bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein synthesis and the cell cycle: centrosome reproduction in sea urchin eggs is not under translational control.
Authors: Sluder G , Miller F J , Cole R , Rieder C L
Published: May 2004

The reproduction, or duplication, of the centrosome is an important event in a cell's preparation for mitosis. We sought to determine if centrosome reproduction is regulated by the synthesis and accumulation of cyclin proteins and/or the synthesis of centrosome-specific proteins at each cell cycle. We continuously treat sea urchin eggs, starting before fertilization, with a combination of emetine and anisomycin, drugs that have separate targets in the protein synthetic pathway. These drugs inhibit the postfertilization incorporation of [35S]methionine into precipitable material by 97.3-100%. Autoradiography of SDS-PAGE gels of drug-treated zygotes reveals that [35S]methionine incorporates exclusively into material that does not enter the gel and material that runs at the dye front; no other labeled bands are detected. Fertilization events and syngamy are normal in drug-treated zygotes, but the cell cycle arrests before first mitosis. The sperm aster doubles once in all zygotes to yield two asters. In a variable but significant percentage of zygotes, the asters continue to double. This continued doubling is slower than normal, asynchronous between zygotes, and sometimes asynchronous within individual zygotes. High voltage electron microscopy of serial semithick sections from drug-treated zygotes reveals that 90% of the daughter centrosomes contain two centrioles of normal appearance. From these results, we conclude that centrosome reproduction in sea urchin zygotes is not controlled by the accumulation of cyclin proteins or the synthesis of centrosome-specific proteins at each cell cycle. New centrosomes are assembled from preexisting pools of ready-to-use subunits. Furthermore, our results indicate that centrosomal and nuclear events are regulated by separate pathways.
Centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle.
Authors: Gard D L , Hafezi S , Zhang T , Doxsey S J
Published: May 2004

Cycloheximide (500 micrograms/ml) rapidly arrests cleavage, spindle assembly, and cycles of an M-phase-specific histone kinase in early Xenopus blastulae. 2 h after cycloheximide addition, most cells contained two microtubule asters radiating from perinuclear microtubule organizing centers (MTOCs). In contrast, blastomeres treated with cycloheximide for longer periods (3-6 h) contained numerous microtubule asters and MTOCs. Immunofluorescence with an anticentrosome serum and EM demonstrated that the MTOCs in cycloheximide-treated cells were typical centrosomes, containing centrioles and pericentriolar material. We conclude that centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle. In addition, these observations suggest that Xenopus embryos contain sufficient material to assemble 1,000-2,000 centrosomes in the absence of normal protein synthesis.
Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor.
Authors: Ewert M , Shivers B D , Lüddens H , Möhler H , Seeburg P H
Published: May 2004

mAbs bd 17, bd 24, and bd 28 raised against bovine cerebral gamma-aminobutyric acid (GABAA)/benzodiazepine receptors were analyzed for their ability to detect each of 12 GABAA receptor subunits expressed in cultured mammalian cells. Results showed that mAb bd 17 recognizes epitopes on both beta 2 and beta 3 subunits while mAb bd 24 is selective for the alpha 1 subunit of human and bovine, but not of rat origin. The latter antibody reacts with the rat alpha 1 subunit carrying an engineered Leu at position four, documenting the first epitope mapping of a GABAA receptor subunit-specific mAb. In contrast to mAbs bd 17 and bd 24, mAb bd 28 reacts with all GABAA receptor subunits tested but not with a glycine receptor subunit, suggesting the presence of shared epitopes on subunits of GABA-gated chloride channels.
Rotational diffusion of acetylcholine receptors on cultured rat myotubes.
Authors: Velez M , Barald K F , Axelrod D
Published: May 2004

The rotational mobility of acetylcholine receptors (AChR) in the plasma membrane of living rat myotubes in culture is measured in this study by polarized fluorescence recovery after photobleaching (PFRAP). These AChR are known to exist in two distinct classes, evident by labeling with rhodamine alpha-bungarotoxin; clustered AChR that are aggregated in a pattern of highly concentrated speckles and streaks, with each cluster occupying an area of approximately 1,000 microns 2; and nonclustered AChR that appear as diffuse labeling. PFRAP results reported here show that: (a) most clustered AChR (approximately 86%) are rotationally immobile within a time scale of at least several seconds; and (b) most nonclustered AChR (approximately 76%) are rotationally mobile with characteristic times ranging from less than 50 ms to 0.1 s. External cross-linking with the tetravalent lectin concanavalin A immobilizes many nonclustered AChR. PFRAP experiments in the presence of carbachol or cytochalasin D show that the restraints to rotational motion in clusters are remarkably immune to treatments that disperse clusters or disrupt cytoplasmic actin. The experiments also demonstrate the feasibility of using PFRAP to measure rotational diffusion on selected microscopic areas of living nondeoxygenated cells labeled with standard fluorescence probes over a very wide range of time scales, and they also indicate what technical improvements would make PFRAP even more practicable.
A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction.
Authors: Chen Q , Sealock R , Peng H B
Published: May 2004

The 58K protein is a peripheral membrane protein enriched in the acetylcholine receptor (AChR)-rich postsynaptic membrane of Torpedo electric organ. Because of its coexistence with AChRs in the postsynaptic membrane in both electrocytes and skeletal muscle, it is thought to be involved in the formation and maintenance of AChR clusters. Using an mAb against the 58K protein of Torpedo electric organ, we have identified a single protein band in SDS-PAGE analysis of Xenopus myotomal muscle with an apparent molecular mass of 48 kD. With this antibody, the distribution of this protein was examined in the myotomal muscle fibers with immunofluorescence techniques. We found that the 48K protein is concentrated at the myotendinous junctions (MTJs) of these muscle fibers. The MTJ is also enriched in talin and vinculin. By double labeling muscle fibers with antibodies against talin and the 48K protein, these two proteins were found to colocalize at the membrane invaginations of the MTJ. In cultured myotomal muscle cells, the 48K protein and talin are also colocalized at sites of membrane-myofibril interaction. The 48K protein is, however, not found at focal adhesion sites in nonmuscle cells, which are enriched in talin. These data suggest that the 48K protein is specifically involved in the interaction of myofibrillar actin filaments with the plasma membrane at the MTJ. In addition to the MTJ localization, 48K protein is also present at AChR clusters both in vivo and in vitro. Thus, this protein is shared by both the MTJ and the neuromuscular junction.
Distinct and different effects of the oncogenes v-myc and v-src on avian sympathetic neurons: retroviral transfer of v-myc stimulates neuronal proliferation whereas v-src transfer enhances neuronal differentiation.
Authors: Haltmeier H , Rohrer H
Published: May 2004

Immature avian sympathetic neurons are able to proliferate in culture for a limited number of divisions albeit expressing several neuron-specific properties. The effect of avian retroviral transfer of oncogenes on proliferation and differentiation of sympathetic neurons was investigated. Primary cultures of 6-d-old quail sympathetic ganglia, consisting of 90% neuronal cells, were infected by Myelocytomatosis virus (MC29), which contains the oncogene v-myc, and by the v-src-containing Rous sarcoma virus (RSV). RSV infection, in contrast to findings in other cellular systems, resulted in a reduction of neuronal proliferation as determined by 3H-thymidine incorporation (50% of control 4 d after infection) and in increased morphological differentiation. This is reflected by increased neurite production, cell size, and expression of neurofilament protein. In addition, RSV-infected neurons, unlike uninfected cells, are able to survive in culture for time periods up to 14 d in the absence of added neurotrophic factors. In contrast, retroviral transfer of v-myc stimulated the proliferation of immature sympathetic neurons preserving many properties of uninfected cells. The neuron-specific cell surface antigen Q211 and the adrenergic marker enzyme tyrosine hydroxylase were maintained in MC29-infected cells and in the presence of chick embryo extract the cells could be propagated over several weeks and five passages. Within 7 d after infection, the number of Q211-positive neurons increased approximately 100-fold. These data demonstrate distinct and different effects of v-src and v-myc-containing retroviruses on proliferation and differentiation of sympathetic neurons: v-src transfer results in increased differentiation, whereas v-myc transfer maintains an immature status reflected by proliferation, immature morphology, and complex growth requirements. The possibility of expanding immature neuronal populations by transfer of v-myc will be of considerable importance for the molecular analysis of neuronal proliferation and differentiation.
Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8.
Authors: Sonnenberg A , Linders C J , Modderman P W , Damsky C H , Aumailley M , Timpl R
Published: May 2004

The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules, possibly proteoglycans, not belonging to the integrin family.
Isolation of a novel integrin receptor mediating Arg-Gly-Asp-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells. Association of a novel beta 1-related subunit with alpha v.
Authors: Dedhar S , Gray V
Published: May 2004

We report the isolation from two human neuroblastoma cell lines of an Arg-Gly-Asp-dependent integrin complex capable of binding to vitronectin, fibronectin, and type I collagen. The two neuroblastoma cell lines, SK-N-SH and IMR-32, exhibit specific attachment to fibronectin and type I collagen. SK-N-SH cells exhibit a much stronger attachment to vitronectin than the IMR-32 cells, which attach poorly to this substrate. Affinity chromatography of octylglucoside extracts of 125I surface-labeled cells on GRGDSPK-Sepharose columns resulted in the specific binding and elution with GRGDSP of three radiolabeled polypeptides with relative molecular masses of 135, 115, and 90 kD when analyzed by SDS-PAGE under nonreducing conditions. In the SK-N-SH cells the 135- and 90-kD polypeptides were more abundant whereas in the IMR-32 cells the 135- and 115-kD polypeptides were more highly expressed. Liposomes prepared from fractions containing all three polypeptides bound to vitronectin, fibronectin, and type I collagen, whereas liposomes prepared from the 135- and 115-kD polypeptides bound only to fibronectin and type I collagen. Polyclonal antibodies against the alpha/beta complexes of both the vitronectin receptor and the fibronectin receptor immunoprecipitated all three polypeptides. A monoclonal antibody against beta 1 immunoprecipitated only the 135- and the 115-kD polypeptides, whereas a monoclonal antibody against beta 3 subunit immunoprecipitated the 135- and 90-kD polypeptides. Although, the 115-kD polypeptide could be recognized by an anti-beta 1 antibody, a comparison of peptide maps generated by V8 protease digestion of the 115-kD polypeptide and beta 1 subunit immunoprecipitated from GRGDSPK-Sepharose flow-through material indicated that these two polypeptides are distinct. Depletion of the 90-kD polypeptide with an anti-beta 3 monoclonal antibody did not effect the ability of the 115- and 135-kD polypeptides to bind to GRGDSPK-Sepharose. These data indicate that the SK-N-SH and IMR-32 neuroblastoma cells express a novel "beta 1-like" integrin subunit that can associate with alpha v and can bind to RGD. We propose to name this beta 1-like subunit beta n. The data reported here thus demonstrate that in these two cell lines alpha v associates with two beta subunits, beta n and beta 3, forming two heterodimers. The alpha v beta n complex mediates binding to fibronectin and type I collagen, whereas the alpha v beta 3 complex mediates binding to vitronectin.
The permeability of the nuclear envelope in dividing and nondividing cell cultures.
Authors: Feldherr C M , Akin D
Published: May 2004

The objective of this study was to determine whether the permeability characteristics of the nuclear envelope vary during different phases of cellular activity. Both passive diffusion and signal-mediated transport across the envelope were analyzed during the HeLa cell cycle, and also in dividing, confluent (growth-arrested), and differentiated 3T3-L1 cultures. Colloidal gold stabilized with BSA was used to study diffusion, whereas transport was investigated using gold particles coated with nucleoplasmin, a karyophilic Xenopus oocyte protein. The gold tracers were microinjected into the cytoplasm, and subsequently localized within the cells by electron microscopy. The rates of diffusion in HeLa cells were greatest during the first and fifth hours after the onset of anaphase. These results correlate directly with the known rates of pore formation, suggesting that pores are more permeable during or just after reformation. Signal-mediated transport in HeLa cells occurs through channels that are located within the pore complexes and have functional diameters up to 230-250 A. Unlike diffusion, no significant differences in transport were observed during different phases of the cell cycle. A comparison of dividing and confluent 3T3-L1 cultures revealed highly significant differences in the transport of nucleoplasmin-gold across the envelope. The nuclei of dividing cells not only incorporated larger particles (230 A versus 190 A in diameter, including the protein coat), but the relative uptake of the tracer was about seven times greater than that in growth-arrested cells. Differentiation of confluent cells to adipocytes was accompanied by an increase in the maximum diameter of the transport channel to approximately 230 A.
A test of microtubule translocation during neurite elongation.
Authors: Lim S S , Edson K J , Letourneau P C , Borisy G G
Published: May 2004

In a previous study using PC-12 cells (Lim, S. S., P. J. Sammak, and G. G. Borisy, 1989. J. Cell Biol. 109:253-263), we presented evidence that the microtubule component of the neuronal cytoskeleton is differentially dynamic but stationary. However, neurites of PC-12 cells grow slowly, hindering a stringent test of slow axonal transport mechanisms under conditions where growth was substantial. We therefore extended our studies to primary cultures of dorsal root ganglion cells where the rate of neurite outgrowth is rapid. Cells were microinjected with X-rhodamine-labeled tubulin 7-16 h after plating. After a further incubation for 6-18 h, the cells were photobleached with an argon ion laser. Using a cooled charged couple device and video microscopy, the cells were monitored for growth of the neurite and movement and recovery of fluorescence in the bleached zone. As for PC-12 cells, all bleached zones in the neurite recovered their fluorescence, indicating that incorporation of tubulin occurred along the neurite. Despite increases in neurite length of up to 70 microns, and periods of observation of up to 5 h, no movement of bleached zones was observed. We conclude that neurite elongation cannot be accounted for by the transport of a microtubule network assembled only at the cell body. Rather, microtubules turn over all along the length of the neurite and neurite elongation occurs by net assembly at the tip.
CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae.
Authors: Adams A E , Johnson D I , Longnecker R M , Sloat B F , Pringle J R
Published: May 2004

Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton. Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events. To gain further insights into these problems, we sought additional mutants with defects in budding. We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells. Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindles), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle. The arrested mutant cells display delocalized cell-surface deposition associated with a loss of asymmetry of the actin cytoskeleton. CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.
Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity.
Authors: Johnson D I , Pringle J R
Published: May 2004

The Saccharomyces cerevisiae CDC42 gene product is involved in the morphogenetic events of the cell division cycle; temperature-sensitive cdc42 mutants are unable to form buds and display delocalized cell-surface deposition at the restrictive temperature (Adams, A. E. M., D. I. Johnson, R. M. Longnecker, B. F. Sloat, and J. R. Pringle. 1990. J. Cell Biol. 111:131-142). To begin a molecular analysis of CDC42 function, we have isolated the CDC42 gene from a yeast genomic DNA library. The use of the cloned DNA to create a deletion of CDC42 confirmed that the gene is essential. Overexpression of CDC42 under control of the GAL10 promoter was not grossly deleterious to cell growth but did perturb the normal pattern of selection of budding sites. Determination of the DNA and predicted amino acid sequences of CDC42 revealed a high degree of similarity in amino acid sequence to the ras and rho (Madaule, P., R. Axel, and A. M. Myers. 1987. Proc. Natl. Acad. Sci. 84:779-783) families of gene products. The similarities to ras proteins (approximately 40% identical or related amino acids overall) were most pronounced in the regions that have been implicated in GTP binding and hydrolysis and in the COOH-terminal modifications leading to membrane association, suggesting that CDC42 function also involves these biochemical properties. The similarities to the rho proteins (approximately 60% identical or related amino acids overall) were more widely distributed through the coding region, suggesting more extensive similarities in as yet undefined biochemical properties and functions.
Elucidating the early stages of keratin filament assembly.
Authors: Coulombe P A , Fuchs E
Published: May 2004

Because of extraordinarily tight coiled-coil associations of type I and type II keratins, the composition and structure of keratin subunits has been difficult to determine. We report here the use of novel genetic and biochemical methods to explore the early stages of keratin filament assembly. Using bacterially expressed humans K5 and K14, we show that remarkably, these keratins behave as 1:1 complexes even in 9 M urea and in the presence of a reducing agent. Gel filtration chromatography and chemical cross-linking were used to identify heterodimers and heterotetramers as the most stable building blocks of keratin filament assembly. EM suggested that the dimer consists of a coiled-coil of K5 and K14 aligned in register and in parallel fashion, and the tetramer consists of two dimers in antiparallel fashion, without polarity. In 4 M urea, both end-to-end and lateral packing of tetramers occurred, leading to a variety of larger heteromeric complexes. The coexistence of multiple, higher-ordered associations under strongly denaturing conditions suggests that there may not be a serial sequence of events leading to the assembly of keratin intermediate filaments, but rather a number of associations may take place in parallel.
Regulation of anaphase chromosome motion in Tradescantia stamen hair cells by calcium and related signaling agents.
Authors: Zhang D H , Callaham D A , Hepler P K
Published: May 2004

Several lines of evidence support the idea that increases in the intracellular free calcium concentration [( Ca2+]i) regulate chromosome motion. To directly test this we have iontophoretically injected Ca2+ or related signaling agents into Tradescantia stamen hair cells during anaphase and measured their effect on chromosome motion and on the Ca2+ levels. Ca2+ at (+)1 nA for 10 s (approximately 1 microM) causes a transient (20 s) twofold increase in the rate of chromosome motion, while at higher levels it slows or completely stops motion. Ca2+ buffers, EGTA, and 5,5'-dibromo-1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, which transiently suppress the ion level, also momentarily stop motion. Injection of K+, Cl-, or Mg2+, as controls, have no effect on motion. The injection of GTP gamma S, and to a lesser extent GTP, enhances motion similarly to a low level of Ca2+. However, inositol 1,4,5-trisphosphate, ATP gamma S, ATP, and GDP beta S have no effect. Measurement of the [Ca2+]i with indo-1 reveals that the direct injections of Ca2+ produce the expected increases. GTP gamma S, on the other hand, causes only a small [Ca2+]i rise, which by itself is insufficient to increase the rate of chromosome motion. Further studies reveal that any negative ion injection, presumably through hyperpolarization of the membrane potential, generates a similar small pulse of Ca2+, yet these agents have no effect on motion. Two major conclusions from these studies are as follows. (a) Increased [Ca2+]i can enhance the rate of motion, if administered in a narrow physiological window around 1 microM; concentrations above 1 microM or below the physiological resting level will slow or stop chromosomes. (b) GTP gamma S enhances motion by a mechanism that does not cause a sustained uniform rise of [Ca2+]i in the spindle; this effect may be mediated through very localized [Ca2+]i changes or Ca2(+)-independent effectors.
Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts.
Authors: Kao J P , Alderton J M , Tsien R Y , Steinhardt R A
Published: May 2004

Global Ca2+ transients have been observed to precede nuclear envelope breakdown and the onset of anaphase in Swiss 3T3 fibroblasts in 8% (vol/vol) FBS. The occurrence of these Ca2+ transients was dependent on intracellular stores. These Ca2+ transients could be (a) abolished by serum removal without halting mitosis, and (b) eliminated by increasing intracellular Ca2+ buffering capacity through loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffer, via the tetra(acetoxymethyl) ester, without hindering the transition into anaphase. Microinjection of sufficient concentrations of BAPTA buffer could block nuclear envelope breakdown. Pulses of Ca2+ generated by flash photolysis of intracellularly trapped nitr-5, a "caged" Ca2+, could precipitate precocious nuclear envelope breakdown in prophase cells. In metaphase cells, photochemically generated Ca2+ pulses could cause changes in the appearance of the chromosomes, but the length of time required for cells to make the transition from metaphase to anaphase remained essentially unchanged regardless of whether a Ca2+ pulse was photoreleased during metaphase. The results from these photorelease experiments were not dependent on the presence of serum in the medium. Discharging intracellular Ca2+ stores with ionomycin in the presence of 1.8 mM extracellular Ca2+ doubled the time for cells to pass from late metaphase into anaphase, whereas severe Ca2+ deprivation by treatment with ionomycin in EGTA-containing medium halted mitosis. Our results collectively indicate that Ca2+ is actively involved in nuclear envelope breakdown, but Ca2+ signals are likely unnecessary for the metaphase-anaphase transition in Swiss 3T3 fibroblasts. Additional studies of intracellular Ca2+ concentrations in mitotic REF52 and PtK1 cells revealed that Ca2+ transients are not observed at all mitotic stages in all cells. The absence of observable global Ca2+ transients, where calcium buffers can block and pulses of Ca2+ can advance mitotic stages, may imply that the relevant Ca2+ movements are too local to be detected.
Auxilin, a newly identified clathrin-associated protein in coated vesicles from bovine brain.
Authors: Ahle S , Ungewickell E
Published: May 2004

We have identified a new coat protein in clathrin-coated vesicles from bovine brain by urea-SDS gel electrophoresis. The protein was purified from Tris-solubilized coat proteins either by combination of hydroxyapatite chromatography and gel filtration or more rapidly in a single step by immunoaffinity chromatography. The purified protein binds to clathrin triskelia and thereby promotes clathrin assembly into regular 50-100-nm cages. We propose for the new protein the name auxilin (Latin auxilium, meaning support). Auxilin migrates as a 110-kD polypeptide in standard type SDS-PAGE, but in the presence of 6 M urea shifts to a position corresponding to 126 kD. Gel filtration in 6 M guanidinium hydrochloride gives a molecular weight of approximately 86,000. The native protein is monomeric in 0.5 M Tris. Antigenic reactivity and two-dimensional peptide maps gave no evidence of gross similarities between auxilin and any of the other known coated vesicle-associated proteins. Since the structural organization of auxilin does not resemble that of the ubiquitous heterotetrameric HA1 and HA2 adaptor complexes, that are believed to connect clathrin to receptors, it is unlikely that it functions as an adaptor. Immunoblotting did not reveal the presence of auxilin in tissues other than brain. If auxilin and AP 180 are indeed both confined to neuronal cells, as the immunochemical evidence suggests, it might be inferred that both serve to adapt clathrin-coated vesicles to an as yet undisclosed function unique to this cell type.
Cell cycle commitment of rat muscle satellite cells.
Authors: Bischoff R
Published: May 2004

Satellite cells of adult muscle are quiescent myogenic stem cells that can be induced to enter the cell cycle by an extract of crushed muscle (Bischoff, R. 1986. Dev. Biol. 115:140-147). Here, evidence is presented that the extract acts transiently to commit cells to enter the cell cycle. Satellite cells associated with both live and killed rat myofibers in culture were briefly exposed to muscle extract and the increase in cell number was determined at 48 h in vitro, before the onset of fusion. An 8-12-h exposure to extract with killed, but not live, myofibers was sufficient to produce maximum proliferation of satellite cells. Continuous exposure for over 40 h was needed to sustain proliferation of satellite cells on live myofibers. The role of serum factors was also studied. Neither serum nor muscle extract alone was able to induce proliferation of satellite cells. In the presence of muscle extract, however, satellite cell proliferation was directly proportional to the concentration of serum in the medium. These results suggest that mitogens released from crushed muscle produce long-lasting effects that commit quiescent satellite cells to divide, whereas serum factors are needed to maintain progression through the cell cycle. Contact with a viable myofiber modulates the response of satellite cells to growth factors.
Regulation of immunoreactive GAP-43 expression in rat cortical macroglia is cell type specific.
Authors: da Cunha A , Vitković L
Published: May 2004

Growth-associated protein 43 (GAP-43) is an abundant, intensely investigated membrane phosphoprotein of the nervous system (Benowitz, L.I., and A. Routtenberg. 1987. Trends Neurosci. 10:527-532; Skene, J. H. P. 1989. Annu. Rev. Neurosci. 12:127-156), with a hitherto unknown function. We have previously demonstrated that astrocytes, brain macroglial cells, contain GAP-43 (Steisslinger, H. W., V. J. Aloyo, and L. Vitković, 1987. Brain Res. 415:375-379; Vitković, L., H. W. Steisslinger, V. J. Aloyo, and M. Mersel. 1988. Proc. Natl. Acad. Sci. USA. 85:8296-8300; Vitković L., and M. Mersel. 1989. Metab. Brain Dis. 4:47-53). Results from double immunofluorescent labeling experiments presented here show that oligodendrocytes also contain GAP-43 immunoreactivity (GAP-43ir). Thus, all three macroglial cell types of the central nervous system (type I and type 2 astrocytes and oligodendrocytes) contain GAP-43. Whereas immunoreactive GAP-43 is expressed by progenitors of all macroglial cell types, the developmental regulation of its expression is cell type specific. Immunoreactive GAP-43 is downregulated in type 1 astrocytes, and constitutively expressed in both type 2 astrocytes and oligodendrocytes. These results may be relevant to potential function(s) of GAP-43.
The embryonic environment strongly attenuates v-src oncogenesis in mesenchymal and epithelial tissues, but not in endothelia.
Authors: Stoker A W , Hatier C , Bissell M J
Published: May 2004

We demonstrate that the behavior of cells expressing v-src, a tyrosine kinase oncogene, differs profoundly between the embryonic and culture environments. V-src was introduced into avian embryo cells both in culture and in stage-24 embryo limbs, using replication-defective retroviral vectors. These vectors were used as single-hit, cellular markers to determine the environmental influences imposed by normal cells and tissues on clonal cell growth. The marker gene lacZ was coexpressed with v-src in order to locate the descendent cells. In culture, v-src induced rapid morphological transformation and anchorage-independent growth of embryo fibroblasts; the vectors were also tumorigenic in hatchling chickens. In contrast, most of the cell clones expressing v-src in the embryo grew normally without neoplasia. Expression of v-src vectors could be found in a wide range of cell types, demonstrating not only that neoplastic transformation is attenuated in ovo, but also that differentiation commitment in many lineages can be maintained concurrently with oncogene expression. Significantly, the embryonic control of cell growth could be perturbed by v-src under certain conditions. Rare, marked clones showed hyperplasia or dysplasia, and the primitive endothelium could succumb to rapid neoplasia; thus, these embryonic tissues are not inherently deficient in transformation factors. We propose that the environmental conditions imposed on cells in ovo are critical for the attenuation of neoplasia, while cultured cells lose this requisite environment.
Expression of annexins as a function of cellular growth state.
Authors: Schlaepfer D D , Haigler H T
Published: May 2004

Annexins are a structurally related family of Ca2+ binding proteins of undertermined biological function. Annexin I (also called lipocortin 1) is a substrate for the EGF-stimulated tyrosine kinase and is postulated to be involved in mitogenic signal transduction. To investigate further the involvement of lipocortin 1 in cell proliferation, we measured lipocortin 1 levels in normal diploid human foreskin fibroblasts (HFF) to determine whether its expression changed as a function of growth status. For comparison, the expression of annexin V (also called endonexin II) was measured in HFF cells. Endonexin II is a protein with similar Ca2+ and phospholipid binding properties as lipocortin 1, but it is not a substrate for tyrosine kinases. Quiescent HFF cell cultures were induced to proliferate by either subculture to lower cell density, EGF stimulation, or serum stimulation. In all three protocols, proliferating HFF cells contained three- to fourfold higher levels of lipocortin 1 and three- to fourfold lower levels of endonexin II than quiescent HFF cells. In contrast, the expression of annexin II (also called calpactin I) and annexin IV (also called endonexin I) remained relatively unchanged in growing and quiescent HFF cells. Lipocortin 1 synthesis rate was eightfold higher and its turnover rate was 1.5-fold slower in proliferating compared to quiescent HFF cells. Endonexin II synthesis rate remained constant but its turnover rate was 2.2-fold faster in proliferating compared to quiescent HFF cells. In a separate set of experiments, annexin expression levels were measured in cultures of rat PC-12 cells, a pheochromocytoma that ceases proliferation and undergoes reversible differentiation into nondividing neuronlike cells in response to nerve growth factor (NGF). After NGF treatment, PC-12 cells expressed fivefold higher levels of endonexin II and 32-fold higher levels of calpactin 1. Lipocortin 1 and endonexin I were not expressed in PC-12 cells. In summary, lipocortin 1 expression exhibited a positive correlation with cell proliferation in HFF cells. The increased expression of endonexin II in quiescent HFF cells and differentiating PC-12 cells implies that this protein may play a more prominent role in nondividing cells.
Role of PDGF-A expression in the control of vascular smooth muscle cell growth by transforming growth factor-beta.
Authors: Majack R A , Majesky M W , Goodman L V
Published: May 2004

Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory peptide that can inhibit or promote the proliferation of cultured vascular smooth muscle cells (SMCs), depending on cell density (Majack, R. A. 1987. J. Cell Biol. 105:465-471). In this study, we have examined the mechanisms underlying the growth-promoting effects of TGF-beta in confluent SMC cultures. In mitogenesis assays using confluent cells, TGF-beta was found to potentiate the stimulatory effects of serum, PDGF, and basic fibroblast growth factor (bFGF), and was shown to act individually as a mitogen for SMC. In gene and protein expression experiments, TGF-beta was found to regulate the expression of PDGF-A and thrombospondin, two potential mediators of SMC proliferative events. The induction of thrombospondin protein and mRNA was density-dependent, delayed relative to its induction by PDGF and, based on cycloheximide experiments, appeared to depend on the de novo synthesis of an intermediary protein (probably PDGF-A). The relationship between PDGF-A expression and TGF-beta-mediated mitogenesis was investigated, and it was determined that a PDGF-like activity (probably PDGF-A) was the biological mediator of the growth-stimulatory effects of TGF-beta on confluent SMC. The effects of purified homodimers of PDGF-A on SMC replication were investigated, and it was determined that PDGF-AA was mitogenic for cultured SMC, particularly when used in combination with other growth factors such as bFGF and PDGF-BB. The data suggest several molecular mechanisms that may account for the ability of TGF-beta to promote the growth of confluent SMC in culture.
Resting chondrocytes in culture survive without growth factors, but are sensitive to toxic oxygen metabolites.
Authors: Tschan T , Höerler I , Houze Y , Winterhalter K H , Richter C , Bruckner P
Published: May 2004

Chondrocytes in dense suspension culture in agarose survive in serum-free DME because they secrete low molecular mass compounds supporting their own viability. This activity can be replaced by pyruvate, or sulfhydryl compounds, e.g., cysteine or dithioerythritol. Catalase, an enzyme decomposing H2O2, also protects the cells, whereas superoxide dismutase has no effect. Therefore, chondrocytes in culture are sensitive to toxic compounds derived from molecular oxygen, i.e., hydroxyl radicals or hydrogen peroxide spontaneously generated in DME containing ascorbate and ferrous ions. Poly-ADP-ribosylation is an important step in the cascade of events triggered by these compounds. To survive, chondrocytes do not require stimulation by growth factors. They remain resting cells in fully defined, serum-free culture also at low density. Proliferation and hypertrophy can be induced by serum, but not by low cell density alone.
Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein.
Authors: Spriggs M K , Collins P L
Published: May 2004

Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.
Anchorage of secretion-competent dense granules on the plasma membrane of bovine platelets in the absence of secretory stimulation.
Authors: Morimoto T , Ogihara S , Takisawa H
Published: May 2004

The ultrastructural changes in electropermeabilized bovine platelets that accompany the Ca2(+)-induced secretion of serotonin were investigated in ultra-thin sections of chemically fixed cells. Such preparations permitted us to study both the localization of and the structures associated with serotonin-containing dense granules. Localization of dense granules within cells was examined by measuring the shortest distances between the granular membranes and the plasma membrane. About 40% of total granules were located close to the plasma membrane at an average distance of 10.8 +/- 1.6 nm. 71% of the total number of granules were localized at a similar average distance of 12.5 +/- 2.7 nm in intact platelets. The percentage of granules apposed to the plasma membrane corresponded closely to the percentage of total serotonin that was maximally secreted after stimulation of the permeabilized (38 +/- 4.9%) and the intact platelets (72 +/- 3.6%). Furthermore, the percentage of granules anchored to the membrane, but not of those in other regions of permeabilized cells, decreased markedly when cells were stimulated for 30 s by extracellularly added Ca2+. The decrease in the numbers of granules in the vicinity of the plasma membrane corresponded to approximately 22% of the total number of dense granules that were used for measurements of the distances between the two membranes and corresponded roughly to the overall decrease (15%) in the average number of the granules per cell. Most dense granules were found to be associated with meshwork structures of microfilaments. Upon secretory stimulation, nonfilamentous, amorphous structures found between the plasma membrane and the apposed granules formed a bridge-like structure that connected both membranes without any obvious accompanying changes in the microfilament structures. These results suggest that the dense granules that are susceptible to secretory stimulation are anchored to the plasma membrane before stimulation, and that the formation of the bridge-like structure may participate in the Ca2(+)-regulated exocytosis.
c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts display more decondensed nucleosomal organization than normal fibroblasts.
Authors: Laitinen J , Sistonen L , Alitalo K , Hölttä E
Published: May 2004

We have compared the nucleosomal organization of c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts with that of normal fibroblasts by using micrococcal nuclease (MNase) as a probe for the chromatin structure. The bulk chromatin from asynchronously and exponentially growing ras-transformed cells was much more sensitive to MNase digestion than chromatin from the normal cells. Southern hybridization analyses of the MNase digests with probes specific for the ornithine decarboxylase (odc) and c-myc genes showed that the coding and/or 3' end regions of these growth-inducible genes carry a nucleosomal organization both in ras-transformed and normal cells. Studies with cells synchronized by serum starvation showed that in both cell lines the nucleosomal organization of chromatin is relatively condensed at the quiescent state, becomes highly decondensed during the late G1 phase of the cell cycle, and starts again to condense during the S phase. However, in ras-transformed cells the decondensation state stayed much longer than in normal cells. Moreover, irrespective of the phase of the cell cycle the bulk chromatin as well as that of the odc and c-myc genes was more sensitive to MNase digestion in the ras-transformed cell than in the normal fibroblast. Decondensation of the chromatin was also observed in the normal c-Ha-ras protooncogene-transfected cells, but to a lesser extent than in the mutant ras-transformed cells. Whether the increased degree of chromatin decondensation plays a regulatory role in the increased expression of many growth-related genes in the ras-transformed cells remains an interesting object of further study.
The yeast RNA1 gene product necessary for RNA processing is located in the cytosol and apparently excluded from the nucleus.
Authors: Hopper A K , Traglia H M , Dunst R W
Published: May 2004

The yeast RNA1 gene is required for RNA processing and nuclear transport of RNA. The rna1-1 mutation of this locus causes defects in pre-tRNA splicing, processing of the primary pre-rRNA transcript, production of mRNA and export of RNA from the nucleus to the cytosol. To understand how this gene product can pleiotropically affect these processes, we sought to determine the intracellular location of the RNA1 protein. As determined by indirect immunofluorescence localization and organelle fractionation, the RNA1 antigen is found exclusively or primarily in the cytoplasm. Only a tiny fraction of the endogenous protein could be localized to and functional in the nucleus. Furthermore, the RNA1 antigen does not localize differently under stress conditions. These findings suggest that the RNA1 protein is not directly involved in RNA processing but may modify nuclear proteins or otherwise transmit a signal from the cytosol to the nucleus or play a role in maintaining the integrity of the nucleus.