bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of intermediate vesicles in the adsorptive endocytosis and transport of ligand to lysosomes by human fibroblasts.
Authors: Merion M , Sly W S
Published: May 2004

Recent work from several laboratories has suggested the participation of intermediate structures in the delivery of adsorbed ligands from the plasma membrane to lysosomes. This report presents subcellular fractionation studies bearing on the role of these structures in adsorptive pinocytosis of epidermal growth factor (EGF), beta-hexosaminidase, and low density lipoprotein (LDL) by human fibroblasts. Using a two-step Percoll density gradient fractionation, we identified newly internalized (5 min) EGF in two intermediate density structures that are essentially negative for plasma membrane marker, and more bouyant than secondary lysosomes. Continued incubation for 20 min resulted in transfer to (or conversion to) vesicles sedimenting with secondary lysosomes. Internalized beta-hexosaminidase and LDL behaved similarly, appearing first in structures of intermediate density, and later appearing in association with secondary lysosomes. Two drugs, NH4Cl and monensin, were found to inhibit ligand transfer to the secondary lysosome peak, although they did not inhibit entry of bound ligands into intermediate density structures. Upon removal of both inhibitors, internalized ligands were quickly transferred to the secondary lysosome peak. This "transfer process" was faster for EGF, than for the other two ligands studied. We interpret these data to indicate that the endocytosis of these three ligands, and their delivery to lysosomes in fibroblasts, proceeds through a common pathway, involving intermediate nonlysosomal structures.
Structure and molecular weight of the dynein ATPase
Authors: Johnson KA , Wall JS
Published: May 2004

Dynein has been examined by scanning transmission electron microscopy (STEM). Samples of 30S dynein from tetrahymena cilia were applied to carbon films and either were freeze- dried and examined as unstained, unfixed specimens, or were negatively stained with uranyl sulfate. A totally new image of the dynein molecule was revealed showing three globular heads connected by three separate strands to a common base. Two of the heads appeared to be identical and exhibited a diameter of 10 nm while the third head was somewhat larger (approximately 12 nm). The overall length of the particle was 35 nm. Mass analysis, based upon the integration of electron scattering intensities for unstained particles, gave a molecular weight of 1.95 (+/-)0.24) megadaltons. Mass per unit length analysis was performed using bovine brain microtubules decorated with dynein under conditions where the dynein shows a linear repeat of 24 nm with seven dynein molecules surrounding a microtubule made up of 14 protofilaments. Undecorated microtubules gave a molecular weight per unit length of 21,000+/-1,900 daltons/A, compared to a value of 84,400+/-2,200 daltons/A for the fully decorated microtubules. Taken together, these data give a molecular weight of 2.17 (+/- 0.14) megadaltons per dynein molecule, in agreement with measurements on the isolated particles. Mass analysis of individual globular heads observed in isolated particles gave a molecular weight distribution with a mean of 416+/- 76 kdaltons. These data could also be viewed as the sum of two populations of head with two-thirds of the heads at approximately 400 kdaltons and one-third at approximately 550 kdaltons, although more precise data will be required to distinguish two classes of heads with confidence. The mass of the dynein-microtubule complex as a function of distance from the midline of the particle was analysed to distinguish which end of the dynein molecule was bound to the microtubule. The projected mass distribution was consistent with a model where the three dynein heads were oriented toward the microtubule and clearly not consistent with the opposite orientation. These data indicate that the three globular heads form the ATP-sensitive site in this heterologous dynein-microtubule system and suggest that the rootlike base of the dynein molecule forms the structural attachment site to the A-subfiber of the outer doublet in cilia and flagella. The structure and function of the dynein are dicussed in terms of these new results.
Inhibition of contraction of cultured muscle fibers results in increased turnover of myofibrillar proteins but not of intermediate-filament proteins.
Authors: Crisona N J , Strohman R C
Published: May 2004

Muscle fibers are maintained in culture in a fully contractile state and are relaxed by the addition of 10(-7) M tetrodotoxin (TTX). This toxin binds to muscle membrane Na+- channels, abolishes spontaneous contractions and causes failure of the fiber to accumulate myosin heavy chains. These effects are reversible on removal of TTX. Synthesis and accumulation kinetics have been obtained for myofibrillar and for cytoplasmic filament proteins in normal, active muscle and in TTX-relaxed muscle fibers in culture. In relaxed fibers the synthesis of most proteins remained normal or slightly elevated. However, the accumulation of all myofibrillar proteins examined was markedly inhibited in TTX-treated cultures, whereas the accumulation of cytoplasmic filament proteins was normal or slightly elevated. Myofibrillar proteins examined were alpha-actin, troponin-C, myosin fast light chain 1, myosin fast light chain 2, alpha, beta-tropomyosins and the phosphorylated forms of tropomyosin and fast light chain 2. Cytoplasmic filament proteins studied were vimentin, alpha, beta-desmin and beta, alpha-actin. We also examined the synthesis and accumulation of six unidentified muscle-specific proteins and nine unidentified nonmuscle-specific proteins. Most of these proteins showed a normal accumulation pattern in TTX-relaxed fibers. We concluded that muscle fibers made inactive by TTX display an increased instability of all myofibrillar proteins while cytoplasmic filament proteins and cytoplasmic proteins in general are relatively unaffected. We suggest that TTX interferes, in a manner as yet unidentified, with assembly and normal stability of myofibrils. Decreased assembly and/or increased instability of myofibrils would lead to increased rates of myofibrillar protein degradation.
Distribution of actin in spreading macrophages: a comparative study on living and fixed cells
Authors: Amato PA , Unanue ER , Taylor DL
Published: May 2004

The distribution of actin in proteose peptone-elicited murine peritoneal macrophages is examined with fluorescent analog cytochemistry (FAC), immunofluorescence, and electron microscopy (EM). Living adherent macrophages, microinjected with 5- iodoacetamidofluorescence-labeled actin, show a rather uniform distribution of actin with punctuate and linear fluorescence in the thin peripheral areas of the cell. Apparent incorporation of a portion of linear fluorescence in the thin peripheral areas of the cell. Apparent incorporation of a portion of the microinjected actin into the cell's actin cytoskeleton is also demonstrated when microinjected cells are subsequently examined for fluorescein fluorescence after fixation and extraction. However, a substantial perinuclear pool of actin, observed with FAC, is lost when microinjected cells are prepared for immunofluorescence using standard fixation methods. These results suggest that part of the cellular actin, possibly nonfilamentous or oligomeric, can be extracted during the normal preparative steps for immunofluorescence. When the dynamic distributin of actin structures is examined in living cells, extension of the cell's periphery is associated with the formation of punctuate structures. The distribution of the most stable, nonextractable actin structures in fixed cells at different stages of spreading is quantified using rhodamine-labeled phalloidin and antiactin indirect immunofluorescence. At early stages, the rounded cells show cortical bands of fluorescence surrounding the nuclear region with punctuate structures directly above the plane of the attached plasma membrane. At later time periods, fully spread cells contain both punctuate and linear fluorescent structures. Adherent macrophage membranes, a preparation in which the attached membrane and membrane-cortex are isolated by shearing away the unattached plasma membrane and underlying cytoplasm, show punctuate and linear fluorescence when stained with rhodamine-labeled phalloidin. When the same cell remnant is negatively stained and examined with EM, the fluorescent punctuate structures coincide with electron-dense foci and associated radiating thin filaments. We suggest that the optimal approach for elucidating the distribution of cytoskeletal and contractile proteins involved in motile processes is a combined approach using all three techniques. Although each technique is subject to potential artifacts and limitations, the use of FAC can permit the visualization of both the soluble and stabilized components of the cytoskeleton in living, functional cells. A qualitative method for determining differences in local concentrations of proteins is also presented.
Microtubule-organizing centers abnormal in number, structure, and nucleating activity in x-irradiated mammalian cells.
Authors: Sato C , Kuriyama R , Nishizawa K
Published: May 2004

Microtubule-organizing centers (MTOCs) in x-irradiated cells were visualized by immunofluorescence using antibody against tubulin. From two to ten reassembly sites of microtubules appeared after microtubule depolymerization at low temperature in an irradiated mitotic cell, in contrast to nonirradiated mitotic cells, which predominantly show 2 MTOCs. A time-course examination of MTOCs in synchronously cultured cells revealed that the multiple MTOCs appeared not immediately after irradiation but at the time of mitosis. Those multiple MTOCs formed at mitosis were inherited by the daughter cells in the next generation. The structure and capacity of the centrosomes to nucleate microtubules in vitro were then examined by electron microscopy of whole-mount preparations as well as by dark-field microscopy. About 70-80% of the centrosomes derived from nonirradiated cells were composed of a pair of centrioles and pericentriolar material, which initiated greater than 100 microtubules. The fraction of fully active complete centrosomes decreased with time of incubation after irradiation. These were replaced by disintegrated centrosomal components such as dissociated centrioles and pericentriolar cloud, a nucleating site with a single centriole, or only an amorphous structure of pericentriolar cloud. Assembly of less than 20 microtubules onto the amorphous cloud without centrioles was seen in 54% of the initiating sites in mitotic cells 2 d after irradiation. These results suggest that x-irradiation causes disintegration of centrosomes at mitosis when the structural and functional reorganization of centrosomes is believed to occur.
Estimation of the membrane potential of cultured macrophages from the fast potential transient upon microelectrode entry
Authors: Ince C , Ypey DL , Van Furth R , Verveen AA
Published: May 2004

Analysis of membrane potential recordings upon microelectrode impalement of four types of macrophages (cell lines P388D1 and PU5-1.8, cultured mouse peritoneal macrophages, and cultured human monocytes) reveals that these cells have membrane potentials at least two times more negative than sustained potential values (E(s)) frequently reported. Upon microelectrode entry into the cell (P388D1), the recorded potential drops to a peak value (E(p)) (mean -37 mV for 50 cells, range -15 to -70 mV) within 2 ms, after which it decays to a depolarized potential (E(n)) (mean -12 mV) in about 20 ms. Thereafter, the membrane develops one or a series of slow hyperpolarizations before a final sustained membrane potential (E(s)) (mean -14 mV, range -5 to -40) is established. The mean value of the peak of the first hyperpolarization (E(h)) is -30 mV (range -10 to -55 mV). The initial fast peak transient, measured upon microelectrode entry, was first described and analyzed by Lassen et al. (Lassen, U.V., A.M. T. Nielson, L. Pape, and L. O. Simonsen, 1971, J. Membr. Biol. 6:269-288 for other change in the membrane potential from its real value before impalement to a sustained depolarized value. This was shown to be true for macrophages by two-electrode impalements of single cells. Values of E(p), E(n), E(h), E(s), and membrane resistance (R(m)) measured for the other macrophages were similar to those of P388D1. From these results we conclude that E(p) is a better estimate of the true membrane potential of macrophages than E(s), and that the slow hyperpolarizations upon impalement should be regarded as transient repolarizations back to the original membrane potentials. Thus, analysis of the initial fast impalement transient can be a valuable aid in the estimation of the membrane potential of various sorts of small isolated cells by microelectrodes.
Actin filaments, stereocilia, and hair cells of the bird cochlea. I. Length, number, width, and distribution of stereocilia of each hair cell are related to the position of the hair cell on the cochlea.
Authors: Tilney L G , Saunders J C
Published: May 2004

Located on the sensory epithelium of the sickle-shaped cochlea of a 7- to 10-d-old chick are approximately 5,000 hair cells. When the apical surface of these cell is examined by scanning microscopy, we find that the length, number, width, and distribution of the stereocilia on each hair cell are predetermined. Thus, a hair cell located at the distal end of the cochlea has 50 stereocilia, the longest of which are 5.5 microns in length and 0.12 microns in width, while those at the proximal end number 300 and are maximally 1.5 microns in length and 0.2 micron in width. In fact, if we travel along the cochlea from its distal to proximal end, we see that the stereocilia on successive hair cells gradually increase in number and width, yet decrease in length. Also, if we look transversely across the cochlea where adjacent hair cells have the same length and number of stereocilia (they are the same distance from the distal end of the cochlea), we find that the stereocilia of successive hair cells become thinner and that the apical surface area of the hair cell proper, not including the stereocilia, decreases from a maximum of 80 microns2 to 15 microns2. Thus, if we are told the length of the longest stereocilium on a hair cell and the width of that stereocilium, we can pinpoint the position of that hair cell on the cochlea in two axes. Likewise, if we are told the number of stereocilia and the apical surface of a hair cell, we can pinpoint the location of that cell in two axes. The distribution of the stereocilia on the apical surface of the cell is also precisely determined. More specifically, the stereocilia are hexagonally packed and this hexagonal lattice is precisely positioned relative to the kinocilium. Because of the precision with which individual hair cells regulate the length, width, number, and distribution of their cell extensions, we have a magnificent object with which to ask questions about how actin filaments that are present within the cell are regulated. Equally interesting is that the gradient in stereociliary length, number, width, and distribution may play an important role in frequency discrimination in the cochlea. This conclusion is amplified by the information presented in the accompanying paper (Tilney, L.G., E.H. Egelman, D.J. DeRosier, and J.C. Saunders, 1983, J. Cell Biol., 96:822-834) on the packing of actin filaments in this stereocilia.
Actin filaments, stereocilia, and hair cells of the bird cochlea. II. Packing of actin filaments in the stereocilia and in the cuticular plate and what happens to the organization when the stereocilia are bent.
Authors: Tilney L G , Egelman E H , DeRosier D J , Saunder J C
Published: May 2004

A comparison of hair cells from different parts of the cochlea reveals the same organization of actin filaments; the elements that vary are the length and number of the filaments. Thin sections of stereocilia reveal that the actin filaments are hexagonally packed and from diffraction patterns of these sections we found that the actin filaments are aligned such that the crossover points of adjacent actin filaments are in register. As a result, the cross-bridges that connect adjacent actin filaments are easily seen in longitudinal sections. The cross-bridges appear as regularly spaced bands that are perpendicular to the axis of the stereocilium. Particularly interesting is that, unlike what one might predict, when a stereocilium is bent or displaced, as might occur during stimulation by sound, the actin filaments are not compressed or stretched but slide past one another so that the bridges become tilted relative to the long axis of the actin filament bundle. In the images of bent bundles, the bands of cross-bridges are then tilted off perpendicular to the stereocilium axis. When the stereocilium is bent at its base, all cross-bridges in the stereocilium are affected. Thus, resistance to bending or displacement must be property of the number of bridges present, which in turn is a function of the number of actin filaments present and their respective lengths. Since hair cells in different parts of the cochlea have stereocilia of different, yet predictable lengths and widths, this means that the force needed to displace the stereocilia of hair cells located at different regions of the cochlea will not be the same. This suggests that fine tuning of the hair cells must be a built-in property of the stereocilia. Perhaps its physiological vulnerability may result from changes of stereociliary structure.
Effect of colchicine on internalization of prolactin in female rat liver: an in vivo radioautographic study.
Authors: Bergeron J J , Resch L , Rachubinski R , Patel B A , Posner B I
Published: May 2004

Binding and internalization of 125I-ovine prolactin into hepatocytes of female rats was visualized by the in vivo radioautographic method (Bergeron, J. J. M., G. Levine, R. Sikstrom, D. O'Shaughnessey, B. Kopriwa, N. J. Nadler, and B. I. Posner, 1977, Proc. Natl. Acad. Sci. USA, 745:051-5055). Receptor-mediated internalization of label was observed into lipoprotein-filled vesicles in the Golgi/bile canalicular region of the hepatocyte. Colchicine treatment had no effect on the internalization of label into the lipoprotein-filled vesicles. However, the location of the radio-labeled lipoprotein-filled vesicles was altered from the Golgi/bile canalicular region to subsinusoidal. Radioactive content of hepatocytes decreased as a function of time after injection of 125I-prolactin; however, colchicine treatment markedly retarded this loss of label. Subcellular fractionation experiments indicated that colchicine treatment led to decreased levels of 125I-prolactin accumulation in microsomes but augmented the accumulation of label in the L fraction. It is concluded that in normal female rats prolactin is internalized into lipoprotein-filled vesicles in the Golgi region before degradation of the hormone. Colchicine treatment accumulates labeled lipoprotein-containing vesicles in a subsinusoidal region and retards hormone catabolism. The labeled vesicles observed after colchicine treatment may correspond to the unique vesicles previously observed in the L fraction and found to be enriched in prolactin receptors (Khan, M. N., B. I. Posner, A. K. Verma, R. J. Khan, and J. J. M. Bergeron, 1981, Proc. Natl. Acad. Sci. USA, 78:4980-4981).
Mitotic architecture of the cell: the filament networks of the nucleus and cytoplasm.
Authors: Capco D G , Penman S
Published: May 2004

The skeletal framework of cells at the various stages of mitosis are prepared by extraction with nonionic detergent and examined by stereoscopic whole mount electron microscopy. The insoluble filament network remaining after the detergent-extraction and the depolymerization of microtubules is shown. The nonchromatin filament network of the nucleus, or nuclear matrix, becomes visible as the chromatin condenses at prophase. Filaments are associated with the chromosomes throughout mitosis. Parts of the chromosomes are associated with or are near the nuclear lamina at early stages. The nuclear lamina disappears at metaphase while chromosomes remain associated with filaments now continuous with the cytoplasmic network. Microtubules appear to be unnecessary for maintaining the chromosome position in these preparations since comparison of cells with and without microtubules shows no gross change in chromosome arrangement. The cellular filament network at metaphase and anaphase appears continuous from the plasma lamina to the chromosomes. The filament networks visualized here may be responsible for the prometaphase chromosome movement and participate in the formation of the midbody.
Purification of morphologically intact triad structures from skeletal muscle.
Authors: Mitchell R D , Palade P , Fleischer S
Published: May 2004

A procedure has been devised for isolation of triads (t-tubule/sarcoplasmic reticulum (SR) junctional complexes) from rabbit skeletal muscle. The procedure consists of preparation of a heavy microsomal fraction followed by two sequential 90-min sucrose gradient centrifugations to enrich the triads. A pyrophosphate/phosphate/magnesium buffer system was introduced to decrease aggregation in order to achieve effective separation. The preparation time is 12 h. Some differences between purified triads isolated by two variants of this method are noted. The purity of the triad fractions has been estimated by particle counting to be in the vicinity of 50%. There is good retention of morphology and Ca++-loading activity and enrichment in Na+,K+-ATPase and adenylate cyclase. The triads are practically devoid of contractile elements, mitochondria, and free plasmalemma, and low in content of light SR. The method for obtaining enriched triads is reproducible, and sufficient yields are obtained for structural, biochemical, and functional characterization.
Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. II. Cell surface localization of granule membrane and content proteins before and after degranulation
Authors: Brown WJ , Shannon WA , Snell WJ
Published: May 2004

The compositional relationship between the cell surface of rabbit polymorphonuclear leukocytes (PMNs) and the membranes of PMN cytoplasmic granules has been investigated. Heterophilic PMNs obtained from peritoneal exudates contained 13 cell surface polypeptides ranging in molecular weight from 220,000 to 12,000 daltons as determined by lactoperoxidase-catalyzed protein iodination and gel electrophoresis. Of these, four polypeptides co-migrated with proteins identified as the major constituents of specific (SpG) and azurophilic (AzG) granule membranes. The most notable of these were cell surface proteins of 145,000 and 96,000 daltons that co-migrated with proteins identified as granule content proteins released from PMNs during exocytosis. Extensive washing did not remove these proteins from the cell surface.

Iodination of PMNs after the release of SpG and AzG contents by calcium ionophore- induced exocytosis revealed that there was not a dramatic quantitative change in the proteins on the cell surface. Instead, there were large, quantitative increases in the relative amounts of (125)I that were incorporated into several pre-existing cell surface proteins; all of these cell surface proteins co-migrated as a set with those polypeptides identified as either granule membrane or content proteins. Although nearly all of the major polypeptides of SpG and AzG had counterparts on the cell surface of freshly isolated peritoneal exudates PMNs, there were several polypeptides that were unique to the cell surface. Thus, the PMN has at least three membrane compartments with strikingly different protein compositions.
Quantitative analysis of modulations in numerical and lateral distribution of intramembrane particles during the cell cycle of neuroblastoma cells.
Authors: de Laat S W , Tertoolen L G , van der Saag P T , Bluemink J G
Published: May 2004

Modulations in the internal structure of the plasma membrane during the cell cycle of mouse C1300 neuroblastoma cells (clone Neuro-2A) have been studied by freeze-fracture electron microscopy. Both the numerical and lateral distributions of the intramembrane particles (IMP) of the P face of the medium-exposed plasma membrane were determined as a function of the IMP diameter. The lateral IMP-distribution was quantified by a differential density distribution analysis, that could distinguish between random, aggregated, and dispersed distributions of IMP-subpopulations at various levels of spatial organization. Nonrandom lateral IMP-distribution was considered to indicate significant directional constraints on the lateral mobility of the represented molecules. The analysis demonstrated that the density, the size distribution, and the lateral distribution of the IMP are modulated during the cell cycle, such that characteristic structural and dynamic membrane properties can be attributed to the various cell cycle phases (M, G1, S, and G2). The results are interpreted in terms of asynchronous assembly of different membrane components and dynamic reorganizations within the plasma membrane during the cell cycle. Furthermore, they provide a structural manifestation of earlier observed changes in the dynamic properties of membrane proteins and lipids, and functional membrane transport properties in these neuroblastoma cells.
Mutant Chinese hamster ovary cells pleiotropically defective in receptor-mediated endocytosis.
Authors: Robbins A R , Peng S S , Marshall J L
Published: May 2004

Populations of Chinese hamster ovary cells selected for resistance to diphtheria toxin were found to be highly enriched for mutants deficient in the uptake of lysosomal hydrolases via the mannose 6-phosphate receptor. One doubly defective mutant, DTF 1-5-1, exhibited increased resistance to Sindbis virus, although it was able to bind and internalize virus normally. Normal production of virus was obtained when, subsequent to virus binding, the mutant was exposed for 2 min to acidic pH. Similarly, a shift to acidic pH increased the sensitivity of DTF 1-5-1 to diphtheria toxin 12-fold. Decreased uptake of lysosomal hydrolases by the mutant correlated with decreased mannose 6-phosphate receptor activity at the cell surface; results of lactoperoxidase-catalyzed iodination indicated that the surface-associated receptor was present but inactive on DTF 1-5-1. Total mannose 6-phosphate receptor activity was also decreased in the mutant and this decrease was reflected by increased secretion of lysosomal hydrolases. The phenotype of DTF 1-5-1 resembles in many ways that of cells treated with ammonia. We suggest that the defect in DTF 1-5-1 stems from an inability to deliver virus, diphtheria toxin, and lysosomal hydrolases to an acidic compartment. Other ligands may be endocytosed through a different pathway since the defect of DTF 1-5-1 did not decrease the endocytosis of ricin, modeccin, or Pseudomonas toxin and had minimal effects on uptake and degradation of low density lipoprotein.
Low angle x-ray diffraction studies of HeLa metaphase chromosomes: effects of histone phosphorylation and chromosome isolation procedure.
Authors: Paulson J R , Langmore J P
Published: May 2004

To test whether gross changes in chromatin structure occur during the cell cycle, we compared HeLa mitotic metaphase chromosomes and interphase nuclei by low angle x-ray diffraction. Interphase nuclei and metaphase chromosomes differ only in the 30-40-nm packing reflection, but not in the higher angle part of the x-ray diffraction pattern. Our interpretation of these results is that the transition to metaphase affects only the packing of chromatin fibers and not, to the resolution of our method, the internal structure of nucleosomes or the pattern of nucleosome packing within chromatin fibers. In particular, phosphorylation of histones H1 and H3 at mitosis does not affect chromatin fiber structure, since the same x-ray results are obtained whether or not histone dephosphorylation is prevented by isolating metaphase chromosomes in the presence of 5,5'-dithiobis(2-nitrobenzoate) or low concentrations of p-chloromercuriphenylsulfonate (ClHgPhSO3). We also compared metaphase chromosomes isolated by several different published procedures, and found that the isolation procedure can significantly affect the x-ray diffraction pattern. High concentrations of ClHgPhSO3 can also profoundly affect the pattern.
Production of monoclonal antibodies against calmodulin by in vitro immunization of spleen cells.
Authors: Pardue R L , Brady R C , Perry G W , Dedman J R
Published: May 2004

Monoclonal antibodies against the highly conserved ubiquitous calcium-binding protein, calmodulin (CaM), were produced by immunization of mouse primary spleen cell cultures. Dissociated spleen cells were cultured for 5 d in the presence of mixed thymocyte culture conditioned media (TCM) and purified bovine testes CaM (50 ng-1 mg). Following immunization, cells were fused with mouse myeloma cells (SP2/0, Ag 8.653) and cultured for 2-3 wk before initial screening for antibody. In five independent immunizations there was a range of 25-44% of the initial polyclonal cultures which produced antibodies reacting with purified CaM as determined by immunoassay. 80% of the cloned hybridoma produced IgM immunoglobulins while the remaining clones were IgG producers. This ratio was changed to 50% IgM and 50% IgG by subsequent extension of the in vitro immunization periods and reduced amounts of antigen and extended in vitro culturing. In vitro immunization introduces a new dimension to monoclonal antibody production where limited antigen or poorly antigenic proteins are of interest. The monoclonal antibodies produced in this study have enabled us to to selectively localize CaM in association with distinct subcellular structures, mitochondria, stress fibers, centrioles, and the mitotic spindle.
Pattern and time course of rhodamine-actin incorporation in cardiac myocytes.
Authors: Glacy S D
Published: May 2004

Microinjection of skeletal actin labeled with rhodamine into cultured cardiac myocytes was followed by rapid incorporation of fluorescence into myofibrils of the cells. Myocytes examined as shortly as 5 min postinjection displayed fluorescent bands corresponding to the sarcomeres. By 10 min, distinct alternating wide and narrow bands of fluorescence were observed. The wide bands appeared to correspond to the full breadth of the I-bands, whereas the narrow bands of fluorescence corresponded to the M-lines. This pattern of fluorescence remained essentially unchanged for at least 15 h postinjection. The myofibrils of cardiac myocytes were functional after rhodamine-actin incorporation as judged by their ability to contract. The results of this study suggest that cardiac myofibrils are morphologically stable structures which, nonetheless, exhibit extensive exchange of actin subunits.
Cell surface localization and tissue distribution of a hepatocyte cell-cell adhesion glycoprotein (cell-CAM 105)
Authors: Ocklind C , Forsum U , Obrink B
Published: May 2004

We recently identified a 105,000-dalton plasma membrane glycoprotein, denoted cell-CAM 105 (CAM, cell adhesion molecule), that is involved in intercellular adhesion of reaggregating rat hepatocytes (Ocklind, C., and B. Obrink, 1982, J. Biol. Chem., 257:6788-6795). In this communication we used a monospecific rabbit antiserum against cell-CAM 105 to localize the antigen by indirect immunofluorescence on isolated rat cells and on frozen rat tissue sections. This antiserum stained the surface of freshly isolated hepatocytes. In liver sections, however, the fluorescence seemed to be located exclusively along the bile canaliculi. In addition, cell-CAM 105 showed a very specific tissue distribution. Thus a specific fluorescence was seen only in the epithelia of the stomach, the small intestine, the large intestine, the glandular epithelium of the parotid gland, and the tubules of the kidney. No specific fluorescence was found in variety of other tissues, including cartilage, interstitial connective tissue, smooth muscle, skeletal muscle, heart muscle, eye, brain, skin, the epithelia of oesophagus, bladder, uterin mucosa, thyroid follicles, prostate gland, or collecting ducts of the kidney. In the simple epithelia of the intestine and the kidney tubules the fluorescence was confined to the apical, luminal portion. Thus, both in these epithelia and in liver, cell-CAM 105 was confined to the apical, luminal portion. Thus, both in these epithelia and in liver, cell-CAM 105 was located where the typical junctional complexes between cells are found. These findings taken together with the fact that cell-CAM 105 is involved in intercellular adhesion between hepatocytes suggest with the fac that cell-CAM 105 is involved in intercellular adhesion between hepatocytes suggest that cell-CAM 105 is a member of the junctional complexes of hepatocytes and some simple epithelia.
A role for adherons in neural retina cell adhesion.
Authors: Schubert D , LaCorbiere M , Klier F G , Birdwell C
Published: May 2004

Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion; they also stimulate the rate of cell-cell aggregation. Adheron-stimulated adhesion is tissue specific, and the spontaneous aggregation of neural retina cells is inhibited by monovalent Fab' fragments prepared from an antiserum against neural retina adherons. Therefore cell surface antigenic determinants shared with adherons are involved in normal cell-cell adhesions. The particles from the heterogeneous neural retina population contain many proteins and several glycosaminoglycans. The adherons migrate as a symmetrical 12S peak on sucrose gradients and are predominantly 15-nm spheres when examined by electron microscopy. Finally, the specific activity of neural retina adherons increases from embryonic days 7 through 12 and then declines. These results suggest that glycoprotein particles may be involved in some of the adhesive interactions between neural retina cells and between the cells and their environment.
The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture.
Authors: Tougard C , Louvard D , Picart R , Tixier-Vidal A
Published: May 2004

Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin.
Behavior of cells seeded in isolated fibronectin matrices.
Authors: Hsieh P , Chen L B
Published: May 2004

Cell-free fibronectin matrix (FN-matrix) isolated from chick embryo fibroblasts was used to study cell-matrix interaction. After 24 h, most fibroblastic cells, including those without cell surface fibronectin, adopted bipolar fusiform morphology. Cells grew in parallel arrays and aligned with each other apparently along FN-matrix. Since the orientation of fibronectin fibers was determined by chick embryo fibroblasts, our results suggested that intercellular organization of "matrix-using" cell type may be influenced by "matrix-producing" cell type. Whereas the elongation and alignment effects induced by FN-matrix have been detected in fibroblasts (both normal and transformed), myoblast, aortic endothelial cells, neural cell lines (B103 and RT4D1), and cardiac muscle cells, similar effects are not detected in bone marrow hemopoietic cells, circulating lymphocytic T and B cells, and sympathetic neurons. For epithelial cells, FN-matrix has varying effects. Elongation and alignment effects are detected only in transformed epithelial cells with a great reduction in keratin expression. The morphology of normal or transformed epithelial cells with abundant keratin appears unaffected by FN-matrix. FN-matrix reduced the growth of several transformed fibroblastic lines up to 25%, but did not restore the appearance of actin stress fibers and the normal migratory activities of Rous sarcoma virus-transformed rat cells.
Role of sodium in ADP- and thrombin-induced megakaryocyte spreading.
Authors: Leven R M , Mullikin W H , Nachmias V T
Published: May 2004

We investigated the role of sodium in megakaryocyte spreading induced by thrombin and ADP. We found that if extracellular sodium was replaced by lithium, potassium, or choline, spreading was inhibited. When extracellular sodium was present, amiloride or tetrodotoxin inhibited spreading. Using intracellular recording we found spreading to be associated with a permanent membrane depolarization. The extent and rate of thrombin-induced depolarization was reduced when lithium replaced sodium. Unspread cells had an average membrane potential of -44.8 mV. Spread cells had an average membrane potential of -18.46 mV. When choline replaced sodium, or when in the presence of tetrodotoxin and amiloride, the spread cells repolarized, indicating that the depolarization is due to an increase in sodium permeability. Similar treatments did not change the membrane potential of unspread cells. Incubation of megakaryocytes with A23187 together with monensin or methylamine induced spreading. Methylamine occasionally caused spreading by itself, but neither ionophore alone caused spreading. These results indicate that megakaryocyte spreading induced by ADP and thrombin depends on an increase in sodium conductance.
Firm structural associations between migratory pigment granules and microtubules in crayfish retinula cells.
Authors: Frixione E
Published: May 2004

The morphology of associations between mobile pigment granules and microtubules of the crayfish retinula cells was examined with transmission electron microscopy. Many pigment granules were found associated with microtubules through linkages of fuzzy appearance in thin sections. The linkages were revealed as discrete strands of variable shape in rotary-shadowed replicas of freeze-fractured and deep-etched specimens. The only feature of constant morphology among these connections consisted of 2-4-nm filaments projecting laterally from the microtubules. The firmness of the pigment granule-microtubule associations was judged by their ability to hold up during cell disruption procedures of increasing disaggregation effects in a low-Ca++ stabilization buffer. The results of these tests were inspected with scanning electron microscopy and with transmission electron microscopy of negatively stained preparations. Numerous pigment granules remained associated with a stable microtubule framework after the plasma membrane had been stripped away. Moreover, granule-microtubule attachments survived breakdown of this framework into free fascicles of microtubules. The pigment granules were associated with the free microtubules either individually or as clusters entangled in a fibrous material interwoven with 10-nm filaments. These findings attest that many pigment granules are bound to microtubules through linkages that constitute effective attachments. Further, it is demonstrated that a highly cohesive substance associates the pigment granules with one another. These conclusions are discussed in terms of a pigment transport mechanism in which a network of interconnected granules would establish firm transient interactions with a supporting skeleton of microtubules.
Microtubule-organizing centers and cell migration: effect of inhibition of migration and microtubule disruption in endothelial cells.
Authors: Gotlieb A I , Subrahmanyan L , Kalnins V I
Published: May 2004

We have previously shown that microtubule-organizing centers (MTOC's) become preferentially oriented towards the leading edge of migrating endothelial cells (EC's) at the margin of an experimentally induced wound made in a confluent EC monolayer. To learn more about the mechanism responsible for the reorientation of MTOC's and to determine whether a similar reorientation takes place when cell migration is inhibited, we incubated the wounded cultures with colcemid (C) and cytochalasin B (CB), which disrupt microtubules (MT's) and microfilaments (MF's), respectively. The results obtained showed that the MTOC reorientation can occur independent of cell migration since MTOC's reoriented preferentially toward the wound edge in the CB-treated cultures, even though forward migration of the EC was inhibited. In addition, the MTOC reorientation is inhibited by C, indicating that it requires an intact system of MT's and/or other intracellular structures whose distribution is dependent on that of MT's.
Filipin-cholesterol complexes form in uncoated vesicle membrane derived from coated vesicles during receptor-mediated endocytosis of low density lipoprotein.
Authors: McGookey D J , Fagerberg K , Anderson R G
Published: May 2004

Filipin has been widely used as an electron microscopic probe to detect 3-beta-hydroxysterols, principally cholesterol, in cellular membranes. When it complexes with sterol, it forms globular deposits that disrupt the planar organization of the membrane. Previous studies have shown that coated pits and coated vesicles, specialized membranes involved in receptor-mediated endocytosis, do not appear to bind filipin. This has led to the suggestion that these membranes are low in cholesterol compared with the remainder of the plasma membrane. Since coated endocytic vesicles become uncoated vesicles during the transport of internalized ligands to the lysosome, we have carried out studies to determine whether or not the membranes that surround these transport vesicles are unable to bind filipin and therefore, are also low in cholesterol. Cells were incubated with ferritin-conjugated ligands that bind to low density lipoprotein (LDL) receptors in coated pits. After allowing internalization of the conjugates, we fixed the cells in either the presence or absence of filipin. This permitted us to identify all of the vesicles involved in the transport of LDL to the lysosome and to determine whether the membranes of these vesicles were able to bind filipin. We found that, coordinate with the dissociation of the clathrin coat from the endocytic vesicles, the membranes became sensitive to the formation of filipin-sterol complexes. Furthermore, all of the uncoated endocytic vesicle membranes, as well as the lysosomal membranes, bound filipin. This suggests either that coated membrane contains normal cholesterol levels, which is not easily detected with filipin, or that cholesterol rapidly moves into endocytic vesicles after the clathrin coat dissociates from the membrane.
Isolation of a cell surface receptor protein for laminin from murine fibrosarcoma cells.
Authors: Malinoff H L , Wicha M S
Published: May 2004

We used affinity chromatography to isolate a specific laminin-binding protein from murine fibrosarcoma cells. These cells bind exogenous laminin to their surface with high affinity (Kd = 2 X 10(-9)M for laminin) with approximately 5 X 10(4) sites per cell. Laminin affinity chromatography of [35S]methionine-labeled cell extracts produced two distinct proteins. One was identified as Type IV (basement membrane) collagen based on its migration pattern on SDS gels and bacterial collagenase sensitivity. The other protein, which migrates as a single band or closely spaced doublet on reduced SDS gels, has a reduced molecular weight of 69,000. Using a nitrocellulose filter disk assay, we found that the latter protein specifically bound 125I-laminin with the same high affinity (Kd = 2 X 10(-9)M for laminin) as did intact fibrosarcoma cells. By iodinating intact cells, we demonstrated that this laminin-binding protein is on the cell surface. We conclude that this protein with reduced molecular weight of 69,000 is a subunit or component of a larger cell surface receptor protein for laminin in this fibrosarcoma model. This laminin receptor may mediate the interaction of the cell with its extracellular matrix.
ATP serves two distinct roles in protein degradation in reticulocytes, one requiring and one independent of ubiquitin.
Authors: Tanaka K , Waxman L , Goldberg A L
Published: May 2004

Protein degradation in rabbit reticulocytes is a nonlysosomal process requiring ATP. Recently, appreciable evidence has been presented that ATP is required for the covalent binding of the polypeptide ubiquitin to epsilon-amino groups on protein substrates. To test whether linkage of ubiquitin to substrates is required for ATP-dependent proteolysis, the amino groups of 3H-methyl-casein and denatured 125I-bovine serum albumin (BSA) were completely (93-99%) blocked by methylation, acetylation, carbamylation, or succinylation. In each case, the proteins lacking amino groups were still degraded by an ATP-stimulated process, although these various treatments altered absolute rates of proteolysis and reduced the magnitude of the ATP stimulation (two- to fourfold) below that seen measured with the unmodified substrates. When ubiquitin was removed by ion exchange chromatography, ATP still stimulated breakdown of casein and carbamylated casein twofold. The addition of ubiquitin in the presence of ATP caused a further twofold increase in the hydrolysis of unmodified casein but did not affect the degradation of casein lacking amino groups. Thus ubiquitin conjugation to substrates appears important in the breakdown of certain substrates (especially of BSA), but this reaction is not essential for ATP-stimulated proteolysis. The ATP-activated step that is independent of ubiquitin probably is also involved in the degradation of unblocked proteins, since both processes require Mg++ and ATP hydrolysis and are inhibited by hemin but not by protoporphyrin IX. These results suggest that ATP has distinct roles at different steps in the degradative pathway.
Two temperature-sensitive mutants of Saccharomyces cerevisiae with altered expression of mating-type functions.
Authors: Manney T R , Jackson P , Meade J
Published: May 2004

Two mutants of Saccharomyces cerevisiae have been isolated from normal haploid MAT alpha strains and characterized as having temperature-sensitive, pleiotropic phenotypes for functions associated with mating. At the permissive temperature, 23 degrees C, they were found to behave as normal MAT alpha haploids with respect to mating efficiency, sporulation in diploids formed with MAT a strains, secretion of alpha-factor, and failure to secrete the MATa-specific products, a-factor and Barrier. At higher temperatures they were found to decline in mating and sporulation efficiency and to express the a-specific functions. Genetic analysis established that one of these mutants, PE34, carries a temperature-sensitive allele of the MAT alpha 2 gene and that the other, PD7, carries a temperature-sensitive allele of the TUP1 gene.