bluuebunny/crossref_metadata_2025 huggingface dataset:
A soluble form of the F3 neuronal cell adhesion molecule promotes neurite outgrowth
Authors: Durbec P , Gennarini G , Goridis C , Rougon G
Published: May 2004

The F3 molecule is a member of the immunoglobulin superfamily anchored to membranes by a glycane-phosphatidylinositol, and is predominantly expressed on subsets of axons of the central and peripheral nervous system. In a previous paper (Gennarini, G., P. Durbec, A. Boned, G. Rougon, and C. Goridis. 1991. Neuron. 6:595-606), we have established that F3 fulfills the operational definition of a cell adhesion molecule and that it stimulates neurite outgrowth when presented to sensory neurons as a surface component of transfected CHO cells. In the present study the question as to whether soluble forms of F3 would be functionally active was addressed in vitro on cultures of mouse dorsal root ganglion neurons. We observed that preparations enriched in soluble F3 had no effect on neuron attachment but enhanced neurite initiation and neurite outgrowth in a dose-dependent manner. By contrast, soluble NCAM-120 does not have any measurable effect on these phenomena. Addition of anti-F3 monovalent antibodies reduced the number of process-bearing neurons and the neuritic output per neuron to control values. Addition of cerebrospinal fluid, a natural source of soluble F3, also stimulated neurite extension, and this effect was partially blocked by anti-F3 antibodies. Our results suggest that the soluble forms of adhesive proteins with neurite outgrowth-promoting properties could act at a distance from their site of release in a way reminiscent of growth and trophic factors.
Expression of a human hepatocyte growth factor/scatter factor cDNA in MDCK epithelial cells influences cell morphology, motility, and anchorage-independent growth
Authors: Uehara Y , Kitamura N
Published: May 2004

The addition of exogenous hepatocyte growth factor (HGF)/scatter factor (SF) to MDCK epithelial cells results in fibroblastic morphology and cell motility. We generated HGF/SF producing MDCK cells by transfection with an expression plasmid containing human HGF/SF cDNA. Production of HGF/SF by these cells induced a change from an epithelial to a fibroblastic morphology and increased cell motility. In addition, the HGF/SF producing cells acquired efficient anchorage-independent growth in soft agar but did not form tumors in nude mice. The morphological change and the stimulation of the anchorage-independent growth were prevented by anti-HGF/SF antibody, suggesting that the factor is secreted and then exerts its effects through cell surface receptors.
The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide
Authors: Foxall C , Watson SR , Dowbenko D , Fennie C , Lasky LA , Kiso M , Hasegawa A , Asa D , Brandley BK
Published: May 2004

The selectins (lectin-EGF-complement binding-cell adhesion molecules [LEC-CAMs]) are a family of mammalian receptors implicated in the initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three known selectins, L-selectin (leukocyte adhesion molecule-1 [LECAM-1]), E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-selectin (GMP-140) share structural features that include a calcium-dependent lectin domain. The sialyl Lewis(x) carbohydrate epitope has been reported as a ligand for both E- and P-selectins. Although L-selectin has been demonstrated to bind to carbohydrates, structural features of potential mammalian carbohydrate ligand(s) have not been well defined. Using an ELISA developed with a sialyl Lewis(x)-containing glycolipid and an E-selectin-IgG chimera, we have demonstrated the direct binding of the L-selectin-IgG chimera to sialyl Lewis(x). This recognition was calcium dependent, and could be blocked by Mel-14 antibody but not by other antibodies. Recognition was confirmed by the ability of cells expressing the native L-selectin to adhere to immobilized sialyl Lewis(x). These data suggest that the sialyl Lewis(x) oligosaccharide may form the basis of a recognition domain common to all three selectins.
Involvement of the stress protein HSP47 in procollagen processing in the endoplasmic reticulum
Authors: Nakai A , Satoh M , Hirayoshi K , Nagata K
Published: May 2004

The 47,000-D collagen-binding glycoprotein, heat shock protein 47 (HSP47), is a stress-inducible protein localized in the ER of collagen-secreting cells. The location and collagen-binding activity of this protein led to speculation that HSP47 might participate in collagen processing. Chemical crosslinking studies were used to test this hypothesis both before and after the perturbation of procollagen processing. The association of procollagen with HSP47 was demonstrated using cleavable bifunctional crosslinking reagents. HSP47 and procollagen were shown to be coprecipitated by the treatment of intact cells with anti-HSP47 or with anticollagen antibodies. Furthermore, several proteins residing in the ER were noted to be crosslinked to and coprecipitated with HSP47, suggesting that these ER-resident proteins may form a large complex in the ER. When cells were heat shocked, or when stable triple-helix formation was inhibited by treatment with alpha,alpha'-dipyridyl, coprecipitation of procollagen with HSP47 was increased. This increase was due to the inhibition of procollagen secretion and to the accumulation of procollagen in the ER. Pulse label and chase experiments revealed that coprecipitated procollagen was detectable as long as procollagen was present in the endoplasmic reticulum of alpha,alpha'-dipyridyl-treated cells. Under normal growth conditions, coprecipitated procollagen was observed to decrease after a chase period of 10-15 min, whereas total procollagen decreased only after 20-25 min. In addition, the intracellular association between HSP47 and procollagen was shown to be disrupted by a change in physiological pH, suggesting that the dissociation of procollagen from HSP47 is pH dependent. These findings support a specific role for HSP47 in the intracellular processing of procollagen, and provide evidence of a new category of "molecular chaperones" in terms of its substrate specificity and the dissociation mechanism.
Lattice defects in microtubules: protofilament numbers vary within individual microtubules
Authors: Chrétien D , Metoz F , Verde F , Karsenti E , Wade RH
Published: May 2004

We have used cryo-electron microscopy of vitrified specimens to study microtubules assembled both from three cycle purified tubulin (3x-tubulin) and in cell free extracts of Xenopus eggs. In vitro assembled 3x-tubulin samples have a majority of microtubules with 14 protofilaments whereas in cell extracts most microtubules have 13 protofilaments. Microtubule polymorphism was observed in both cases. The number of protofilaments can change abruptly along individual microtubules usually by single increments but double increments also occur. For 3x-tubulin, increasing the magnesium concentration decreases the proportion of 14 protofilament microtubules and decreases the average separation between transitions in these microtubules. Protofilament discontinuities may correspond to dislocation-like defects in the microtubule surface lattice.
Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210
Authors: Hamaguchi JR , Tobey RA , Pines J , Crissman HA , Hunter T , Bradbury EM
Published: May 2004

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.
Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein
Authors: Amatruda JF , Cooper JA
Published: May 2004

Capping protein binds the barbed ends of actin filaments and nucleates actin filament assembly in vitro. We purified capping protein from Saccharomyces cervisiae. One of the two subunits is the product of the CAP2 gene, which we previously identified as the gene encoding the beta subunit of capping protein based on its sequence similarity to capping protein beta subunits in chicken and Dictyostelium (Amatruda, J. F., J. F. Cannon, K. Tatchell, C. Hug, and J. A. Cooper. 1990. Nature (Lond.) 344:352-354). Yeast capping protein has activity in critical concentration and low-shear viscometry assays consistent with barbed-end capping activity. Like chicken capping protein, yeast capping protein is inhibited by PIP2. By immunofluorescence microscopy yeast capping protein colocalizes with cortical actin spots at the site of bud emergence and at the tips of growing buds and shmoos. In contrast, capping protein does not colocalize with actin cables or with actin rings at the site of cytokinesis.
Neurite outgrowth in peripherin-depleted PC12 cells
Authors: Troy CM , Greene LA , Shelanski ML
Published: May 2004

Peripherin is the major neuronal intermediate filament (IF) protein in PC12 cells and both its synthesis and amount increase during nerve growth factor (NGF) promoted neuronal differentiation. To address the question of the biological function of peripherin in neurite initiation we have used an antisense oligonucleotide complementary to the 5' region of peripherin mRNA to specifically inhibit its transcription. The oligonucleotide blocks both the synthesis of peripherin and its increase in response to NGF. Peripherin was found to be a stable protein with a cellular half-life of approximately 7 d. 6 wk of incubation with the oligonucleotide decreases peripherin to 11% of the level in naive control cells and to 3% of that in NGF-treated control cells. Despite the depletion, NGF elicits apparently normal neurite outgrowth from the oligonucleotide-treated cells. As evaluated by EM, there are few IFs in these cells, either in the cell bodies or neurites. There is no compensatory increase in NF-M, NF-L, or vimentin levels as a result of the inhibition of peripherin synthesis. These findings suggest that peripherin is not required for neurite formation, but is necessary for the formation of a cellular IF network which could be involved in process stability. They also demonstrate the utility of antisense oligonucleotides for the study of proteins with long half-lives.
Ganglioside modulation of neural cell adhesion molecule and N-cadherin-dependent neurite outgrowth
Authors: Doherty P , Ashton SV , Skaper SD , Leon A , Walsh FS
Published: May 2004

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human neural cell adhesion molecule (NCAM) or chick N-cadherin as a culture substrate for PC12 cells. NCAM and N-cadherin in the monolayer directly promote neurite outgrowth from PC12 cells via a G-protein-dependent activation of neuronal calcium channels. In the present study we show that ganglioside GM1 does not directly activate this pathway in PC12 cells. However, the presence of GM1 (12.5-100 micrograms/ml) in the co-culture was associated with a potentiation of NCAM and N-cadherin-dependent neurite outgrowth. Treatment of PC12 cells with GM1 (100 micrograms/ml) for 90 min led to trypsin-stable increases in both beta-cholera toxin binding to PC12 cells and an enhanced neurite outgrowth response to N-cadherin. The ganglioside response could be fully inhibited by treatment with pertussis toxin. These data are consistent with exogenous gangliosides enhancing neuritic growth by promoting cell adhesion molecule-induced calcium influx into neurons.
Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast
Authors: Klein F , Laroche T , Cardenas ME , Hofmann JF , Schweizer D , Gasser SM
Published: May 2004

Topoisomerase II (topoII) and RAP1 (Repressor Activator Protein 1) are two abundant nuclear proteins with proposed structural roles in the higher-order organization of chromosomes. Both proteins co-fractionate as components of nuclear scaffolds from vegetatively growing yeast cells, and both proteins are present as components of pachytene chromosome, co-fractionating with an insoluble subfraction of meiotic nuclei. Immunolocalization using antibodies specific for topoII shows staining of an axial core of the yeast meiotic chromosome, extending the length of the synaptonemal complex. RAP1, on the other hand, is located at the ends of the paired bivalent chromosomes, consistent with its ability to bind telomeric sequences in vitro. In interphase nuclei, again in contrast to anti-topoII, anti-RAP1 gives a distinctly punctate staining that is located primarily at the nuclear periphery. Approximately 16 brightly staining foci can be identified in a diploid nucleus stained with anti-RAP1 antibodies, suggesting that telomeres are grouped together, perhaps through interaction with the nuclear envelope.
Immunological evidence for eight spans in the membrane domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum
Authors: Roitelman J , Olender EH , Bar-Nun S , Dunn WA , Simoni RD
Published: May 2004

We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. Finer-Moore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli beta-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg224 through Leu242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr284 through Glu302 (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys248 and Asp276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.
A single mRNA, transcribed from an alternative, erythroid-specific, promoter, codes for two non-myristylated forms of NADH-cytochrome b5 reductase
Authors: Pietrini G , Aggujaro D , Carrera P , Malyszko J , Vitale A , Borgese N
Published: May 2004

Two forms of NADH-cytochrome b5 reductase are produced from one gene: a myristylated membrane-bound enzyme, expressed in all tissues, and a soluble, erythrocyte-specific, isoform. The two forms are identical in a large cytoplasmic domain (Mr approximately 30,000) and differ at the NH2-terminus, which, in the membrane form, is responsible for binding to the bilayer, and which contains the myristylation consensus sequence and an additional 14 uncharged amino acids. To investigate how the two differently targeted forms of the reductase are produced, we cloned a reductase transcript from reticulocytes, and studied its relationship to the previously cloned liver cDNA. The reticulocyte transcript differs from the liver transcript in the 5' non-coding portion and at the beginning of the coding portion, where the seven codons specifying the myristoylation consensus are replaced by a reticulocyte-specific sequence which codes for 13 non-charged amino acids. Analysis of genomic reductase clones indicated that the ubiquitous transcript is generated from an upstream "housekeeping" type promoter, while the reticulocyte transcript originates from a downstream, erythroid-specific, promoter. In vitro translation of the reticulocyte-specific mRNA generated two products: a minor one originating from the first AUG, and a major one starting from a downstream AUG, as indicated by mutational analysis. Both the AUGs used as initiation codons were in an unfavorable sequence context. The major, lower relative molecular mass product behaved as a soluble protein, while the NH2-terminally extended minor product interacted with microsomes in vitro. The generation of soluble reductase from a downstream AUG was confirmed in vivo, in Xenopus oocytes. Thus, differently localized products, with respect both to tissues and to subcellular compartments, are generated from the same gene by a combination of transcriptional and translational mechanisms.
Examining the function and regulation of hsp 70 in cells subjected to metabolic stress
Authors: Beckmann RP , Lovett M , Welch WJ
Published: May 2004

Members of the heat-shock protein (hsp) 70 family, distributed within various cellular compartments, have been implicated in facilitating protein maturation events. In particular, related hsp 70 family members appear to bind nascent polypeptides which are in the course of synthesis and/or translocation into organelles. We previously reported that in normal, unstressed cells, cytosolic hsp 70 (hsp 72/73) interacted transiently with nascent polypeptides. We suspect that such interactions function to prevent or slow down the folding of the nascent polypeptide chain. Once synthesis is complete, and now with all of the information for folding present, the newly synthesized protein appears to commence along its folding pathway, accompanied by the ATP-dependent release of hsp 72/73. Herein, we examined how these events occur in cells subjected to different types of metabolic stress. In cells exposed to either an amino acid analog or sodium arsenite, two potent inducers of the stress response, newly synthesized proteins bind to but are not released from hsp 70. Under these conditions of metabolic stress, we suspect that the newly synthesized proteins are unable to commence proper folding and consequently remain bound to their hsp 70 chaperone. In cells subjected to heat shock, a large number of both newly synthesized as well as mature proteins are rendered insoluble. Within this insoluble material are appreciable amounts of hsp 72/73. Finally, we show that in cells depleted of ATP, the release of hsp 70 from maturing proteins is inhibited. Thus, in cells experiencing metabolic stress, newly synthesized proteins unable to properly fold, as will as mature proteins which begin to unfold become stably bound to hsp 72/73. As a consequence and over time, the free or available levels of pre-existing hsp 72/73 are reduced. We propose that this reduction in the available levels of hsp 72/73 is the trigger by which the stress response is initiated.
Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp70 as a potential autoregulatory factor
Authors: Baler R , Welch WJ , Voellmy R
Published: May 2004

Heat shock genes encode proteins (hsp's) that play important structural roles under normal circumstances and are essential to the cells' ability to survive environmental insults. Evidence is presented herein that transcriptional regulation of hsp gene expression is linked with the regulation of overall protein synthesis as well as with the accumulation of proteins denatured by stressful events. The factor that connects the three processes appears to be one of the hsp's, presumably a member(s) of the hsp70 family. Biochemical experiments demonstrate that complexes containing hsp70 and heat shock transcription factor, the specific regulator of hsp gene activity, are formed in the cells.
Biosynthesis of lipoprotein: location of nascent apoAI and apoB in the rough endoplasmic reticulum of chicken hepatocytes
Authors: Dixon JL , Chattapadhyay R , Huima T , Redman CM , Banerjee D
Published: May 2004

Our previous studies showed that in hepatic RER of young chickens, nascent apoAI is not associated with lipoprotein particles and only becomes part of these lipoprotein structures in the Golgi. In this study, we have used three different methodologies to determine the locations of apoAI and apoB in the RER and compared them to that of albumin. Immunoelectron microscopic examination of the RER cell fractions showed that both apoAI and apoB were associated only with the RER membrane whereas albumin was located both within the lumen and on the limiting membrane of the vesicles. To examine the possibility of membrane integration of nascent apoAI and apoB in the RER, we administered L-[3H]leucine to young chickens for 10 min, isolated RER, treated this cell fraction with buffers of varying pH, and measured the release of radioactive albumin, apoAI, and apoB. The majority of nascent apoAI (64%), nascent apoB (100%), and nascent albumin (97%) was released from RER vesicles at pH 11.2, suggesting that, like albumin, apolipoproteins are not integrated within the membrane. To determine if nascent apoproteins are exposed to the cytoplasmic surface, we administered L-[3H]leucine to young chickens and at various times isolated RER and Golgi cell fractions. Radioactive RER and Golgi cell fractions were treated with exogenous protease and the percent of nascent apoAI and apoB accessible to proteolysis was determined and compared to that of albumin. At 5, 10, and 20 min of labeling, 35-56% of nascent apoAI and 60-75% of apoB in RER were degraded, while albumin was refractive to this treatment. At all times both apolipoproteins and albumin present in Golgi cell fractions were protected from proteolysis. These biochemical and morphological findings indicate that apoAI and apoB are associated with the rough microsomal membrane and are partially exposed to the cytoplasmic surface at early stages of secretion. They may later enter the luminal side of the ER and, on entering the Golgi, form lipoprotein particles.
Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-L1 adipocytes: effects of ATP insulin, and GTP gamma S and localization of GLUT4 to clathrin lattices
Authors: Robinson LJ , Pang S , Harris DS , Heuser J , James DE
Published: May 2004

Insulin stimulates the movement of two glucose transporter isoforms (GLUT1 and GLUT4) to the plasma membrane (PM) in adipocytes. To study this process we have prepared highly purified PM fragments by gently sonicating 3T3-L1 adipocytes grown on glass coverslips. Using confocal laser immunofluorescence microscopy we observed increased PM labeling for GLUT1 (2.3-fold) and GLUT4 (eightfold) after insulin treatment in intact cells. EM immunolabeling of PM fragments indicated that in the nonstimulated state GLUT4 was mainly localized to flat clathrin lattices. Whereas GLUT4 labeling of clathrin lattices was only slightly increased after insulin treatment, labeling of uncoated PM regions was markedly increased with insulin. These data suggest that GLUT4 recycles from the cell surface both in the presence and absence of insulin. In streptolysin-O permeabilized adipocytes, insulin, and GTP gamma S increased PM levels of GLUT4 to a similar extent as observed with insulin in intact cells. In the absence of an exogenous ATP source the magnitude of these effects was considerably reduced. Removal of ATP per se caused a significant increase in cell surface levels of GLUT4 suggesting that ATP may be required for intracellular sequestration of these transporters. When insulin and GTP gamma S were added together, in the presence of ATP, PM GLUT4 levels were similar to levels observed when either insulin or GTP gamma S was added individually. Addition of GTP gamma S was able to overcome this ATP dependence of insulin-stimulated GLUT4 movement. GTP gamma S had no effect on constitutive secretion of adipsin in permeabilized cells. In addition, there was no effect of insulin or GTP gamma S on GLUT4 movement to the PM in noninsulin sensitive streptolysin-O-permeabilized 3T3-L1 fibroblasts overexpressing GLUT4. We conclude that the insulin-stimulated movement of GLUT4 to the cell surface in adipocytes may require ATP early in the insulin signaling pathway and a GTP-binding protein(s) at a later step(s). We propose that the association of GLUT4 with clathrin lattices may be important in maintaining the exclusive intracellular location of this transporter in the absence of insulin.
Proteolysis of the major yolk glycoproteins is regulated by acidification of the yolk platelets in sea urchin embryos
Authors: Mallya SK , Partin JS , Valdizan MC , Lennarz WJ
Published: May 2004

The precise function of the yolk platelets of sea urchin embryos during early development is unknown. We have shown previously that the chemical composition of the yolk platelets remains unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and total protein content after fertilization and early development. However, the platelet is not entirely static because the major 160-kD yolk glycoprotein YP-160 undergoes limited, step-wise proteolytic cleavage during early development. Based on previous studies by us and others, it has been postulated that yolk platelets become acidified during development, leading to the activation of a cathepsin B-like yolk proteinase that is believed to be responsible for the degradation of the major yolk glycoprotein. To investigate this possibility, we studied the effect of addition of chloroquine, which prevents acidification of lysosomes. Consistent with the postulated requirement for acidification, it was found that chloroquine blocked YP-160 breakdown but had no effect on embryonic development. To directly test the possibility that acidification of the yolk platelets over the course of development temporally correlated with YP-160 proteolysis, we added 3-(2,4-dinitroanilo)-3-amino-N-methyldipropylamine (DAMP) to eggs or embryos. This compound localizes to acidic organelles and can be detected in these organelles by EM. The results of these studies revealed that yolk platelets did, in fact, become transiently acidified during development. This acidification occurred at the same time as yolk protein proteolysis, i.e., at 6 h after fertilization (64-cell stage) in Strongylocentrotus purpuratus and at 48 h after fertilization (late gastrula) in L. pictus. Furthermore, the pH value at the point of maximal acidification of the yolk platelets in vivo was equal to the pH optimum of the enzyme measured in vitro, indicating that this acidification is sufficient to activate the enzyme. For both S. purpuratus and Lytechinus pictus, the observed decrease in the pH was approximately 0.8 U, from 7.0 to 6.2. The trypsin inhibitor benzamidine was found to inhibit the yolk proteinase in vivo. By virtue of the fact that this inhibitor was reversible we established that the activity of the yolk proteinase is developmentally regulated even though the enzyme is present throughout the course of development. These findings indicate that acidification of yolk platelets is a developmentally regulated process that is a prerequisite to initiation of the catabolism of the major yolk glycoprotein.
Gas2, a growth arrest-specific protein, is a component of the microfilament network system
Authors: Brancolini C , Bottega S , Schneider C
Published: May 2004

In this report we analyze the protein product of a growth arrest-specific gene, gas2, by means of an affinity-purified antibody raised against the protein produced in bacteria. The regulation of Gas2 biosynthesis reflects the pattern of mRNA expression (Schneider, C., R. King, and L. Philipson. 1988. Cell. 54:787-793): its relative level is tightly associated with growth arrest. Gas2 seems to be regulated also at the posttranslational level via a phosphorylation mechanism. Gas2 is well conserved during the evolution with the same apparent molecular mass (36 kD) between mouse and human. We also demonstrate that Gas2 is a component of the microfilament system. It colocalizes with actin fiber, at the cell border and also along the stress fiber, in growth-arrested NIH 3T3 cells. The pattern of distribution, detected in arrested cells, can also be observed in growing cells when they are microinjected with the purified GST-Gas2 protein. In none of the analyzed oncogene-transformed NIH 3T3 cell lines was Gas2 expression induced under serum starvation.
Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells
Authors: Navone F , Niclas J , Hom-Booher N , Sparks L , Bernstein HD , McCaffrey G , Vale RD
Published: May 2004

To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOH-terminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesin-like protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with translational regulation in certain mRNAs. After transient expression in CV-1 cells, the kinesin heavy chain showed both a diffuse distribution and a filamentous staining pattern that coaligned with microtubules but not vimentin intermediate filaments. Altering the number and distribution of microtubules with taxol or nocodazole produced corresponding changes in the localization of the expressed kinesin heavy chain. The expressed NH2-terminal motor and the COOH-terminal tail domains, but not the alpha-helical coiled coil rod domain, also colocalized with microtubules. The finding that both the kinesin motor and tail domains can interact with cytoplasmic microtubules raises the possibility that kinesin could crossbridge and induce sliding between microtubules under certain circumstances.
Flagellar microtubule dynamics in Chlamydomonas: cytochalasin D induces periods of microtubule shortening and elongation; and colchicine induces disassembly of the distal, but not proximal, half of the flagellum
Authors: Dentler WL , Adams C
Published: May 2004

To study the mechanisms responsible for the regulation of flagellar length, we examined the effects of colchicine and Cytochalasin D (CD) on the growth and maintenance of Chlamydomonas flagella on motile wild type cells as well as on pf 18 cells, whose flagella lack the central microtubules and are immobile. CD had no effect on the regeneration of flagella after deflagellation but it induced fully assembled flagella to shorten at an average rate of 0.03 microns-min. Cells remained fully motile in CD and even stubby flagella continued to move, indicating that flagellar shortening did not selectively disrupt machinery necessary for motility. To observe the effects of the drug on individual cells, pf 18 cells were treated with CD and flagella on cells were monitored by direct observation over a 5-hour period. Flagella on control pf 18 cells maintained their initial lengths throughout the experiment but flagella on CD-treated cells exhibited periods of elongation, shortening, and regrowth suggestive of the dynamic behavior of cytoplasmic microtubules observed in vitro and in vitro. Cells behaved individually, with no two cells exhibiting the same flagellar behavior at any given time although both flagella on any single cell behaved identically. The rate of drug-induced flagellar shortening and elongation in pf 18 cells varied from 0.08 to 0.17 microns-min-1, with each event occurring over 10-60-min periods. Addition of colchicine to wild type and pf 18 cells induced flagella to shorten at an average rate of 0.06 microns-min-1 until the flagella reached an average of 73% of their initial length, after which they exhibited no further shortening or elongation. Cells treated with colchicine and CD exhibited nearly complete flagellar resorption, with little variation in flagellar length among cells. The effects of these drugs were reversible and flagella grew to normal stable lengths after drug removal. Taken together, these results show that the distal half to one-third of the Chlamydomonas flagellum is relatively unstable in the presence of colchicine but that the proximal half to two-thirds of the flagellum is stable to this drug. In contrast to colchicine, CD can induce nearly complete flagellar microtubule disassembly as well as flagellar assembly. Flagellar microtubules must, therefore, be inherently unstable, and flagellar length is stabilized by factors that are sensitive, either directly or indirectly, to the effects of CD.
Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung
Authors: Hennemann H , Suchyna T , Lichtenberg-Fraté H , Jungbluth S , Dahl E , Schwarz J , Nicholson BJ , Willecke K
Published: May 2004

From a mouse genomic library, a clone has been isolated that codes for a connexin-homologous sequence of 358 amino acids. Because of its theoretical molecular mass of 40.418 kD it is named connexin40 (Cx40). Based on both protein and nucleotide sequence, mouse Cx40 is more closely related to mouse Cx43 (alpha subgroup of connexins) than to mouse Cx32 (beta subgroup). The highest overall homology detected, however, was to chick Cx42 (67% amino acid and 86% nucleotide identity), raising the possibility that Cx40 may be the mouse analogue. The coding region of Cx40 is uninterrupted by introns and is detected as a single copy gene in the mouse genome. High stringency hybridization of Northern blots with the coding sequence of Cx40 identified a single transcript of 3.5 kb that is at least 16-fold more abundant in lung-similar to mouse Cx37-than in other adult tissues (kidney, heart, and skin). In embryonic kidney, skin, and liver the level of the Cx40 transcript is two- to fourfold higher than in the corresponding adult tissues. Microinjection of Cx40 cRNA into Xenopus oocytes induced functional cell-to-cell channels between pairs. These channels show a symmetrical and markedly cooperative closure in response to transjunctional voltage (Boltzmann parameters of Vo = +/- 35 mV; A = 0.32) which is also fast relative to other connexin channels recorded similarly (tau = 580 ms at Vj of +/- 50 mV). Although Cx40-expressing oocytes did not couple efficiently with oocytes expressing endogenous connexins, they did couple well to Cx37-expressing oocytes. The heterotypic channels which formed had voltage-gating properties modified from those of the original homotypic forms. Transfection of mouse Cx40 DNA, under control of the SV-40 early promoter, into coupling-deficient human HeLa or SK-Hep-1 cells resulted in expression of the expected transcript and restoration of fluorescent dye transfer in transfected clones.
Identification of amino acid sequences in the integrin beta 1 cytoplasmic domain implicated in cytoskeletal association
Authors: Reszka AA , Hayashi Y , Horwitz AF
Published: May 2004

Wild-type and mutant chicken integrin beta 1 subunit (beta 1c) cDNAs were expressed in NIH 3T3 cells and assayed for localization in focal adhesions of cells plated on fibronectin substrates. Focal adhesion localization in stable transfected cells was assayed by indirect immunofluorescent staining with chicken-specific anti-beta 1c antibodies. Mutant beta 1c integrins containing internal deletions of 13 amino acids adjacent to the membrane, delta 759-771, and 20 centrally located amino acids, delta 771-790, localized in focal adhesions demonstrating that sequences required for direction to focal adhesion structures were not limited to one region of the cytoplasmic domain. Point mutations revealed three clusters of amino acids which contribute to localization in focal adhesions. These three clusters or signals are: cyto-1 (764-774), cyto-2 (785-788), and cyto-3 (797-800). The 11-residue cyto-1 signal is only found on integrin beta subunit sequences, except beta 4. Four residues within this region, D764, F768, F771, and E774, could not be altered without reducing focal adhesion staining intensities, and likely form a signal that occupies one side of an alpha helix. Mutations involving two cyto-1 residues, K770 and F771, also appeared to affect heterodimer affinity and specificity. Cyto-2 (785-788,), NPIY, is an NPXY signal that forms a tight turn motif. Cyto-2 provides a structural conformation, which when perturbed by proline removal or addition, inhibits integrin localization in focal adhesions. Cyto-3 (797-800), NPKY, resembles cyto-2, however, the nonconserved proline residue can be replaced without alteration of the localization phenotype. Cyto-3, therefore, constitutes a unique integrin signal, NXXY. Both serine and tyrosine residues at positions 790 and 788, respectively, which have been implicated in integrin phosphorylation/regulation, were conservatively replaced without detectable effect on focal adhesion localization. However, acidic replacements for these amino acids reduced focal adhesion staining intensities, suggesting that phosphorylation at these sites may negatively regulate integrin function.
Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide
Authors: Drake SL , Klein DJ , Mickelson DJ , Oegema TR , Furcht LT , McCarthy JB
Published: May 2004

Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S-heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S-HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN-C/H II. These data support the hypothesis that a phosphatidylinositol-anchored HSPG on mouse melanoma cells (MPIHP-63) initiates recognition to FN-C/H II, and implicate PI-associated signal transduction pathways in mediating melanoma cell adhesion to this defined ligand.
Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility
Authors: Hardwick C , Hoare K , Owens R , Hohn HP , Hook M , Moore D , Cripps V , Austen L , Nance DM , Turley EA
Published: May 2004

A cDNA encoding a unique hyaluronan receptor has been molecularly cloned from a lambda GT11 3T3 cDNA expression library. Immunoblot analyses of cell lysates, using antibodies to peptides encoded in the cDNA, specifically react with a 58-kD protein. This protein is regulated by the mutant H-ras gene in cells containing a metallothionein promoter H-ras hybrid gene. Further, antibodies to peptide sequences encoded in the cDNA block the increase in locomotion resulting from induction of the mutant H-ras gene in this cell line. In a transblot assay, the bacterially expressed protein binds to biotinylated hyaluronan. Antibodies to peptides encoded in the cDNA react in immunoblot assays with the 58- and 52-kD proteins of a novel hyaluronan receptor complex previously implicated in cell locomotion. Furthermore, antibodies specific to the 58- and 52-kD proteins, which block ras-induced locomotion, also cross-react with the expressed, encoded protein. The gene product described here appears to be a new type of hyaluronan receptor that is involved in cell locomotion. It is named RHAMM, an acronym for receptor for hyaluronan-mediated motility.
Malaria sporozoites and circumsporozoite proteins bind specifically to sulfated glycoconjugates
Authors: Pancake SJ , Holt GD , Mellouk S , Hoffman SL
Published: May 2004

Circumsporozoite (CS) proteins, which densely coat malaria (Plasmodia) sporozoites, contain an amino acid sequence that is homologous to segments in other proteins which bind specifically to sulfated glycoconjugates. The presence of this homology suggests that sporozoites and CS proteins may also bind sulfated glycoconjugates. To test this hypothesis, recombinant P. yoelii CS protein was examined for binding to sulfated glycoconjugate-Sepharoses. CS protein bound avidly to heparin-, fucoidan-, and dextran sulfate-Sepharose, but bound comparatively poorly to chondroitin sulfate A- or C-Sepharose. CS protein also bound with significantly lower affinity to a heparan sulfate biosynthesis-deficient mutant cell line compared with the wild-type line, consistent with the possibility that the protein also binds to sulfated glycoconjugates on the surfaces of cells. This possibility is consistent with the observation that CS protein binding to hepatocytes, cells invaded by sporozoites during the primary stage of malaria infection, was inhibited by fucoidan, pentosan polysulfate, and heparin. The effects of sulfated glycoconjugates on sporozoite infectivity were also determined. P. berghei sporozoites bound specifically to sulfatide (galactosyl[3-sulfate]beta 1-1ceramide), but not to comparable levels of cholesterol-3-sulfate, or several examples of neutral glycosphingolipids, gangliosides, or phospholipids. Sporozoite invasion into hepatocytes was inhibited by fucoidan, heparin, and dextran sulfate, paralleling the observed binding of CS protein to the corresponding Sepharose derivatives. These sulfated glycoconjugates blocked invasion by inhibiting an event occurring within 3 h of combining sporozoites and hepatocytes. Sporozoite infectivity in mice was significantly inhibited by dextran sulfate 500,000 and fucoidan. Taken together, these data indicate that CS proteins bind selectively to certain sulfated glycoconjugates, that sporozoite infectivity can be inhibited by such compounds, and that invasion of host hepatocytes by sporozoites may involve interactions with these types of compounds.
Export of mRNA from microinjected nuclei of Xenopus laevis oocytes.
Authors: Dargemont C , Kühn L C
Published: May 2004

Export of mRNA from the nucleus to the cytoplasm was studied in mature Xenopus laevis oocytes. In vitro transcribed, capped 32P-labeled mRNA was microinjected into nuclei, and its appearance in the cytoplasm measured by counting radioactivity or by RNA extraction and gel electrophoresis. Both for a 5.0-kb transferrin receptor mRNA and a 2.0-kb 4F2 antigen heavy chain mRNA we found saturable transport with an apparent Km of 3.6 x 10(8) molecules per oocyte nucleus. Under non-saturating conditions the half-time for mRNA export from the nucleus was approximately 2 min at 20 degrees C. At higher concentrations of injected mRNA this half-time was prolonged, and the maximal transport rate was reached at approximately 1.6 x 10(8) molecules/min. mRNA transport showed properties of an energy-dependent mechanism, since it was inhibited at 4 degrees C or by ATP depletion. Co-injection of the cap dinucleotide m7GpppG blocked the export effectively, suggesting a role for the cap in this process. The export was also inhibited by the pre-injection of wheat germ agglutinin. The effect of the lectin was specific and abolished by co-injection of N-acetylglucosamine. Finally, we found significant competitive inhibition in mRNA export by the presence of tRNA. Our results suggest that mRNA transport is a facilitated process which may share common steps with tRNA transport. Preliminary gel retardation experiments show that injected mRNA associates with endogenous nuclear proteins and suggest an exchange of some of the bound components during the transport to the cytoplasm.
Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly.
Authors: Hoyt M A , He L , Loo K K , Saunders W S
Published: May 2004

Two Saccharomyces cerevisiae genes, CIN8 and KIP1 (a.k.a. CIN9), were identified by their requirement for normal chromosome segregation. Both genes encode polypeptides related to the heavy chain of the microtubule-based force-generating enzyme kinesin. Cin8p was found to be required for pole separation during mitotic spindle assembly at 37 degrees C, although overproduced Kip1p could substitute. At lower temperatures, the activity of at least one of these proteins was required for cell viability, indicating that they perform an essential but redundant function. Cin8p was observed to be a component of the mitotic spindle, colocalizing with the microtubules that lie between the poles. Taken together, these findings suggest that these proteins interact with spindle microtubules to produce an outwardly directed force acting upon the poles.