bluuebunny/crossref_metadata_2025 huggingface dataset:
The location of blood group antigen a on cultured rabbit kidney cells as revealed by ferritin-labelled antibody
Authors: Dimmock Elizabeth , Franks D. , Glauert Audrey M.
Published: April 2021

Fluorescein- and ferritin-labelled antibodies were used to locate blood group antigen A on the surfaces of cultured rabbit kidney cells (RK 13). Fluorescent anti-A revealed the antigen on some cells, but not on others. Controls indicated that specific staining could be obtained. Comparable observations were made with ferritin-labelled antibody, and the comparatively high resolution of this method allowed individual antigen molecules to be located. Measurements were made of the distances between the centres of the ferritin molecules and the cell surface, and the position of the blood group antigen relative to the visible components of the membrane is discussed in relation to this.
Experimental studies on the axons intrinsic to the glomerular layer of the olfactory bulb
Authors: Pinching A. J. , Powell T. P. S.
Published: April 2021

Lesions have been made in the superficial layers of the dorsal surface of the rat olfactory bulb; the resulting axonal degeneration has been studied with light and electron microscopy, in order to determine the mode and extent of interconnexion of the intrinsic interneurons of the glomerular layer. Although the lesions necessarily involve extrinsic pathways, the axonal distribution of each of the intrinsic neurons may be deduced from this evidence and a knowledge of normal and experimental material. Tufted cell collaterals extend for 6–12 glomeruli, periglomerular cell axons for 4–5 glomeruli and superficial short-axon cell axons for 2–3 glomeruli; these figures correspond well with observations of Golgi-impregnated material. The experimental approach to the study of intrinsic connexions and its applicability to other sites is considered, and the results are discussed from the point of view of neural coding and odour specificity.
Synthesis and localization of sulphated mucopolysaccharide in megakaryocytes and platelets of the rat, an analysis by electron-microscope autoradiography
Authors: Macpherson G. G.
Published: April 2021

Electron-microscope autoradiography has been used to investigate the synthesis and localization of sulphated mucopolysaccharide in megakaryocytes and blood platelets. Following 10-min incubation of bone marrow with 35S-sulphate in vitro the majority of the activity in megakaryocytes was associated with the Golgi apparatus, but a substantial proportion was associated with other cytoplasmic organelles, suggesting either rapid transport or sulphation of mucopolysaccharide outside the Golgi apparatus. Three hours after the intravenous injection of 35SO4 only a small proportion of the total activity was associated with the Golgi apparatus, most being associated with demarcation membranes and dense granules, while 12 h after injection almost all the activity was associated with demarcation membranes and granules. A rising proportion of activity localized solely on the demarcation membranes suggested that they may possess some activity of their own.

Autoradiographs of blood platelets prepared 72 h after the injection of 35SO4 were analysed. It was shown that most of the activity was associated with the a-granules, but there was strong evidence that the platelet membrane possessed a low level of activity.
Quantitative cytochemical observations on the control of respiration in polymorphonuclear neutrophil leucocytes of Amphiuma tridactylum
Authors: David G. B. , Mcmullen Jamie M.
Published: April 2021

Quantitative effects of altering oxidative phosphorylation and respiration on the activity of the enzyme menadione reductase (NAD[P]H,:2-methyl-1,4-naphthoquinone oxidoreductase, E.C. 1.6.5.2), in stabilized polymorphonuclear neutrophil leucocytes of Amphiunia tridactylum, were studied by amplitude-contrast microscopy and microspectrophotometry.

In a general way, the rate of enzymic activity was proportional to ADP concentration and inversely proportional to the concentration of ATP. Terminal respiratory blocking by azide produced selective subtotal inhibition. Uncoupling of phosphorylation by dinitrophenol produced complex results.

Neutrophils of A. tridactylum, irrespective of their stage of maturation in the circulating blood, could be subdivided into 3 metabolic classes: Class I cells, of low enzymic activity’ (predominantly mitochondrial), greatly activated by ADP, somewhat activated by ATP, and only slightly inhibited by dinitrophenol; Class II cells, twice as active as Class I (in which the endoplasmic reticulum and idiozome were as active as the mitochondria), further activated by ADP used alone or with dinitrophenol, and unaffected by ATP or dinitrophenol; Class III, hyperactive cells (enzymic localization identical with that in Class II), inhibited by ATP and dinitrophenol, and not activated by ADP. Some of the mitochondria of Class III neutrophils retained nearly a third of their reductase activity when the reaction mixture contained 10−1M azide.

There is reason to believe that Class I neutrophils may form a reserve population of vegetative cells; in vitro, they can be transformed into Class II cells when a high concentration of ADP is added. Class II and Class III cells are potentially capable of amoeboid movement and phagocytosis. The metabolic mobilization of neutrophils could be interpreted as being controlled by 2 different feedback mechanisms: activation by ADP in cells of Classes I and II, and inhibition by ATP in cells of Class III.
Aggregation of burkitt lymphoma cells in stationary culture: experimental and theoretical analysis
Authors: Mayhew E. , Blumenson L. E.
Published: April 2021

Cellular motility plays an important role in natural aggregative phenomena but previously’ has been difficult to quantitate. We here describe a general method which can be used to determine the importance of cell motility in cell aggregation behaviour.

When actively moving Burkitt lymphoma cells cultured in microtest plate wells come into contact they adhere to one another forming an aggregate. The aggregate increases in size when more cells come in contact with it. The final size and number of aggregates per well was found to be dependent on the number of cells added per well. With increasing cell numbers added per well the number of aggregates formed increased until it reached a peak of 47–57 aggregates per well 20 h after the addition of 180–710 cells per well. At higher cell concentrations the number of aggregates formed decreased. The system was analysed theoretically by programming a computer to simulate the experimental system. This simulation showed it was probable that the experimental results obtained were due to (a) random dropping of the cells at zero time and (b) the adhesion of the cells when they made contact and (c) unrestricted random movement of cells in the well when they reached the well surface. The computer simulation is such that given the experimentally determined cell concentration per well and rate of cell movement we can predict the number of aggregates formed for different probabilities that the adhesions formed after the cells come in contact are permanent. This experimental approach along with the computer simulation can be used to quantitate the role of cell motility and permanence of contact in cell aggregation.
Studies on chemically induced cell fusion
Authors: Ahkong Q. F. , Cramp F. C. , Fisher D. , Howell J. I. , Lucy J. A.
Published: April 2021

Hen erythrocytes that were fixed after treatment with lysolecithin in aqueous solution for 30 s at 37 °C showed evidence of bridge formation between adjacent lysed cells. Generally, the homokaryons that were produced using lysolecithin in this way contained large numbers of nuclei. These giant syncytia had damaged nuclear membranes and unstable plasma membranes; complete disintegration of the syncytia occurred within 1 min of adding lysolecithin to the erythrocytes.

In order to localize the action of lysolecithin, the fusing agent was incorporated into microdroplets of lipid. Cell fusion following the addition of lysolecithin in an aqueous glyceride-lecithin emulsion was slower than with lysolecithin in aqueous solution, taking 10–30 min, and it was accompanied by considerably less damage to the plasma and nuclear membranes. The fused erythrocytes, which usually contained only two or three nuclei, lysed slowly during the 45 min following fusion, and lysis could be arrested by cooling the fused cells. The plasma membranes of lysed, multinucleated cells remained intact at 37 °C for at least 90 h.

Mouse fibroblast-hen erythrocyte heterokaryons formed with the aid of the emulsion were more stable than those produced with lysolecithin in solution, but the hybrid cells nevertheless had damaged subcellular organelles. Viable clones of hybrid mouse-hamster fibroblast cells were obtained using the emulsion although, possibly owing to reduced viability of the lysole-cithin-treated cells, only at twice the frequency of spontaneously produced hybrids.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Production of matrix metalloproteinase 3 (stromelysin) by cultured ovine endometrial cells
Authors: Salamonsen Lois A. , Nagase Hideaki , Woolley David E.
Published: April 2021

Ovine endometrial cells (stromal plus epithelial) in primary culture release matrix metalloproteinase 3 (stromelysin, MMP-3), the enzyme that degrades various extracellular components including the basement membrane components collagen IV and laminin. The enzyme was detected in tissue culture medium by specific enzyme assay. A low level of MMP-3 activity was released from the cells (0.12±0.02 unit per 106 cells per 48 h; 1 unit degrades l/<g of reduced, carboxymethylated transferrin min−1 at 37 °C), but following stimulation by phorbol myristate acetate (PMA) a mean 4.4-fold increase in MMP-3 production was observed (to 0.53±0.13 unit per 108 cells per 48 h). The enzyme was released primarily in the form of its proenzyme, which could be activated in vitro by (4-aminophenyl)mercuric acetate (APMA).

Stimulation of enzyme release from cells by PMA was not seen before 24 h and required PMA at a dose of 10 nM or higher. This finding was substantiated by Western blot analysis. Immunocytochemistry of cells that had been treated with PMA for 48 h, and with the ionophore, monensin, for 4 h, confirmed the potential of the endometrial cells to produce stromelysin. The release of MMP-3 from ovine endometrial cells suggests that this enzyme may have a role in assisting the passage of syncytial processes through the basement membrane during the process of implantation.
Basket scales of the green alga, Mesostigma viride: Chemistry and ultrastructure
Authors: Domozych David S. , Wells Brian , Shaw Peter J.
Published: April 2021

The unicellular green algal flagellate, Mesostigma viride, possesses an extracellular matrix consisting of three layers of highly distinct scales. This study focused upon the elaborate basket scales. The basket scale is approximately 450–500 nm in height and consists of a solid base, two distinct lattices, two upper rims and various struts and roots. Pure preparations of isolated basket scales were obtained and used for subsequent chemical and immunological analyses. X-ray microanalyses revealed the scale as being mineralized with both calcium and phosphorus. Fourier Transform Infrared Spectroscopic analyses revealed the presence of phosphate, suggesting in turn that the basket scale is complexed with calcium phosphate. SDS-gel electrophoresis of partially digested scales revealed the presence of two protein components, a high Mr, non-migrating form and a form with a Mr of approximately 48 000. Further biochemical analyses of scales showed the presence of two major sugars: glucose and the unusual keto sugar acid, 3-deoxy-lyxo-2-heptulosaric acid, or DHA. Antibodies to scales were raised in rats and used in immunolabeling studies. It is estimated from immunofluorescence studies that a cell of 8/cm contains about 800 basket scales. The scales are in a close-packed arrangement resulting in quasi-crystalline arrays upon the cell surface.
Bone-marrow-derived chondrogenesis in vitro
Authors: Berry L. , Grant M. E. , McClure J. , Rooney P.
Published: April 2021

Bone marrow stromal cells from embryonic, neo-natal and adult chickens were grown in vitro over a 21-day period. Marrow stromal cells from embryonic and neonatal chicks produced clonally derived chondrocytic colonies. The cells within the colonies were surrounded by a refractile, Alcian-blue-positive matrix and their cartilagenous nature was shown biochemically and immunocytochemically by the synthesis of collagen types n and X. The ability of chick bone marrow cells to form chondrocytic colonies decreased during development and was lost by adulthood. In addition to chondrocytic colonies, fat cells and fibroblasts were also observed in the cultures.

Our data demonstrate that chick bone marrow stroma contains cells that are capable of differentiating along different pathways Within the same culture, providing further evidence for the presence in bone marrow of a stromal stem cell.
Vimentin expression as a late event in the in vitro differentiation of human promonocytic cells
Authors: Rius Carlos , Aller Patricio
Published: April 2021

The administration of either 12-O-tetradeeanoyl phor-bol-13-acetate (TPA, 3xlO−8M), sodium butyrate (SB, 10−3M), A6,2’-O-dibutyryladenosine-3’:5’-cyclic monophosphate (dbcAMP, 10−3M), cytosine arabinoside (ara-C, 10−7M), amsacrine (mAMSA, 10−7M) or retinoic acid (RA, 10−6M) inhibits the growth activity of human promonocytic U-937 cells, by arresting them at G1 or at the G1/S border (SB, RA, ara-C), at G2 (mAMSA) or at G1 and G2 (dbcAMP). All these agents trigger cell differentiation, as proved by the increased expression of the maturation-associated CDllb and CD 11c surface antigens, and induce the expression of the vimentin gene at both the protein and the mRNA levels. TPA, SB and dbcAMP behave as “early” inducers, in the sense that vimentin mRNA levels are rapidly increased (hour 6) upon drug administration. In contrast, mAMSA and RA behave as “late” inducers, since they do not increase vimentin mRNA levels until 48 to 72 hours, following the stimulation of surface antigen expression. The action of RA is characterized by an initial inhibition period, in which the basal level of vimentin mRNA is abolished (hour 24). Nevertheless, this RNA is later re-induced, to reach at 72 hours higher levels than in untreated cells. Moreover, RA is capable of delaying the early induction of vimentin expression caused by TPA and SB, without affecting the normal expression of differentiation markers. Taken together, these results strongly suggest that vimentin expression is not required at the initial stages of promonocytic cell differentiation, although it could play a role at an advanced stage.
Organisation of glycoproteins into plasma membrane domains on Fucus serratas eggs
Authors: Stafford C. J. , Green J. R. , Callow J. A.
Published: April 2021

Fertilisation in the brown alga Fucus involves the interaction of motile, biflagellate sperm with spherical eggs. The eggs differ from those of animals in not having the equivalent of a vitelline layer, jelly coat or zona pellucida outside the plasma membrane, and in addition they are not surrounded by a cell wall. Previous studies on Fucus eggs have shown that the lectin Concanavalin A (ConA) binds in patches on the egg surface, suggesting that there is a non-uniform distribution of plasma membrane glycoproteins. In this paper we have further investigated the occurrence of domains on the Fucus egg plasma membrane using monoclonal antibodies (mAbs) and the lectins ConA and Fucose Binding Protein (FBP). Confocal laser scanning microscopy (CLSM) has been used to observe the binding of probes to the Fucus egg cell surface. Four mAbs (FS2, FS4, FS5 and FS9) raised to Fucus serratas sperm have previously been shown to cross-react with crude egg membrane vesicles. Three of these mAbs (FS2, FS4 and FS5) have now been shown to bind to the egg cell surface and they recognise glycoproteins which are organised into domains. mAb FS4 labels large areas of the egg surface, whereas mAbs FS2 and FS5 bind to smaller patches. The lectins ConA and FBP also bind to smaller, discrete domains. Western blotting results and competition binding assays have shown that mAbs FS2 and FS5 compete for binding to the same set of glycoproteins, and FS5 is used in subsequent experiments; FS4 also binds to several glycoproteins but produces a different pattern of labelling on Western blots compared to FS5, though there may be some common components. ConA labels a subset of the glycoproteins recognised by mAb FS5, and FBP recognises one major glycoprotein which is also recognised by ConA and FS5. Double labelling experiments using the CLSM, with FITC- and Au-labelled probes, have shown that the regions on the egg surface labelled by FS4 and FS5 are mainly exclusive, with small areas of overlap. There are also areas which are not labelled by either of these antibodies. The domains recognised by mAb FS5 contain smaller areas which are labelled by ConA. Overall the results show that the Fucus egg surface is heterogeneous with different sets of glycoproteins being organised into domains. With the probes used it is possible to distinguish between FS4+ FS5−, FS4− FS5+, FS4+ FS5+ and FS4− FS5” regions. Within the FS5+ domains smaller sets of glycoproteins are recognised by ConA and within these latter regions there are glycoproteins recognised by FBP.
Histamine decreases the permeability of an endothelial cell monolayer by stimulating cyclic AMP production through the H2-receptor
Authors: Takeda Taichi , Yamashita Yuko , Shimazaki Syuji , Mitsui Youji
Published: April 2021

To determine if histamine acts directly on the vascular endothelium, the effect of histamine on the permeability of cultured human endothelial cell monolayers and the role of second messengers were examined. The addition of 10−6 to 10−4 M histamine to the culture medium decreased the endothelial cell monolayer permeability and increased both cyclic AMP and free-calcium levels. The decrease in permeability and the increase in cyclic AMP mediated by histamine were prevented by an H2-blocker (famotidine) while the increase in free-calcium was inhibited by an H1-blocker (diphenhydramine). These results suggest that histamine decreases the permeability of endothelial cell monolayers through the H2-receptor, and cyclic AMP plays a more important role than calcium ion as a second messenger.
A monoclonal antibody recognizing nuclear matrix-associated nuclear bodies
Authors: Stuurman Nico , Graaf Arjan De , Floore Arno , Josso Arthur , Humbel Bruno , Jong Luitzen De , Driel Roel Van
Published: April 2021

We have isolated a monoclonal antibody, 5E10, that labels discrete spots in the interphase nucleus. By immunoblotting mAb SE10 recognized predominantly a 126 kDa polypeptide with an isoelectric point of 5.5. Indirect immunofluorescence showed that mAb 5E10 labeled spots in many cell lines and tissues from rat or human origin, but not in cells from mouse, chicken, African green monkey, or the lower eukaryotes Saccharomyces and Dictyostelium. In the human bladder carcinoma cell line T24 the number of nuclear spots was found to be 21 ± 10 (n=132). In many cells spots were found also in the cytoplasm. In a small fraction of T24 cells the mAb revealed thread-like structures in addition to spots. Throughout mitosis the antigen was found to be clustered in the cytoplasm, not associated with metaphase chromosomes. The spherical structures that contain the antigen were tightly bound to the nuclear matrix. Immunogold labeling with mAb 5E10 showed that the antigen is localized in 0.3 /mi diameter spherical, electron-dense structures, reminiscent of nuclear bodies. Double-labeling experiments showed that these spots do not colocalize with U1 snRNPs and centromeres. The spots did colocalize with nuclear speckles recognized by a primary biliary cirrhosis autoimmune serum, which is thought to recognize nuclear bodies. On the basis of these observations we conclude that mAb 5E10 recognizes discrete nuclear substructures, most likely nuclear bodies.
Tubulin in sea urchin embryonic cilia: post-translational modifications during regeneration
Authors: Stephens R. E.
Published: April 2021

Tubulin is the major protein found in the membrane/periaxonemal matrix fraction of mature sea urchin embryonic cilia but its distribution and possible function during ciliary assembly are unknown. Hypertonic salt may be used to deciliate the embryos, allowing synchronous regrowth of cilia and subsequent deciliation of the regenerating embryos at various times. During the earliest stages of regeneration, the amounts of tubulin in the axoneme and membrane/matrix fractions are nearly equal, but the proportion of tubulin in the axoneme fraction increases coincident with the quasi-linear growth phase while the membrane/matrix tubulin remains constant. Antibodies to tyrosinated and detyro-sinated α-tubulin show that both the membrane/matrix and axonemal tubulin fractions are primarily unmodified (i.e. tyrosinated) at the earliest stages of regeneration but are progressively and equally detyrosinated coincident with regeneration, approaching a final level of 50% C-terminal Glu. A monoclonal antibody to acetylated a-tubulin reveals that both tubulin fractions are equally and maximally acetylated at relatively early stages of regeneration. In contrast, three-times-repoly-merized tubulin from either unfertilized eggs or mid-gastrula embryos is primarily tyrosinated (>97%) and not detectably acetylated. These data suggest that membrane/matrix tubulin is a precursor to axonemal tubulin and that acetylation and detyrosination may be involved in partitioning tubulin among cytoplasmic, ciliary membrane/matrix, and 9+2 compartments.
Tripeptide RGD-dependent adhesion of articular chondrocytes to synovial fibroblasts
Authors: Ramachandrula Anand , Tiku Katherine , Tiku Moti L.
Published: April 2021

Cell-cell interactions play an important role in the development of cartilage. Heterologous and homologous cell-cell interactions are critical for chondrogenic differentiation during development. Cell-cell interactions in the formation of fracture callus and cartilage neoplasia also invoke the process of cartilage differentation. We have investigated cell-cell interactions between articular chondrocytes and synovial fibroblasts and show that there was enhanced binding between these two cell types compared to background binding of the labelled cells to the tissue culture plastic surface. The binding of chondrocytes to fibroblasts was temperature- and calcium-dependent, suggesting ligand-integrin involvement. The peptide, GRGDSP, which competes with the ligand-integrin through the tripeptide RGD (arginineglycine-aspartic acid), almost completely inhibited chondrocyte attachment to synovial fibroblasts. The control peptide, GRGESP, had no Inhibitory effect on binding. Antibodies to fibronectin (Fn) inhibited chondrocyte attachment by about 50%. Monoclonal antibodies to the α and β chains of the fibronectin receptor (FnR) interfered with the attachment of chondrocytes to synovial fibroblasts. A combination of antibodies to Fn and to FnR did not completely abrogate chondrocyte binding, suggesting that other ligand-receptors were involved in the adhesion process. Chondrocytes and fibroblasts were shown to express membrane-associated Fn and FnR, by immunofluorescence. The α and β chains of FnR, migrating at 110 and 140 kDa, respectively, could be immunoprecipitated from [35S]methionine-labelled synovial fibroblasts and chondrocytes. Northern blots showed the presence of mRNA for the α and β chains of fibronectin receptors in fibroblasts and chondrocytes. Changes in cell shape were observed in chondrocytes on attachment to fibroblasts, i.e. the chondrocytes appeared fibroblast-like, suggesting that the chondrocytes had dedifferentiated. These studies suggest that chondrocytes specifically bind to synovial fibroblasts through RGD-dependent receptors. β1 Integrins are involved in this adhesion process and these heterlogous cell interactions appear to have a negative influence on chondrogenic differentiation.