bluuebunny/crossref_metadata_2025 huggingface dataset:
Widespread distribution of a 210,000 mol wt microtubule-associated protein in cells and tissues of primates.
Authors: Bulinski J C , Borisy G G
Published: May 2004

Antisera prepared against a 210,000 mol wt microtubule-associated protein (210k MAP) isolated from the human cell line, HeLa, were used to survey a variety of cells and tissues for the presence of immunologically related proteins. The antisera were employed to test extracts of the cells and tissues, using a sensitive indirect immunofluorescence technique applied to polyacrylamide gels. Cross-reactive material of 210,000 mol wt was found in 10 kinds of cells and tissues derived from humans and four lines of cells from monkeys. Indirect immunofluorescent staining was also carried out on fixed cells and showed that the cross-reactive material was localized to interphase and mitotic microtubules as assayed in nine human and seven monkey cell lines. No protein that cross-reacted with 210k MAP antisera was detected in cells and tissues derived from two rodents, an ungulate, a marsupial, or a chicken. Therefore, the 210k MAP isolated from HeLa cells is present in a wide variety of cells and tissues of humans and other primates but is antigenically distinct from MAPs present in lower organisms.
Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells.
Authors: Mooseker M S , Graves T A , Wharton K A , Falco N , Howe C L
Published: May 2004

The bundle of filaments within microvilli of intestinal epithelial cells contains five major proteins including actin, calmodulin, and subunits of 105-, 95-, and 70-kdaltons. It has been previously shown (Howe, C. L., M. S. Mooseker, and T. A. Graves. 1980. Brush-border calmodulin: a major component of the isolated microvillus core. J. Cell Biol. 85: 916-923) that the addition of Ca++ (> 10(-6) M) to microvillus cores causes a rapid, drastic, but at least partially reversible disruption of this actin filament bundle. High-speed centrifugation of microvillus cores treated with Ca++ indicates that several core proteins are solubilized, including 30-50% of the actin and calmodulin, along with much of the 95- and 70-kdalton subunits. Gel filtration of such Ca++ extracts in the presence and absence of Ca++ indicates that microvillar actin "solated" by Ca++ is in an oligomeric state probably complexed with the 95-kdalton subunit. Removal of Ca++ results in the reassembly of F-actin, probably still complexed with 95-kdalton subunit, as determined by gel filtration, cosedimentation, viscometry, and electron microscopy. The 95-kdalton subunit (95K) was purified from Ca++ extracts by DEAE-Sephadex chromatography and its interaction with actin characterized by viscometry, cosedimentation, and EM in the presence and absence of Ca++. In the presence, but not absence, of Ca++, 95K inhibits actin assembly (50% inhibition at 1:50-60 95K to actin) and also reduces the viscosity of F-actin solutions. Similarly, sedimentation of actin is inhibited by 95K, but a small, presumably oligomeric actin- 95K complex formed in the presence of Ca++ is pelletable after long-term centrifugation. In the absence of Ca++, 95K cosediments with F-actin. EM of 95K-actin mixtures reveals that 95K "breaks" actin into small, filamentous fragments in the presence of Ca++. Reassembly of filaments occurs once Ca++ is removed. In the absence of Ca++, 95K has no effect on filament structure and, at relatively high ratios (1:2-6) of 95K to actin, this core protein will aggregate actin filaments into bundles.
Human dynein and sperm pathology.
Authors: Baccetti B , Burrini A G , Pallini V , Renieri T
Published: May 2004

Human spermatozoa with normal structure and with different axonemal deficiencies (absence of axoneme, of arms, or of central structures) were studied by electron microscopy, SDS-polyacrylamide gel electrophoresis, and ATPase activity measurements. Normal human sperm possess a complement of high molecular weight polypeptides with an electrophoretic migration similar to that of sea urchin and other mammalian sperm dyneins. Human high molecular weight bands are numbered one to four in order of increasing of electrophoretic mobility; all of them are absent in spermatozoa that lack axoneme. The absence of doublet arms, coincides with the absence of bands 2, 3, and 4; the absence of central structures coincides with a reduction in intensity of band 2. In the latter two abnormal conditions, band 1 has an increased intensity. The data are tentatively interpreted by attributing the polypeptides forming bands 3 and 4 to the arm structure, whereas band 2 is supposed to contain a mixture of polypeptides localized in the arms and in the central structures; these abnormal sperm contain modified polypeptides which gather in band 1. Histochemical ATPase stainings indicate that this enzyme is localized mainly in the doublet arms and, to a minor extent, in the central structures.
Studies on cell adhesion and recognition. I. Extent and specificity of cell adhesion triggered by carbohydrate-reactive proteins (glycosidases and lectins) and by fibronectin.
Authors: Rauvala H , Carter W G , Hakomori S I
Published: May 2004

The extent and the specificity of the initial cell attachment induced by various proteins coated on plastic surfaces have been studied with the following results: (a) Cell adhesion on the surfaces coated with sialidase and beta-galactosidase was as strong as on concanavalin A and limulus lectin-coated surfaces and the reactions were strongly inhibited by glycosidase inhibitors or by competitive substrates. The adhesion on sialidase was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid and by polysialoganglioside (GT1b) at low concentration (0.05-0.1 mM). The cell adhesion on beta-galactosidase coat was inhibited by 1,4-D-galactonolactone and beta-methylgalactoside but not by alpha-methylgalactoside. Thus, the initiation of cell adhesion on glycosidase surfaces could be mediated through the interactions of the specific binding sites of the enzyme surface with the cell surface substrates under physiological conditions. (b) Cell adhesion on various lectins could be blocked by various competing monosaccharides at the concentrations similar to the inhibitory concentrations for binding of lectins from solution to the cells. (c) Cell adhesion on fibronectin surfaces as well as on gelatin-coated surfaces was equally inhibited by GT1b at relatively high concentrations (0.25-0.5 mM). Lower concentrations of GT1b (0.05-0.1 mM) inhibited the cell adhesion on surfaces of Limulus lectin and sialidase. It is suggested that the cell adhesion mediated by fibronectin is based on yet unknown interactions in contrast to a specific cell adhesion through glycosidases and lectins.
Structural evidence that botulinum toxin blocks neuromuscular transmission by impairing the calcium influx that normally accompanies nerve depolarization.
Authors: Hirokawa N , Heuser J E
Published: May 2004

Taking advantage of the fact that nerve terminal mitochondria swell and sequester calcium during repetitive nerve stimulation, we here confirm that this change is caused by calcium influx into the nerve and use this fact to show that botulinum toxin abolishes such calcium influx. The optimal paradigm for producing the mitochondrial changes in normal nerves worked out to be 5 min of stimulation at 25 Hz in frog Ringer's solution containing five time more calcium than normal. Applying this same stimulation paradigm to botulinum-intoxicated nerves produced no mitochondrial changes at all. Only when intoxicated nerves were stimulated in 4-aminopyridine (which grossly exaggerates calcium currents in normal nerves) or when they were soaked in black widow spider venom (which is a nerve-specific calcium ionophore) could nerve mitochondria be induced to swell and accumulate calcium. These results indicate that nerve mitochondria are not damaged directly by the toxin and point instead to a primary inhibition of the normal depolarization-evoked calcium currents that accompany nerve activity. Because these currents normally provide the calcium that triggers transmitter secretion from the nerve, this demonstration of their inhibition helps to explain how botulinum toxin paralyzes.
Clathrin is axonally transported as part of slow component b: the microfilament complex.
Authors: Garner J A , Lasek R J
Published: May 2004

During axonal transport, membranes travel down axons at a rapid rate, whereas the cytoskeletal elements travel in either of two slow components, SCa (with tubulin and neurofilament protein) and SCb (with actin). Clathrin, the highly ordered, structural coat protein of coated vesicles, has recently been shown to be able to interact in vitro with cytoskeletal proteins in addition to membranes. The present study examines whether clathrin travels preferentially with the membrane elements or the cytoskeletal elements when it is axonally transported. Guinea pig visual system was labeled with tritiated amino acids. Radioactive SDS-polyacrylamide gel electrophoresis profiles from the major components of transport were coelectrophoresed with clathrin. Only SCb had a band comigrating with clathrin. In addition, radioactive clathrin was purified from guinea pig brain containing only radioactive SCb polypeptides. Kinetic analysis of the putative clathrin band in SCb revealed that it travels entirely within the SCb wave. Thus we conclude that clathrin travels preferentially with the cytoskeletal proteins making up SCb, rather than with the membranes and membrane-associated proteins in the fast component.
Secretory protein decondensation as a distinct, Ca2+-mediated event during the final steps of exocytosis in Paramecium cells.
Authors: Bilinski M , Plattner H , Matt H
Published: May 2004

The contents of secretory vesicles ("trichocysts") were isolated in the condensed state from Paramecium cells. It is well known that the majority portion of trichocysts perform a rapid decondensation process during exocytosis, which is visible in the light microscope. We have analyzed this condensed leads to decondensed transition in vitro and determined some relevant parameters. In the condensed state, free phosphate (and possibly magnesium) ions screen local surplus charges. This is supported by x-ray spectra recorded from individual trichocysts (prepared by physical methods) in a scanning transmission electron microscope. Calcium, as well as other ions that eliminate phosphate by precipitation, produces decondensation in vitro. Under in vivo conditions, Ca2+ enters the vesicle lumen from the outside medium, once an exocytic opening has been formed. Consequently, within the intact cell, membrane fusion and protein decondensation take place with optimal timing. Ca2+ might then trigger decondensation in the same way by precipitating phosphate ions (as it does in vitro) and, indeed, such precipitates (again yielding Ca and P signals in x-ray spectra) can be recognized in situ under trigger conditions. As decondensation is a unidirectional, rapid process in Paramecium cells, it would contribute to drive the discharge of the secretory contents to the outside. Further implications on the energetics of exocytosis are discussed.
Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor
Authors: Huff K , End D , Guroff G
Published: May 2004

PC12 cells, which differentiate morphologically and biochemically into sympathetic neruonlike cells in response to nerve growth fact, also respond to epidermal growth factor. The response to epidermal growth factor is similar in certain respects to the response to nerve growth fact. Both peptides produce rapid increases in cellular adhesion and 2-deoxyglucose uptake and both induce ornithine decarboxylase. But nerve growth factor causes a decreased cell proliferation and a marked hypertrophy of the cells. In contrast, epidermal growth factor enhances cell proliferation and does not cause hypertrophy. Nerve growth factor induces the formation of neuritis; epidermal growth factor does not.

When both factors are presented simultaneously, the cells form neurites. Furthermore, the biological response to epidermal growth fact, as exemplified by the induction of ornithine decarboxylase, is attenuated by prior treatment of the cells with nerve growth factor.

PC12 cells have epidermal growth factor receptors. The binding of epidermal growth factor to these receptors is rapid and specific, and exhibits an equilibrium constant of 1.9 x 10(-9) M. Approximately 80,000 receptors are present per cell, and this number is independent of cell density.

Treatment of the cells with nerve growth factor reduces the amount of epidermal growth factor binding by at least 80 percent. The decrease in receptor binding begins after approximately 12-18 h of nerve growth factor treatment and is complete within 3 d. Scratchard plots indicate that the number of binding sites decreases, not the affinity of the binding sites for epidermal growth factor.
Isolation of 10-nm filaments from astrocytes in the mouse optic nerve.
Authors: Tsukita S , Ishikawa H , Kurokawa M
Published: May 2004

Astroglial filaments approximately 10 nm in diameter were isolated from degenerated mouse optic nerves by Triton X-100 and DNase I treatments followed by sucrose density gradient centrifugation. 2-4 wk after bilateral enucleation, optic nerves contained virtually a single population of 10-nm filaments (astroglial filaments), free from neurofilaments. In negative-staining and thin-section electron microscopy, the isolated filaments were seen as nonbranching linear structures with smooth contour, and were morphologically identical to those in situ. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the isolated filaments to be composed of two major polypeptides with molecular weights of 45,000 and 55,000, present in an approximate molar ratio of 1:1. These findings, together with the results of one-dimensional peptide mapping and solubility study, indicate that the astroglial filaments in the mouse optic nerve are primarily composed of these two polypeptides.
Membrane-bound ribosomes of myeloma cells. VI. Initiation of immunoglobulin mRNA translation occurs on free ribosomes.
Authors: Mechler B
Published: May 2004

Immunoglobulin heavy (Ig H) and light (Ig L) chain mRNA molecules have been released from the endoplasmic reticulum (ER) membranes as free (F) mRNP particles when MOPC 21 (P3K) mouse myeloma cells are exposed to a hypertonic initiation block (HIB). The subsequent fate of these mRNA sequences has been examined when the cells are returned to normal growth medium. Upon return to isotonicity, all previously translated mRNA molecules reassociate with ribosomes and form functional polysomes. Ig H mRNA is found incorporated first into F polysomes and then into membrane-bound (MB) polysomes. Kinetic studies indicate that the time of passage of Ig H mRNA in F polysomes is approximately 30 s, during which a nascent polypeptide chain of approximately 80 amino acids would have been completed. When the rate of polypeptide elongation is depressed with emetine during the recovery from HIB, both Ig H and L mRNA molecules accumulate in small F polysomes. These results indicate that the formation of Ig-synthesizing polysomes proceeds in the sequence: mRNA leads to F polysomes leads to MB polysomes. With the additional observation that during HIB recovery puromycin completely prevents the reassociation of Ig mRNA with the ER, these findings support a model of MB polysome formation in which the specificity of membrane attachment is determined by the nature of the N-terminal amino acid sequence of the nascent polypeptide chain.
Intracellular univalent cations and the regulation of the BALB/c-3T3 cell cycle.
Authors: Frantz C N , Nathan D G , Scher C D
Published: May 2004

Addition of serum to density-arrested BALB/c-3T3 cells causes a rapid increase in uptake of Na+ and K+, followed 12 h later by the onset of DNA synthesis. We explored the role of intracellular univalent cation concentrations in the regulation of BALB/c-3T3 cell growth by serum growth factors. As cells grew to confluence, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) fell from 40 and 180 to 15 and 90 mmol/liter, respectively. Stimulation of growth of density-inhibited cells by the addition of serum growth factors increased [Na]i by 30% and [K+]i by 13-25% in early G0/G1, resulting in an increase in total univalent cation concentration. Addition of ouabain to stimulated cells resulted in a concentration-dependent steady decrease in [K+]i and increase in [Na+]i. Ouabain (100 microM) decreased [K+]i to approximately 60 mmol/liter by 12 h, and also prevented the serum-stimulated increase in 86Rb+ uptake. However, 100 microM ouabain did not inhibit DNA synthesis. A time-course experiment was done to determine the effect of 100 microM ouabain on [K+]i throughout G0/G1 and S phase. The addition of serum growth factors to density-inhibited cells stimulated equal rates of entry into the S phase in the presence or absence of 100 microM ouabain. However, in the presence of ouabain, there was a decrease in [K+]i. Therefore, an increase in [K+]i is not required for entry into S phase; serum growth factors do not regulate cell growth by altering [K+]i. The significance of increased total univalent cation concentration is discussed.
Radial spokes of Chlamydomonas flagella: genetic analysis of assembly and function.
Authors: Huang B , Piperno G , Ramanis Z , Luck D J
Published: May 2004

In addition to the previously studied pf-14 and pf-1 loci in Chlamydomonas reinhardtii, mutations for another five genes (pf-17, pf-24, pf-25, pf-26, and pf-27) have been identified and characterized as specifically affecting the assembly and function of the flagellar radial spokes. Mutants for each of the newly identified loci show selective alterations for one or more of the 17 polypeptides in the molecular weight range of 20,000-130,000 which form the radial spoke structure. In specific instances the molecular defect has been correlated with altered radial spoke morphology. Biochemical analysis of in vivo complementation in mutant X wild-type dikaryons has provided indirect evidence that mutations for four of the five new loci (pf-17, pf-24, pf-25, and pf-26) reside in structural genes for spoke components. In the case of pf-24, the identity of the mutant gene product was supported by analysis of induced intragenic revertants. In contrast to the other radial spoke mutants thus far investigated, evidence suggests that the gene product in pf-27 is extrinsic to the radial spokes and is required for the specific in vivo phosphorylation of spoke polypeptides.
Hypertrophy-induced increase of intermediate filaments in vascular smooth muscle.
Authors: Berner P F , Somlyo A V , Somlyo A P
Published: May 2004

The distribution of filaments was studied in hypertrophied rabbit vascular smooth muscle. Hypertrophy was induced by partial ligation of the portal-anterior mesenteric vein. 14 d after ligation, there was an approximately threefold increase in the number of intermediate filaments per cross-sectional area, as compared to control values. The actin:intermediate:myosin filament ratio was 15:1.1:1 in control and 15:3.5:0.5 in hypertrophied portal-anterior mesentric vein vascular smooth muscle. Comparison of the filament ratios with the increase in volume density of the hypertrophied cells suggests that the number of myosin filaments per cell profile remained approximately the same as in controls, whereas the number of actin filaments increased in proportion to the increase in cell volume.
Application of scanning electron microscopy to x-ray analysis of frozen-hydrated sections. II. Analysis of standard solutions and artificial electrolyte gradients.
Authors: Saubermann A J , Beeuwkes R , Peters P D
Published: May 2004

New specimen handling and analytic techniques for the application of x-ray microanalysis to studies of cell and organ biology have been recently described (Saubermann et al., 1981, J. Cell Biol. 88:257-267). Based on these techniques, absolute quantitative standardization has been established through x-ray analysis of frozen-hydrated and then dried sections of independently measured standard solutions of elements. These experiments demonstrate that the specific techniques employed have a probable error of less than 10%. Artificial electrolyte gradients established in gelatin were subjected to analysis to determine whether there was elemental displacement under non-membrane-limited conditions at the temperatures employed for sectioning (-30 degrees to -40 degrees C). No significant difference was observed between such gradients in serial sections cut at -30 degrees and -80 degrees C. Similarly, no additional ice-crystal-damage artifact was found in sections cut at -30 degrees C when compared with sections cut at -80 degrees C. Thus, in terms of ice-crystal size, gradient maintenance, and compartmental differentiation, cryosectioning at -30 degrees to -40 degrees C was not associated with redistribution incompatible with 1- to 2-micrometers spatial resolution, and absolute measurements of elemental concentration were practical within regions of this size.
Application of scanning electron microscopy to x-ray analysis of frozen-hydrated sections. III. Elemental content of cells in the rat renal papillary tip.
Authors: Bulger R E , Beeuwkes R , Saubermann A J
Published: May 2004

The electrolyte and water content of cellular and interstitial compartments in the renal papilla of the rat was determined by x-ray microanalysis of frozen-hydrated tissue sections. Papillae from rats on ad libitum water were rapidly frozen in a slush of Freon 12, and sectioned in a cryomicrotome at -30 to -40 degrees C. Frozen 0.5-micrometer sections were mounted on carbon-coated nylon film over a Be grid, transferred cold to the scanning microscope, and maintained at -175 degrees C during analysis. The scanning transmission mode was used for imaging. Structural preservation was of good quality and allowed identification of tissue compartments. Tissue mass (solutes + water) was determined by continuum radiation from regions of interest. After drying in the SEM, elemental composition of morphologically defined compartments (solutes) was determined by analysis of specific x-rays, and total dry mass by continuum. Na, K, Cl, and H2O contents in collecting-duct cells (CDC), papillary epithelial cells (PEC), and interstitial cells (IC) and space were measured. Cells had lower water content (mean 58.7%) than interstitium (77.5%). Intracellular K concentrations (millimoles per kilogram wet weight) were unremarkable (79-156 mm/kg wet weight); P was markedly higher in cells than in interstitium. S was the same in all compartments. Intracellular Na levels were extremely high (CDC, 344 +/- 127 SD mm/kg wet weight; PEC, 287 +/- 105; IC, 898 +/- 194). Mean interstitial Na was 590 +/- 119 mm/kg wet weight. CI values paralleled those for Na. If this Na is unbound, then these data suggest that renal papillary interstitial cells adapt to their hyperosmotic environment by a Na-uptake process.
Different polypeptides form the intermediate filaments in bovine hoof and esophageal epithelium and in aortic endothelium.
Authors: Milstone L M , McGuire J
Published: May 2004

Polypeptides that form 10-nm filaments in vitro were isolated from three different bovine tissues: the viable portion of the hoof epithelium, the epithelium of the esophagus, and cultured endothelial cells derived from aorta. The seven polypeptides from hoof, the two from esophagus, and the one from endothelial cells were different with respect to mobility in SDS polyacrylamide gels and/or limited proteolytic digestion. Peptide maps of the different filament-forming polypeptides (FFP's) showed that none of the smaller FFP's was a fragment of any of the larger FFP's. Several isomobile fragments were found in the peptide maps of different FFP's, suggesting that they might contain regions of amino acid sequence homology. We present a hypothesis that suggests how the different 10-nm filament-forming proteins may be related.
Structural changes in lysosomes from cultured human fibroblasts in Duchenne's muscular dystrophy.
Authors: Gelman B B , Davis M H , Morris R E , Gruenstein E
Published: May 2004

We have previously reported a decreased activity of the lysosomal enzyme dipeptidyl aminopeptidase-I (DAP-I) in cultured fibroblasts from patients with Duchenne's muscular dystrophy (DMD). Here we report that electron microscope examination of these cells reveals the presence of abundant lamellar bodies, a morphologic abnormalities commonly associated with impaired lysosomal function. Morphometric analysis of these cytoplasmic figures in dystrophic cells shows a sevenfold increase relative to normal controls (P less than 0.01). Analysis of lysosomal density profiles by density gradient centrifugation reveals similar patterns in normal and DMD cells. Treatment of lysosomes wit the nonionic detergent Triton X-100 causes an activation of DAP-I. This activation, attributable to structure-linked latency, is markedly diminished in DMD cells which show an optimal activation of only 180% compared to 255% for control fibroblasts (P less than 0.01). These data suggest an alteration in the properties of the lysosomal membrane in DMD fibroblasts. This suggestion is also supported by studies on the release of DAP-I from lysosomes by osmotic shock which show it to be a membrane-associated enzyme with membrane-binding characteristics intermediate between those of tightly bound beta-glucosidase and those of unbound N-acetylgalactosaminidase. The latency characteristics of these other lysosomal enzymes are not altered in the DMD cells, indicating that the effect is specific for DAP-I.
Electron microscopic localization of cytoplasmic myosin with ferritin-labeled antibodies.
Authors: Herman I M , Pollard T D
Published: May 2004

We localized myosin in vertebrate nonmuscle cells by electron microscopy using purified antibodies coupled with ferritin. Native and formaldehyde-fixed filaments of purified platelet myosin filaments each consisting of approximately 30 myosin molecules bound an equivalent number of ferritin-antimyosin conjugates. In preparations of crude platelet actomyosin, the ferritin-antimyosin bound exclusively to similar short, 10-15 nm wide filaments. In both cases, binding of the ferritin-antimyosin to the myosin filaments was blocked by preincubation with unlabeled antimyosin. With indirect fluorescent antibody staining at the light microscope level, we found that the ferritin-antimyosin and unlabeled antimyosin stained HeLa cells identically, with the antibodies concentrated in 0.5-microns spots along stress fibers. By electron microscopy, we found that the concentration of ferritin-antimyosin in the dense regions of stress fibers was five to six times that in the intervening less dense regions, 20 times that in the cytoplasmic matrix, and 100 times that in the nucleus. These concentration differences may account for the light microscope antibody staining pattern of spread interphase cells. Some, but certainly not all, of the ferritin-antimyosin was associated with 10-15-nm filaments. In mouse intestinal epithelial cells, ferritin-antimyosin was located almost exclusively in the terminal web. In isolated brush borders exposed to 5 mM MgCl2, ferritin-antimyosin was also concentrated in the terminal web associated with 10-15-nm filaments.
Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells
Authors: Hayman EG , Engvall E , Ruoslahti E
Published: May 2004

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.
Heterogeneous binding of high mobility group chromosomal proteins to nuclei
Authors: Gordon JS , Bruno J , Lucas JJ
Published: May 2004

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs.
Absence of filipin-sterol complexes from the membranes of active zones and acetylcholine receptor aggregates at frog neuromuscular junctions.
Authors: Nakajima Y , Bridgman P C
Published: May 2004

The polyene antibiotic filipin reacts specifically with membrane cholesterol and produces distinctive membrane lesions. We treated frog cutaneous and sartorius muscles with 0.04% filipin in a glutaraldehyde solution with or without prefixation with glutaraldehyde. Freeze-fracture of these muscles revealed numerous 19 to 38-nm protuberances and depressions (filipin-sterol complexes) in most areas of muscle, axon, and Schwann cell membranes. In the presynaptic membrane, however, these filipin-sterol complexes were absent from active zones consisting of ridges bordered with double rows of particles. In the postsynaptic membrane, filipin-sterol complexes were also virtually absent from the areas occupied by aggregates of large particles representing acetylcholine receptors. These results suggest that the membrane regions of active zones and acetylcholine receptor aggregates have a low cholesterol content.
Morphometric analysis of volumes and surface areas in membrane compartments during endocytosis in Acanthamoeba.
Authors: Bowers B , Olszewski T E , Hyde J
Published: May 2004

Stereologic analysis was made of cell surface membrane (PM) and two interrelated cytoplasmic membrane systems, the vacuole membranes (VM) and small vesicle membranes (SVM). Volumes and surface areas of the three membrane compartments were measured during steady-state pinocytosis, when membrane recycling is rapid, and during phagocytosis, when a shift to a lower rate of membrane uptake by endocytosis occurs (B. Bowers, 1977, Exp. Cell Res. 110:409). Total membrane area in the three compartments was 3.2 micrometers 2/micrometers 3 of protoplasmic volume and was constant throughout the experiments. In pinocytosing cells, 32% of the membrane was in the PM, 25% in the vM, and 43% in the SVM. The vacuole compartment occupies approximately 20% of the total cell volume, and the small vesicle, approximately 3%. As the endocytic uptake of membrane from the surface decreased, there was an increase in PM area and a marked decrease in SVM area. The VM area remained constant even though "empty" vacuoles were almost completely replaced by newly formed phagosomes within 45 min. This demonstrates directly a rapid flux of membrane though this compartment. A model, taking into consideration these and other data on Acanthamoeba, is proposed to account for the observed membrane shifts. The data suggest that the vacuolar (digestive) system of Acanthamoeba is central to cellular control of endocytosis and membrane recycling.
Distribution of proteins between nucleus and cytoplasm of amoeba proteus
Authors: Goldstein L , Ko C
Published: May 2004

By transplanting nuclei between labeled and unlabeled cells, we determined the localization of the major proteins of amebas and described certain features of their intracellular distributon. We identified approximately 130 cellular proteins by fluorography of one-dimensional polyacrylamide electrophoretic gels and found that slightly less than half of them (designated NP, for nuclear proteins) are almost exclusively nuclear. About 95 percent of the other proteins (designated CP for cytoplamsic proteins) are roughly equally concentrated in nucleus and cytoplasm, but - because the cytoplasm is 50 times larger than the nucleus - about 98 percent of each of the latter is in the cytoplasm. Of the CP, roughly 5 percent are not detectable in the nucleus. Assuming that these are restricted to the cytoplasm only because, for example, they are in structures too large to enter the nucleus and labeled CP readily exit a nucleus introduced into unlabeled cytoplasm, we conclude that the nuclear envelope does not limit the movement of any nonstructural cellular protein in either direction between the two compartments. Some NP are not found in the cytoplasm (although ostensibly synthesized there) presumably because of preferential binding within the nucleus. Almost one half of the protein mass in nuclei in vivo is CP and apparently only proteins of that group are lost from nuclei when cells are lysed. Thus, while an extracellular environment allows CP to exit isolated nuclei, the nuclear binding affinities for NP are retained.

Further examination of NP distribution shows that many NP species are, in fact, detectable in the cytoplasm (although at only about 1/300 the nuclear concentration), apparently because the nuclear affinity is relatively low. These proteins are electrophoretically distinguishable from the high-affinity NP not found in the cytoplasm.

New experiments show that an earlier suggestion that the nuclear transplantation operation causes an artifactual release of NP to the cytoplasm is largely incorrect. Moreover, we show that cytoplasmic "contamination" of nuclear preparations is not a factor in classifying proteins by these nuclear transplantation experiments.

We speculate the no mechanism has evolved to confine most CP to the cytoplasm (where they presumably function exclusively) because the cytoplasm's large volume ensures that CP will be abundant there. Extending Bonner's idea of "quasi-functional nuclear binding sites" for NP, we suggest that a subset of NP usually have a low affinity for available intranuclear sites because their main function(s) occurs at other intranuclear sites to which they bind tightly only when particular metabolic conditions demand. The other NP (those completely absent from cytoplasm) presumable always are bound with high affinity at their primary functional sites.
Chromosome behavior after laser microirradiation of a single kinetochore in mitotic PtK2 cells.
Authors: McNeill P A , Berns M W
Published: May 2004

The role of the kinetochore in chromosome movement was studied by 532-nm wavelength laser microirradiation of mitotic PtK2 cells. When the kinetochore of a single chromatid is irradiated at mitotic prometaphase or metaphase, the whole chromosome moves towards the pole to which the unirradiated kinetochore is oriented, while the remaining chromosomes congregate on the metaphase plate. The chromatids of the irradiated chromosome remain attached to one another until anaphase, at which time they separate by a distance of 1 or 2 micrometers and remain parallel to each other, not undergoing any poleward separation. Electron microscopy shows that irradiated chromatids exhibit either no recognizable kinetochore structure or a typical inactive kinetochore in which the tri-layer structure is present but has no microtubules associated with it. Graphical analysis of the movement of the irradiated chromosome shows that the chromosome moves to the pole rapidly with a velocity of approximately 3 micrometers/min. If the chromosome is close to one pole at irradiation, and the kinetochore oriented towards that pole is irradiated, the chromosome moves across the spindle to the opposite pole. The chromosome is slowed down as it traverses the equatorial region, but the velocity in both half-spindles is approximately the same as the anaphase velocity of a single chromatid. Thus a single kinetochore moves twice the normal mass of chromatin (two chromatids) at the same velocity with which it moves a single chromatid, showing that the velocity with which a kinetochore moves is independent, within limits, of the mass associated with it.
Electron microscope localization of acetylcholinesterase and butyrylcholinesterase in the superior cervical ganglion of the cat. II. Preganglionically denervated ganglion.
Authors: Davis R , Koelle G B
Published: May 2004

Cat superior cervical ganglia (SCG), denervated preganglionically 6-8 d previously, were stained for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) by the bis-(thioacetoxy)aurate (I), or Au(TA)2, method and compared by electron microscopy with normal SCG described previously (Davis, R., and G. B. Koelle. 1978. J. Cell Biol. 78:785-809). In confirmation of earlier light microscopic findings by the highly specific copper thiocholine method, there was nearly a total disappearance of AChE from the ganglion; no myelinated or unmyelinated axons with AChE-stained axolemmas were found, and only occasional traces of AChE staining were noted at dendritic and perikaryonal plasma membranes. Considerable staining for BuChE persisted at the latter sites, however. As in the normal SCG, physostigmine-resistant staining, caused by noncholinesterase enzymes plus the possible presence of very low concentrations of AChE or BuChE, was noted at external mitochondrial membranes, elements of the endoplasmic reticulum of neurites and Schwann cells, and also in lysosomes. These findings confirm the previous identification of AChE-stained myelinated fibers in the normal SCG as preganglionic and of the unstained myelinated fibers as postganglionic. It is proposed that the maintenance of AChE at postsynaptic sites in normal ganglia is caused by the release of a trophic factor(s) from presynaptic terminals. The source of the postsynaptic BuChE, which is apparently completely absent from the endoplasmic reticulum of the ganglion cells, remains unexplained.
Intramembrane particles and the organization of lymphocyte membrane proteins
Authors: Kuby JM , Wofsy L
Published: May 2004

An experimental system was developed in which the majority of all lymphocyte cell-surface proteins, regardless of antigenic specificity, could be cross-linked and redistributed in the membrane to determine whether this would induce a corresponding redistribution of intramembrane particles (IMP). Mouse spleen cells were treated with P-diazoniumphenyl- β-D-lactoside (lac) to modify all exposed cell-surface proteins. Extensive azo- coupling was achieved without significantly reducing cell viability or compromising cellular function in mitogen- or antigen-stimulated cultures. When the lac-modified cell- surface proteins were capped with a sandwich of rabbit antilactoside antibody and fluorescein-goat anti-rabbit Ig, freeze-fracture preparations obtained from these cells revealed no obvious redistribution of IMP on the majority of fracture faces. However, detailed analysis showed a statistically significant 35 percent decrease (P less than 0.01) in average IMP density in the E face of the lac-capped spleen cells compared with control cells, whereas a few E-face micrographs showed intense IMP aggregation. In contrast, there was no significant alteration of P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP do not present accessible antigenic sites on the lymphocyte surface and do not associate in a stable manner with surface protein antigens. This finding suggests that IMP, as observed in freeze-fracture analysis, may not comprise a representative reflection of lymphocyte transmembrane protein molecules and complexes because other evidence establishes: (a) that at least some common lymphocyte surface antigens are indeed exposed portions of transmembrane proteins and (b) that the aggregation of molecules of any surface antigen results in altered organization of contractile proteins at the cytoplasmic face of the membrane.
A permeabilized cell model for studying cell division: a comparison of anaphase chromosome movement and cleavage furrow constriction in lysed PtK1 cells.
Authors: Cande W Z , McDonald K , Meeusen R L
Published: May 2004

After lysis in a Brij 58-polyethylene glycol medium, PtK1 cells are permeable to small molecules, such as erythrosin B, and to proteins, such as rhodamine-labeled FAB, myosin subfragment-1, and tubulin. Holes are present in the plasma membrane, and the mitochondria are swollen and distorted, but other membrane-bounded organelles of the lysed cell model are not noticeably altered. After lysis, the mitotic apparatus is functional; chromosomes move poleward and the spindle elongates. Cells lysed while in cytokinesis will continue to divide for several minutes. Addition of crude tubulin extracts, MAP-free tubulin, or taxol to the lysis medium retards anaphase chromosome movements but does not affect cleavage. On the other hand, N-ethylmaleimide-modified myosin subfragment-1, phalloidin, and cytochalasin B inhibit cleavage but have no effect on anaphase chromosome movements under identical lysis conditions. These results suggest that actomyosin plays no functional role in anaphase chromosome movement in mammalian tissue culture cells and that microtubule depolymerization is a rate-limiting step for chromosome-to-pole movements.
Consequences of chemotactic peptide receptor modulation for leukocyte orientation.
Authors: Zigmond S H
Published: May 2004

To exhibit chemotaxis, the orientation of locomotion along a chemical gradient cells sense differences in concentrations of a chemotactic factor by detecting some difference in the occupancy of their chemotactic receptors. Thus chemotaxis is sensitive to the number of receptors present and might be used to evaluate the consequences of receptor down-regulation. The ability of rabbit peritoneal polymorphonuclear leukocytes (PMNs) to orient to a standard gradient at various concentrations of N-formylnorleucylleucylphenylalanine (FNLLP) was examined. The observed orientation was compared to that expected if the directional signal were proportional to a difference in the absolute number or the fractional number of receptors occupied. The receptor occupancy in varying gradients was calculated from the binding constant of FNLLP, 2 X 10(-8) M (Zigmond and Sullivan, 1979, J. Cell Biol. 82:517-527), and the receptor number (a) present initially or (b) present after down-regulation (Sullivan and Zigmond, 1980, J. Cell Biol. 85:703-711). The observed concentration dependence of cell orientation is similar to the change in the number of receptors occupied, the receptor number being corrected for down-regulated cells. The net effect of receptor loss appears to be a decreased sensitivity to gradients at high concentrations of peptide.
Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis
Authors: Rao PN , Smith ML
Published: May 2004

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.