/calmodulin complex, can alter the behavior of Mts in lysed carrot protoplasts. Mechanistically, we show that Ca2+/calmodulin most likely interacts with Mts via associations with microtubule associated proteins (MAPs). These results are discussed with reference to how Ca2+ may alter the dynamic behavior of Mts during growth and development.
Biosynthesis and transport of lysosomal α-glucosidase in the human colon carcinoma cell line Caco-2: Secretion from the apical surface
Authors: Klumperman Judith , Fransen Jack A. M. , Boekestijn Tine C. , Oude Elferink Ronald P. J. , Matter Karl , Hauri Hans-Peter , Tager Joseph M. , Ginsel Leo A. 
Published: April 2021

ABSTRACT
The human adenocarcinoma cell line Caco-2 was used for studies on the biosynthesis and transport of lysosomal acid α-glucosidase in polarized epithelial cells. Metabolic labelling revealed that in Caco-2 cells α-glucosidase is synthesized as a precursor form of 110 × 103Mr. This form is converted into a precursor of slightly higher Mr (112×103) by the addition of complex oligosaccharide chains. Via an intermediate form of 95×103Mr, this precursor is processed into a mature form of 76×103Mr.
Combination of metabolic labelling with subcellular fractionation showed that the 112×103Mr precursor of α-glucosidase is transported to the lysosomes. However, the same form is secreted into the culture medium (20% of newly synthesized enzyme after 4h of chase). Immunoprecipitation of α-glucosidase from culture medium derived from either the apical or basolateral site of radiolabelled Caco-2 cells, showed that 70–80% of the total amount of precursor form present in the medium is secreted from the apical membrane. Measurement of enzyme activities also showed that α-glucosidase, unlike other lysosomal enzymes, is mainly secreted via the apical pathway. Furthermore, immunocytochemistry showed the presence of a precursor form of α-glucosidase on the apical, but not the basolateral, membrane of the Caco-2 cells. We conclude that α-glucosidase is, unlike all other secretory proteins studied so far, secreted preferentially from the apical membrane of Caco-2 cells.
SPIF regulates spore coat protein synthesis by Dictyostelium discoideum at the translational level
Authors: Gibson F. P. , Schofield T. , Hames B. D. 
Published: April 2021

ABSTRACT
Spore coat protein synthesis in submerged pseudoplasmodia of Dictyostelium discoideum is dependent on the presence of a low relative molecular mass extracellular factor, SPIF, the activity of which can be mimicked by methionine. In vitro translation and northern blot analysis revealed that the level of spore coat protein mRNA in pseudoplasmodia incubated in the absence of methionine is little different from that in its presence. Furthermore, nogalamycin, a potent inhibitor of RNA synthesis, does not prevent the regulation of spore coat protein synthesis by methionine. These data suggest that the regulatory step is probably at the translational level. The proportion of total ribosomes associated in polysomes in pseudoplasmodia incubated in the absence of methionine is substantially lower than in its presence indicating a relative decrease in the number of translationally active mRNAs. However, measurements of the average polysome size and ribosome transit time in pseudoplasmodia initiated in the presence or absence of methionine show that the initiation rate of protein synthesis is essentially identical in both situations.