bluuebunny/crossref_metadata_2025 huggingface dataset:
Type 2X-myosin heavy chain is coded by a muscle fiber type-specific and developmentally regulated gene.
Authors: DeNardi C , Ausoni S , Moretti P , Gorza L , Velleca M , Buckingham M , Schiaffino S
Published: May 2004

We have previously reported the identification of a distinct myosin heavy chain (MyHC) isoform in a major subpopulation of rat skeletal muscle fibers, referred to as 2X fibers (Schiaffino, S., L. Gorza, S. Sartore, L. Saggin, M. Vianello, K. Gundersen, and T. Lømo. 1989. J. Muscle Res. Cell Motil. 10:197-205). However, it was not known whether 2X-MyHC is the product of posttranslational modification of other MyHCs or is coded by a distinct mRNA. We report here the isolation and characterization of cDNAs coding a MyHC isoform that is expressed in type 2X skeletal muscle fibers. 2X-MyHC transcripts differ from other MyHC transcripts in their restriction map and 3' end sequence and are thus derived from a distinct gene. In situ hybridization analyses show that 2X-MyHC transcripts are expressed at high levels in the diaphragm and fast hindlimb muscles and can be coexpressed either with 2B- or 2A-MyHC transcripts in a number of fibers. At the single fiber level the distribution of each MyHC mRNA closely matches that of the corresponding protein, determined by specific antibodies on serial sections. In hindlimb muscles 2X-, 2A-, and 2B-MyHC transcripts are first detected by postnatal day 2-5 and display from the earliest stages a distinct pattern of distribution in different muscles and different fibers. The emergence of type 2 MyHC isoforms thus defines a distinct neonatal phase of fiber type differentiation during muscle development. The functional significance of MyHC isoforms is discussed with particular reference to the velocity of shortening of skeletal muscle fibers.
Orientation and three-dimensional organization of actin filaments in dividing cultured cells.
Authors: Fishkind D J , Wang Y L
Published: May 2004

The current hypothesis of cytokinesis suggests that contractile forces in the cleavage furrow are generated by a circumferential band of actin filaments. However, relatively little is known about the global organization of actin filaments in dividing cells. To approach this problem we have used fluorescence-detected linear dichroism (FDLD) microscopy to measure filament orientation, and digital optical sectioning microscopy to perform three-dimensional reconstructions of dividing NRK cells stained with rhodamine-phalloidin. During metaphase, actin filaments in the equatorial region show a slight orientation along the spindle axis, while those in adjacent regions appear to be randomly distributed. Upon anaphase onset and through cytokinesis, the filaments become oriented along the equator in the furrow region, and along the spindle axis in adjacent regions. The degree of orientation appears to be dependent on cell-cell and cell-substrate adhesions. By performing digital optical sectioning microscopy on a highly spread NRK subclone, we show that actin filaments organize as a largely isotropic cortical meshwork in metaphase cells and convert into an anisotropic network shortly after anaphase onset, becoming more organized as cytokinesis proceeds. The conversion is most dramatic on the adhering ventral surface which shows little or no cleavage activity, and results in the formation of large bundles along the equator. On the dorsal surface, where cleavage occurs actively, actin filaments remain isotropic, showing only subtle alignment late in cytokinesis. In addition, stereo imaging has led to the discovery of a novel set of filaments that are associated with the cortex and traverse through the cytoplasm. Together, these studies provide important insights into the process of actin remodeling during cell division and point to possible additional mechanisms for force generation.
Cytoplasmic dynein plays a role in mammalian mitotic spindle formation.
Authors: Vaisberg E A , Koonce M P , McIntosh J R
Published: May 2004

The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles, but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP-hydrolytic sites of the cytoplasmic dynein heavy chain from HeLa cells and from Dictyostelium. Monospecific antibodies have been raised to the resulting polypeptides, and these inhibit dynein motor activity in vitro. Their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells become arrested with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles that it may play are redundant. Possible mechanisms of dynein's involvement in mitosis are discussed.
At least two kinases phosphorylate the MPM-2 epitope during Xenopus oocyte maturation.
Authors: Kuang J , Ashorn C L
Published: May 2004

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes (the MPM-2 epitope), are associated with various mitotically important structures. The central mitotic regulator cdc2 kinase has been proposed to induce M-phase by phosphorylating many proteins which might include the MPM-2 antigens. To clarify the relationship of cdc2 kinase and the MPM-2 antigens, we developed an in vitro assay that enabled us to specifically detect the kinases that phosphorylate the MPM-2 epitope (ME kinases) in crude cell extracts. Two different ME kinase activities were identified in unfertilized Xenopus eggs, neither of which was cdc2 kinase, but both appeared to be activated by the introduction of cdc2 kinase into oocytes or oocyte extract. The two ME kinases differed in molecular size, substrate specificity, peptide components, and MPM-2 reactivity. The larger one, ME kinase-H, phosphorylated several MPM-2 antigens, while the smaller one, ME kinase-L, phosphorylated mainly one. We purified ME kinase-L to near homogeneity by sequential chromatography and showed that it has the characteristics of the 42-kD microtubule-associated protein (MAP) kinase. Our results support the previous finding that MAP kinase is activated during Xenopus oocyte maturation and suggest that MAP kinase may contribute to oocyte maturation induction by phosphorylating one subtype of MPM-2 epitope.
Transgenic mouse models that explore the multistep hypothesis of intestinal neoplasia.
Authors: Kim S H , Roth K A , Moser A R , Gordon J I
Published: May 2004

SV-40 T antigen (TAg), human K-rasVal12, and a dominant negative mutant of human p53 (p53Ala143) have been expressed singly and in all possible combinations in postmitotic enterocytes distributed throughout the duodenal-colonic axis of 1-12-mo-old FVB/N transgenic mice to assess the susceptibility of this lineage to gene products implicated in the pathogenesis of human gut neoplasia. SV-40 TAg produces re-entry into the cell cycle. Transgenic pedigrees that produce K-rasVal12 alone, p53Ala143 alone, or K-rasVal12 and p53Ala143 have no detectable phenotypic abnormalities. However, K-rasVal12 cooperates with SV-40 TAg to generate marked proliferative and dysplastic changes in the intestinal epithelium. These abnormalities do not progress to form adenomas or adenocarcinomas over a 9-12-mo period despite sustained expression of the transgenes. Addition of p53Ala143 to enterocytes that synthesize SV-40 TAg and K-rasVal12 does not produce any further changes in proliferation or differentiation. Mice that carry one, two, or three of these transgenes were crossed to animals that carry Min, a fully penetrant, dominant mutation of the Apc gene associated with the development of multiple small intestinal and colonic adenomas. A modest (2-5-fold) increase in tumor number was noted in animals which express SV-40 TAg alone, SV-40 TAg and K-rasVal12, or SV-40 TAg, K-rasVal12 and p53Ala143. However, the histopathologic features of the adenomas were not altered and the gut epithelium located between tumors appeared similar to the epithelium of their single transgenic, bi-transgenic, or tri-transgenic parents without Min. These results suggest that (a) the failure of the dysplastic gut epithelium of SV-40 TAg X K-rasVal12 mice to undergo further progression to adenomas or adenocarcinomas is due to the remarkable protective effect of a continuously and rapidly renewing epithelium, (b) initiation of tumorigenesis in Min mice typically occurs in crypts rather than in villus-associated epithelial cell populations, and (c) transgenic mouse models of neoplasia involving members of the enterocytic lineage may require that gene products implicated in tumorigenesis be directed to crypt stem cells or their immediate descendants. Nonetheless, directing K-rasVal12 production to proliferating and nonproliferating cells in the lower and upper half of small intestinal and colonic crypts does not result in any detectable abnormalities.
Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils.
Authors: McCormick B A , Colgan S P , Delp-Archer C , Miller S I , Madara J L
Published: May 2004

In human intestinal disease induced by Salmonella typhimurium, transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane. In this report, we model those interactions in vitro, using polarized monolayers of the human intestinal epithelial cell, T84, isolated human PMN, and S. typhimurium. We show that Salmonella attachment to T84 cell apical membranes did not alter monolayer integrity as assessed by transepithelial resistance and measurements of ion transport. However, when human neutrophils were subsequently placed on the basolateral surface of monolayers apically colonized by Salmonella, physiologically directed transepithelial PMN migration ensued. In contrast, attachment of a non-pathogenic Escherichia coli strain to the apical membrane of epithelial cells at comparable densities failed to stimulate a directed PMN transepithelial migration. Use of the n-formyl-peptide receptor antagonist N-t-BOC-1-methionyl-1-leucyl-1- phenylalanine (tBOC-MLP) indicated that the Salmonella-induced PMN transepithelial migration response was not attributable to the classical pathway by which bacteria induce directed migration of PMN. Moreover, the PMN transmigration response required Salmonella adhesion to the epithelial apical membrane and subsequent reciprocal protein synthesis in both bacteria and epithelial cells. Among the events stimulated by this interaction was the epithelial synthesis and polarized release of the potent PMN chemotactic peptide interleukin-8 (IL-8). However, IL-8 neutralization, transfer, and induction experiments indicated that this cytokine was not responsible for the elicited PMN transmigration. These data indicate that a novel transcellular pathway exists in which subepithelial PMN respond to lumenal pathogens across a functionally intact epithelium. Based on the known unique characteristics of the intestinal mucosa, we speculate that IL-8 may act in concert with an as yet unidentified transcellular chemotactic factor(s) (TCF) which directs PMN migration across the intestinal epithelium.
Commitment to differentiation and expression of early differentiation markers in murine keratinocytes in vitro are regulated independently of extracellular calcium concentrations.
Authors: Drozdoff V , Pledger W J
Published: May 2004

In the epidermis, one of the earliest characterized events in keratinocyte differentiation is the coordinate induction of a pair of keratins specifically expressed in suprabasal cells, keratin 1 (K1) and keratin 10 (K10). Both in vivo and in vitro, extracellular calcium is necessary for several biochemical and structural changes during keratinocyte differentiation. However, it has been unclear if calcium serves as a differentiation signal in keratinocytes. In these studies, expression of suprabasal keratin mRNA and protein is used to test whether the initial differentiation of primary mouse keratinocytes in vitro is dependent on changes in the concentration of extracellular calcium. K1 mRNA was expressed at low levels in cultures of keratinocytes growing on plastic in 0.05 mM calcium but in attached cells was not further induced by increases in the concentration of extracellular calcium. Suspension of the keratinocytes into semi-solid medium induced a rapid and substantial increase in both expression of K1 mRNA and in the percentage of cells expressing suprabasal keratin proteins. The induction was unaffected by the concentration of calcium in the semi-solid medium and could not be enhanced by exposing attached cells to higher calcium before suspension. The induction of K1 mRNA could be inhibited by exposure of the keratinocytes to either EGF or fibronectin. These results suggest that commitment of mouse keratinocytes to terminal differentiation is independent of extracellular calcium and may be regulated primarily by extracellular factors other than calcium.
Accumulation of actin in subsets of pioneer growth cone filopodia in response to neural and epithelial guidance cues in situ.
Authors: O'Connor T P , Bentley D
Published: May 2004

Directed outgrowth of neural processes must involve transmission of signals from the tips of filopodia to the central region of the growth cone. Here, we report on the distribution and dynamics of one possible element in this process, actin, in live growth cones which are reorienting in response to in situ guidance cues. In grasshopper embryonic limbs, pioneer growth cones respond to at least three types of guidance cues: a limb axis cue, intermediate target cells, and a circumferential band of epithelial cells. With time-lapse imaging of intracellularly injected rhodamine-phalloidin and rhodamine-actin, we monitored the distribution of actin during growth cone responses to these cues. In distal limb regions, accumulation of actin in filopodia and growth cone branches accompanies continued growth, while reduction of actin accompanies withdrawal. Where growth cones are reorienting to intermediate target cells, or along the circumferential epithelial band, actin selectively accumulates in the proximal regions of those filopodia that have contacted target cells or are extending along the band. Actin accumulations can be retrogradely transported along filopodia, and can extend into the central region of the growth cone. These results suggest that regulation and translocation of actin may be a significant element in growth cone steering.
Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits.
Authors: Hell J W , Westenbroek R E , Warner C , Ahlijanian M K , Prystay W , Gilbert M M , Snutch T P , Catterall W A
Published: May 2004

To identify and localize the protein products of genes encoding distinct L-type calcium channels in central neurons, anti-peptide antibodies specific for the class C and class D alpha 1 subunits were produced. Anti-CNC1 directed against class C immunoprecipitated 75% of the L-type channels solubilized from rat cerebral cortex and hippocampus. Anti-CND1 directed against class D immunoprecipitated only 20% of the L-type calcium channels. Immunoblotting revealed two size forms of the class C L-type alpha 1 subunit, LC1 and LC2, and two size forms of the class D L-type alpha 1 subunit, LD1 and LD2. The larger isoforms had apparent molecular masses of approximately 200-210 kD while the smaller isoforms were 180-190 kD, as estimated from electrophoresis in gels polymerized from 5% acrylamide. Immunocytochemical studies using CNC1 and CND1 antibodies revealed that the alpha 1 subunits of both L-type calcium channel subtypes are localized mainly in neuronal cell bodies and proximal dendrites. Relatively dense labeling was observed at the base of major dendrites in many neurons. Staining in more distal dendritic regions was faint or undetectable with CND1, while a more significant level of staining of distal dendrites was observed with CNC1, particularly in the dentate gyrus and the CA2 and CA3 areas of the hippocampus. Class C calcium channels were concentrated in clusters, while class D calcium channels were generally distributed in the cell surface membrane of cell bodies and proximal dendrites. Our results demonstrate multiple size forms and differential localization of two subtypes of L-type calcium channels in the cell bodies and proximal dendrites of central neurons. The differential localization and multiple size forms may allow these two channel subtypes to participate in distinct aspects of electrical signal integration and intracellular calcium signaling in neuronal cell bodies. The preferential localization of these calcium channels in cell bodies and proximal dendrites implies their involvement in regulation of calcium-dependent functions occurring in those cellular compartments such as protein phosphorylation, enzyme activity, and gene expression.
Nuclear organization of splicing snRNPs during differentiation of murine erythroleukemia cells in vitro.
Authors: Antoniou M , Carmo-Fonseca M , Ferreira J , Lamond A I
Published: May 2004

Murine erythroleukemia (MEL) cells are erythroid progenitors that can be induced to undergo terminal erythroid differentiation in culture. We have used MEL cells here as a model system to study the nuclear organization of splicing snRNPs during the physiological changes in gene expression which accompany differentiation. In uninduced MEL cells, snRNPs are widely distributed throughout the nucleoplasm and show an elevated concentration in coiled bodies. Within the first two days after induction of terminal erythroid differentiation, the pattern of gene expression changes, erythroid-specific transcription is activated and transcription of many other genes is repressed. During this early stage splicing snRNPs remain widely distributed through the nucleoplasm and continue to associate with coiled bodies. At later stages of differentiation (four to six days), when total transcription levels have greatly decreased, splicing snRNPs are redistributed. By six days postinduction snRNPs were concentrated in large clusters of interchromatin granules and no longer associated with coiled bodies. At the end-point of erythroid differentiation, just before enucleation, we observe a dramatic segregation of splicing snRNPs from the condensed chromatin. Analysis by EM shows that the snRNPs are packaged into a membrane-associated structure at the nuclear periphery which we term the "SCIM" domain (i.e., SnRNP Clusters Inside a Membrane).
Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation.
Authors: Wang L H , Rothberg K G , Anderson R G
Published: May 2004

The clathrin-coated pit lattice is held onto the plasma membrane by an integral membrane protein that binds the clathrin AP-2 subunit with high affinity. In vitro studies have suggested that this protein controls the assembly of the pit because membrane bound AP-2 is required for lattice assembly. If so, the AP-2 binding site must be a resident protein of the coated pit and recycle with other receptors that enter cells through this pathway. Proper recycling, however, would require the switching off of AP-2 binding to allow the binding site to travel through the endocytic pathway unencumbered. Evidence for this hypothesis has been revealed by the cationic amphiphilic class of drugs (CAD), which have previously been found to inhibit receptor recycling. Incubation of human fibroblasts in the presence of these drugs caused clathrin lattices to assemble on endosomal membranes and at the same time prevented coated pit assembly at the cell surface. These effects suggest that CADs reverse an on/off switch that controls AP-2 binding to membranes. We conclude that cells have a mechanism for switching on and off AP-2 binding during the endocytic cycle.
The subcellular distribution of early endosomes is affected by the annexin II2p11(2) complex.
Authors: Harder T , Gerke V
Published: May 2004

The tyrosine kinase substrate annexin II is a member of a multigene family of Ca2+ and lipid-binding proteins which have been implicated in a number of membrane-related events. We have analyzed the subcellular distribution of annexin II in relation to other cellular components in normal and specifically manipulated MDCK cells. In a polarized monolayer of MDCK cells annexin II and its cellular ligand p11 are restricted almost exclusively to the cortical regions of the cells which also contain peripheral early endosomes. Treatment of the polarized cells with low Ca2+ medium leads to a disintegration of the cortical cytoskeleton and a translocation of both, the annexin II2p11(2) complex and early endosomes, to the cytoplasm. A similar translocation which is however specific for the annexin II2p11(2) complex and early endosomes and does not affect other elements of the cell cortex is observed in cells expressing a trans-dominant annexin II-p11 mutant. This chimeric mutant protein causes the aggregation of endogenous annexin II and p11 and the simultaneous detachment of early endosomes from the cell periphery resulting in the binding of the early endosomes but no other components of the endocytotic or biosynthetic pathways to the annexin II/p11 aggregates. The specificity of this effect argues for the association of the annexin II2p11(2) complex with early endosomes and suggests that this association contributes to establish the peripheral localization of early endosomal structures.
Yeast actin with a mutation in the "hydrophobic plug" between subdomains 3 and 4 (L266D) displays a cold-sensitive polymerization defect.
Authors: Chen X , Cook R K , Rubenstein P A
Published: May 2004

Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.] 347: 44-49) hypothesized that between subdomains 3 and 4 of actin is a loop of 10 amino acids including a four residue hydrophobic plug that inserts into a hydrophobic pocket formed by two adjacent monomers on the opposing strand thereby stabilizing the F-actin helix. To test this hypothesis we created a mutant yeast actin (L266D) by substituting Asp for Leu266 in the plug to disrupt this postulated hydrophobic interaction. Haploid cells expressing only this mutant actin were viable with no obvious altered phenotype at temperatures above 20 degrees C but were moderately cold-sensitive for growth compared with wild-type cells. The critical concentration for polymerization increased 10-fold at 4 degrees C compared with wild-type actin. The length of the nucleation phase of polymerization increased as the temperature decreased. At 4 degrees C nucleation was barely detectable. Addition of phalloidin-stabilized F-actin nuclei and phalloidin restored L266D actin's ability to polymerize at 4 degrees C. This mutation also affects the overall rate of elongation during polymerization. Small effects of the mutation were observed on the exchange rate of ATP from G-actin, the G-actin intrinsic ATPase activity, and the activation of myosin S1 ATPase activity. Circular dichroism measurements showed a 15 degrees C decrease in melting temperature for the mutant actin from 57 degrees C to 42 degrees C. Our results are consistent with the model of Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.]. 347:44-49) involving the role of the hydrophobic plug in actin filament stabilization.
Axonal regulation of Schwann cell integrin expression suggests a role for alpha 6 beta 4 in myelination.
Authors: Einheber S , Milner T A , Giancotti F , Salzer J L
Published: May 2004

Ensheathment and myelination of axons by Schwann cells in the peripheral nervous system requires contact with a basal lamina. The molecular mechanism(s) by which the basal lamina promotes myelination is not known but is likely to reflect the activity of integrins expressed by Schwann cells. To initiate studies on the role of integrins during myelination, we characterized the expression of two integrin subunits, beta 1 and beta 4, in an in vitro myelination system and compared their expression to that of the glial adhesion molecule, the myelin-associated glycoprotein (MAG). In the absence of neurons, Schwann cells express significant levels of beta 1 but virtually no beta 4 or MAG. When Schwann cells are cocultured with dorsal root ganglia neurons under conditions promoting myelination, expression of beta 4 and MAG increased dramatically in myelinating cells, whereas beta 1 levels remained essentially unchanged. (In general agreement with these findings, during peripheral nerve development in vivo, beta 4 levels also increase during the period of myelination in sharp contrast to beta 1 levels which show a striking decrease.) In cocultures of neurons and Schwann cells, beta 4 and MAG appear to colocalize in nascent myelin sheaths but have distinct distributions in mature sheaths, with beta 4 concentrated in the outer plasma membrane of the Schwann cell and MAG localized to the inner (periaxonal) membrane. Surprisingly, beta 4 is also present at high levels with MAG in Schmidt-Lanterman incisures. Immunoprecipitation studies demonstrated that primary Schwann cells express beta 1 in association with the alpha 1 and alpha 6 subunits, while myelinating Schwann cells express alpha 6 beta 4 and possibly alpha 1 beta 1. beta 4 is also downregulated during Wallerian degeneration in vitro, indicating that its expression requires continuous Schwann cell contact with the axon. These results indicate that axonal contact induces the expression of beta 4 during Schwann cell myelination and suggest that alpha 6 beta 4 is an important mediator of the interactions of myelinating Schwann cells with the basal lamina.
Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding.
Authors: Pan T C , Sasaki T , Zhang R Z , Fässler R , Timpl R , Chu M L
Published: May 2004

A new protein, fibulin-2, was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library. This protein consists of a 1195-residue polypeptide preceded by a 26-residue signal peptide. The COOH-terminal region of 787 amino acids contained three anaphylatoxin-related segments (domain I), 11 EGF-like repeats (domain II), 10 of which had a consensus motif for calcium-binding, and a 115-residue globular domain III. Except for two additional EGF-like repeats, this COOH-terminal region showed 43% sequence identity with the previously described fibulin-1 (BM-90). The NH2-terminal 408 residues, unique to fibulin-2, showed no sequence homology to other known proteins and presumably form two additional domains that differ in their cysteine content. Recombinant fibulin-2 was produced and secreted by human cell clones as a disulfide-bonded trimer. Rotary shadowing visualized the protein as three 40-45 nm long rods which are connected at one end in a globe-like structure. No significant immunological cross-reaction could be detected between fibulin-1 and fibulin-2. Production of the fibulin-2 was demonstrated by Northern blots and radioimmunoassay in fibroblasts but not in several tumor cell lines. Together with the observation that the serum level of fibulin-2 is 1,000-fold lower than that of fibulin-1, the data indicate that these two isoforms are not always coordinately expressed. This is also suggested by Northern blots of tissue mRNAs and by immunofluorescence localizations using mouse tissues. The latter studies also demonstrated an extracellular localization for fibulin-2 in basement membranes and other connective tissue compartments.
Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating trans-activation.
Authors: Ruhl M , Himmelspach M , Bahr G M , Hammerschmid F , Jaksche H , Wolff B , Aschauer H , Farrington G K , Probst H , Bevec D
Published: May 2004

Expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the presence of the viral trans-activator protein Rev. Rev is localized in the nucleus and binds specifically to the Rev response element (RRE) sequence in viral RNA. Furthermore, the interaction of the Rev activation domain with a cellular cofactor is essential for Rev function in vivo. Using cross-linking experiments and Biospecific Interaction Analysis (BIA) we identify eukaryotic initiation factor 5A (eIF-5A) as a cellular factor binding specifically to the HIV-1 Rev activation domain. Indirect immunofluorescence studies demonstrate that a significant fraction of eIF-5A localizes to the nucleus. We also provide evidence that Rev transactivation is functionally mediated by eIF-5A in Xenopus oocytes. Furthermore, we are able to block Rev function in mammalian cells by antisense inhibition of eIF-5A gene expression. Thus, regulation of HIV-1 gene expression by Rev involves the targeting of RRE-containing RNA to components of the cellular translation initiation complex.
A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome.
Authors: Brodsky J L , Schekman R
Published: May 2004

Reconstituted proteoliposomes derived from solubilized yeast microsomes are able to translocate a secreted yeast mating pheromone precursor (Brodsky, J. L., S. Hamamoto, D. Feldheim, and R. Schekman. 1993. J. Cell Biol. 120:95-107). Reconstituted proteoliposomes prepared from strains with mutations in the SEC63 or KAR2 genes are defective for translocation; the kar2 defect can be overcome by the addition of purified BiP (encoded by the KAR2 gene). We now show that addition of BiP to wild-type reconstituted vesicles increases their translocation efficiency three-fold. To identify other ER components that are required for translocation, we purified a microsomal membrane protein complex that contains Sec63p. We found that the complex also includes BiP, Sec66p (gp31.5), and Sec67p (p23). The Sec63p complex restores translocation activity to reconstituted vesicles that are prepared from a sec63-1 strain, or from cells in which the SEC66 or SEC67 genes are disrupted. BiP dissociates from the complex when the purification is performed in the presence of ATP gamma S or when the starting membranes are from yeast containing the sec63-1 mutation. We conclude that the purified Sec63p complex is active and required for protein translocation, and that the association of BiP with the complex may be regulated in vivo.
Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane.
Authors: Kean L S , Fuller R S , Nichols J W
Published: May 2004

Digital, video-enhanced fluorescence microscopy and spectrofluorometry were used to follow the internalization into the yeast Saccharomyces cerevisiae of phosphatidylcholine molecules labeled on one acyl chain with the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). Two pathways were found: (1) transport by endocytosis to the vacuole and (2) transport by a non-endocytic pathway to the nuclear envelope and mitochondria. The endocytic pathway was inhibited at low temperature (< 2 degrees C) and by ATP depletion. Mutations in secretory (SEC) genes that are necessary for membrane traffic through the secretory pathway (including SEC1, SEC2, SEC4, SEC6, SEC7, SEC12, SEC14, SEC17, SEC18, and SEC21) almost completely blocked endocytic uptake. In contrast, mutations in the SEC63, SEC65, or SEC11 genes, required for translocation of nascent secretory polypeptides into the ER or signal peptide processing in the ER, only slightly reduced endocytic uptake. Phospholipid endocytosis was also independent of the gene encoding the clathrin heavy chain, CHC1. The correlation of biochemical analysis with fluorescence microscopy indicated that the fluorescent phosphatidylcholine was degraded in the vacuole and that degradation was, at least in part, dependent on the vacuolar proteolytic cascade. The non-endocytic route functioned with a lower cellular energy charge (ATP levels 80% reduced) and was largely independent of the SEC genes. Non-endocytic transport of NBD-phosphatidylcholine to the nuclear envelope and mitochondria was inhibited by pretreatment of cells with the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzenesulfonic acid, suggesting the existence of protein-mediated transmembrane transfer (flip-flop) of phosphatidylcholine across the yeast plasma membrane. These data establish a link between lipid movement during secretion and endocytosis in yeast and suggest that phospholipids may also gain access to intracellular organelles through non-endocytic, protein-mediated events.
Mechanisms of urinary K+ and H+ excretion: primary structure and functional expression of a novel H,K-ATPase.
Authors: Jaisser F , Horisberger J D , Geering K , Rossier B C
Published: May 2004

The kidney plays an essential role in regulating potassium and acid balance. A major site for these regulations is in the collecting tubule. In the present study, we report the primary sequence of a novel alpha subunit of the P-ATPase gene family, which we isolated from the urinary bladder epithelium of the toad Bufo marinus, the amphibian equivalent of the mammalian collecting tubule. The cDNA encodes a protein of 1,042 amino acids which shares approximately 67% identity with the alpha 1 subunit of the ouabain-inhibitable Na,K-ATPase and approximately 69% identity with the alpha subunit of the SCH28080-inhibitable gastric H,K-ATPase. When coexpressed in Xenopus oocytes with a beta subunit isolated from the same cDNA library, the ATPase is able to transport rubidium (a potassium surrogate) inward, and hydrogen outward, leading to alkalization of the intracellular compartment and acidification of the external medium. The novel ATPase has a unique pharmacological profile showing intermediate sensitivity to both ouabain and SCH28080. Our findings indicate that the bladder ATPase is a member of a new ion motive P-ATPase subfamily. The bladder ATPase is expressed in the urinary tract but not in the stomach or the colon. This H,K-ATPase may be one of the molecules involved in H+ and K+ homeostasis, mediating the transport of these ions across urinary epithelia and therefore regulating their urinary excretion.
Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy.
Authors: Miller D J , Gong X , Decker G , Shur B D
Published: May 2004

The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy. Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment. Therefore, N-acetylglucosaminidase, or a related glycosidase, may be present in cortical granules and be responsible for ZP3's loss of sperm-binding activity at fertilization. Of eight glycosidases assayed in exudates of ionophore-activated eggs, N-acetylglucosaminidase was 10-fold higher than any other activity. The enzyme was localized to cortical granules using immunoelectron microscopy. Approximately 70 or 90% of the enzyme was released from cortical granules after ionophore activation or in vivo fertilization, respectively. The isoform of N-acetylglucosaminidase found in cortical granules was identified as beta-hexosaminidase B, the beta, beta homodimer. Inhibition of N-acetylglucosaminidase released from activated eggs, with either competitive inhibitors or with specific antibodies, resulted in polyspermic binding to the zona pellucida. Another glycosidase inhibitor or nonimmune antibodies had no effect on sperm binding to activated eggs. Therefore, egg cortical granule N-acetylglucosaminidase is released at fertilization, where it inactivates the sperm GalTase-binding site, accounting for the block in sperm binding to the zona pellucida.
Olfactory receptors are displayed on dog mature sperm cells.
Authors: Vanderhaeghen P , Schurmans S , Vassart G , Parmentier M
Published: May 2004

Olfactory receptors constitute a huge family of structurally related G protein-coupled receptors, with up to a thousand members expected. We have shown previously that genes belonging to this family were expressed in the male germ line from both dog and human. The functional significance of this unexpected site of expression was further investigated in the present study. We demonstrate that a few dog genes representative of various subfamilies of olfactory receptors are expressed essentially in testis, with little or no expression in olfactory mucosa. Other randomly selected members of the family show the expected site of expression, restricted to the olfactory system. Antibodies were generated against the deduced amino acid sequence of the most abundantly expressed olfactory receptor gene in dog testis. The purified serum was able to detect the gene product (DTMT receptor) in late round and elongated spermatids, as well as in the cytoplasmic droplet that characterizes the maturation of dog sperm cells, and on the tail midpiece of mature spermatozoa. Western blotting further confirmed the presence of a 40-kD immunoreactive protein in the membrane of mature sperm cells. Altogether , these results demonstrate that the main expression site of a subset of the large olfactory receptor gene family is not olfactory mucosa but testis. This expression correlates with the presence of the corresponding protein during sperm cell maturation, and on mature sperm cells. The pattern of expression is consistent with a role as sensor for unidentified chemicals possibly involved in the control of mammalian sperm maturation, migration, and/or fertilization.
440-kD ankyrinB: structure of the major developmentally regulated domain and selective localization in unmyelinated axons.
Authors: Chan W , Kordeli E , Bennett V
Published: May 2004

440-kD ankyrinB is an alternatively spliced variant of 220-kD ankyrinB, with a predicted 220-kD sequence inserted between the membrane/spectrin binding domains and COOH-terminal domain (Kunimoto, M., E. Otto, and V. Bennett. 1991. J. Cell Biol. 236:1372-1379). This paper presents the sequence of 2085 amino acids comprising the alternatively spliced portion of 440-kD ankyrinB, and provides evidence that much of the inserted sequence has the configuration of an extended random coil. Notable features of the inserted sequence include a hydrophilicity profile that contains few hydrophobic regions, and 220 predicted sites for phosphorylation by protein kinases (casein kinase 2, protein kinase C, and proline-directed protein kinase). Secondary structure and folding of the inserted amino acid residues were deduced from properties of recombinant polypeptides. Frictional ratios of 1.9-2.4 were calculated from Stokes radii and sedimentation coefficients, for polypeptides comprising 70% of the inserted sequence, indicating a highly asymmetric shape. Circular dichroism spectra of these polypeptides indicate a nonglobular structure with negligible alpha-helix or beta sheet folding. These results suggest a ball-and-chain model for 440-kD ankyrinB with a membrane-associated globular head domain and an extended filamentous tail domain encoded by the inserted sequence. Immunofluorescence and immunoblot studies of developing neonatal rat optic nerve indicate that 440-kD ankyrinB is selectively targeted to premyelinated axons, and that 440-kD ankyrinB disappears from these axons coincident with myelination. Hypomyelinated nerve tracts of the myelin-deficient Shiverer mice exhibit elevated levels of 440-kD ankyrinB. 440-kD ankyrinB thus is a specific component of unmyelinated axons and expression of 440-kD ankyrinB may be downregulated as a consequence of myelination.
Regulated docking of nuclear membrane vesicles to vimentin filaments during mitosis.
Authors: Maison C , Horstmann H , Georgatos S D
Published: May 2004

During mitosis, several types of intermediate-sized filaments (IFs) undergo an extensive remodelling in response to phosphorylation by cdc 2 and other protein kinases. However, unlike the nuclear lamins, the cytoplasmic IFs do not seem to follow a fixed disassembly stereotype and often retain their physical continuity without depolymerizing into soluble subunits. To investigate potential interactions between mitotically modified IFs and other cellular structures, we have examined prometaphase-arrested cells expressing the IF protein vimentin. We demonstrate here that vimentin filaments associate in situ and co-fractionate with a distinct population of mitotic vesicles. These vesicles carry on their surfaces nuclear lamin B, the inner nuclear membrane protein p58, and wheat germ agglutinin (WGA)-binding proteins. Consistent with a tight interaction between the IFs and the mitotic membranes, vimentin, nuclear lamin B, and a 180-kD WGA-binding protein are co-isolated when whole mitotic homogenates are incubated with anti-vimentin or anti-lamin B antibodies immobilized on magnetic beads. The vimentin-associated vesicles are essentially depleted of ER, Golgi and endosomal membrane proteins. The interaction of vimentin with lamin B-carrying membranes depends on phosphorylation and is weakened by dephosphorylation during nuclear reassembly in vitro. These observations reveal a novel interaction between IFs and cellular membranes and further suggest that the vimentin filaments may serve as a transient docking site for inner nuclear membrane vesicles during mitosis.
The rod domain of NF-L determines neurofilament architecture, whereas the end domains specify filament assembly and network formation.
Authors: Heins S , Wong P C , Müller S , Goldie K , Cleveland D W , Aebi U
Published: May 2004

Neurofilaments, assembled from NF-L, NF-M, and NF-H subunits, are the most abundant structural elements in myelinated axons. Although all three subunits contain a central, alpha-helical rod domain thought to mediate filament assembly, only NF-L self-assembles into 10-nm filaments in vitro. To explore the roles of the central rod, the NH2-terminal head and the COOH-terminal tail domain in filament assembly, full-length, headless, tailless, and rod only fragments of mouse NF-L were expressed in bacteria, purified, and their structure and assembly properties examined by conventional and scanning transmission electron microscopy (TEM and STEM). These experiments revealed that in vitro assembly of NF-L into bona fide 10-nm filaments requires both end domains: whereas the NH2-terminal head domain promotes lateral association of protofilaments into protofibrils and ultimately 10-nm filaments, the COOH-terminal tail domain controls lateral assembly of protofilaments so that it terminates at the 10-nm filament level. Hence, the two end domains of NF-L have antagonistic effects on the lateral association of protofilaments into higher-order structures, with the effect of the COOH-terminal tail domain being dominant over that of the NH2-terminal head domain. Consideration of the 21-nm axial beading commonly observed with 10-nm filaments, the approximate 21-nm axial periodicity measured on paracrystals, and recent cross-linking data combine to support a molecular model for intermediate filament architecture in which the 44-46-nm long dimer rods overlap by 1-3-nm head-to-tail, whereas laterally they align antiparallel both unstaggered and approximately half-staggered.
Targeted expression of a conditional oncogene in hematopoietic cells of transgenic mice.
Authors: Ravid K , Li Y C , Rayburn H B , Rosenberg R D
Published: May 2004

We have produced two lines of transgenic mice in which the expression of temperature-sensitive SV-40 large T antigen is targeted to bone marrow megakaryocytes via the platelet factor 4 (PF4) tissue-specific promoter. The progeny of these transgenic mice were observed for about 3 mo, and no malignancies were detected over this period of time. The offspring of these transgenic mice, 6- to 12-wk of age, served as a source of bone marrow cells, which upon in vitro cultivation at the permissive temperature yielded immortalized cell lines (MegT). At the permissive temperature, MegT cells exhibit the characteristics of early 2N and 4N megakaryocytes which include the presence of specific gene products such as PF4, glycoprotein IIb, acetylcholinesterase, and CD45 as well as the absence of molecular markers of other cell lineages such as the macrophage marker Mac-1, the T helper cell marker CD4, the mast cell marker IgE, the T cell marker CD2 or the erythroid cell marker alpha-globin. The inactivation of the oncogene by a shift of temperature from 34 degrees to 39.5 degrees C produces a reduction in the frequency of the 2N cells, in conjunction with the appearance of 8N and 16N cells, consisting of 27 and 3% of total cells, respectively. Thus, we have generated hematopoietic cell lines that are trapped in the early stages of megakaryocyte commitment, but able to undergo part of the normal program of terminal differentiation.
Ectopic trkA expression mediates a NGF survival response in NGF-independent sensory neurons but not in parasympathetic neurons.
Authors: Allsopp T E , Robinson M , Wyatt S , Davies A M
Published: May 2004

We have investigated the role of trkA, the tyrosine kinase NGF receptor, in mediating the survival response of embryonic neurons to NGF. Embryonic trigeminal mesencephalic (TMN) neurons, which normally survive in the presence of brain-derived neurotrophic factor (BDNF) but not NGF, become NGF-responsive when microinjected with an expression vector containing trkA cDNA. In contrast, microinjection of ciliary neurotrophic factor (CNTF)-dependent embryonic ciliary neurons with the same construct does not result in the acquisition of NGF responsiveness by these neurons despite de novo expression of trkA mRNA and protein. The failure of trkA to result in an NGF-promoted survival response in ciliary neurons is not due to absence of the low-affinity NGF receptor, p75, in these neurons. Quantitative RT/PCR and immunocytochemistry showed that TMN and ciliary neurons both express p75 mRNA and protein. These findings not only provide the first direct experimental demonstration of trkA mediating a physiological response in an appropriate cell type, namely NGF-promoted survival of embryonic neurons, but indicate that not all neurons are able to respond to a trkA-mediated signal transduction event.