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PubMed1993

Glucocorticoid-regulated trafficking of mouse mammary tumor virus proteins in permeabilized hepatoma cells. Requirements of intracellular membrane transport for maturation of the cytoplasmic phosphorylated polyprotein.

Glucocorticoids coincidentally regulate the localization of mouse mammary tumor virus (MMTV) glycoproteins and maturation of viral phosphoproteins in viral infected rat hepatoma cells. To test for a functional interaction between MMTV transmembrane glycoproteins and cytoplasmic phosphoproteins, the bacterial cytolysin streptolysin-O was utilized to selectively permeabilize the plasma membrane and reconstitute exocytic trafficking. Streptolysin-O-permeabilized M1.54 cells pretreated with glucocorticoids retained the capability for proteolytic processing, cell surface delivery, and externalization of MMTV glycoproteins as determined by immunoprecipitation and immunofluorescence microscopy. The efficient maturation of MMTV phosphoproteins indicated that these viral proteins are properly transported near or to the plasma membrane in permeabilized cells. These maturation events in semi-intact cells were dependent on the addition of cell cytosol and were specifically inhibited by the membrane impermeant GTP analog guanosine 5'3-O-(thio)triphosphate, an agent known to impede vesicular transport of membrane proteins, but which has not previously been shown to alter cytoplasmic protein maturation or transport. The addition of anti-MMTV antibodies directed against the cytoplasmic domain of the glycoprotein precursor to transport competent semi-intact M1.54 cells resulted in the dramatic inhibition of both MMTV glycoprotein and phosphoprotein maturation. These results were not obtained using either preimmune sera or antiserum specific for the luminal portion of the glycoprotein precursor. Our findings suggest that the functional interaction of cytosolic MMTV phosphoproteins with the cytoplasmic domain of the viral membrane glycoprotein is required for the efficient transport and processing of each class of proteins in glucocorticoid-treated cells and provides the first evidence for the involvement of vesicular transport in the delivery and maturation of cytoplasmic viral proteins at the plasma membrane or the pericellular region.

Animals↗
PubMed1996

Successful intracytoplasmic sperm injection without performing cytoplasmic aspiration.

OBJECTIVE: To compare the effect of cytoplasmic aspiration versus no aspiration before intracytoplasmic sperm injection (ICSI) on the rate of oocyte damage, fertilization rate, and embryo quality. DESIGN: A randomized prospective study on sibling oocytes. SETTING: The Egyptian IVF-ET Center, Cairo, Egypt. PARTICIPANTS: Fifty-eight patients who were infertile due to male factor who underwent 60 ICSI cycles. INTERVENTION: Intracytoplasmic sperm injection was performed on randomly allocated metaphase II oocytes with cytoplasmic aspiration in group I and without cytoplasmic aspiration in group II before sperm injection. MAIN OUTCOME MEASURE: Fertilization rate, oocyte damage rate, and embryo quality. RESULTS: Normal fertilization rate per injected oocyte was 61.4% in group I compared with 62.5% in group II. The damage rate per injected oocyte was 16.8% in group I compared with 4.6% in group II. Grade I embryos were 24.5% in group I compared with 48.5% in group II. CONCLUSION: Cytoplasmic aspiration before sperm injection in ICSI is not essential for oocyte activation. It did not improve the rate of normal fertilization. On the other hand, it increased the damaged oocyte rate and the rate of cytoplasmic fragments.

Adult↗
PubMed1996

DNA-containing cytoplasmic bridges in a human breast cancer cell line, MX-1: morphological markers of a highly mobile cell type?

We have used a CREST anti-centromere serum and a DNA-specific fluorescent dye to study the composition of extended cytoplasmic bridges between interphase cells of a human carcinoma cell line, MX-1, grown on coverslips. Under natural conditions, approximately 8% of the cells possessed cytoplasmic bridges up to 60 microns long. Elongated extensions from the cell surface were also observed and were interpreted as severed cytoplasmic bridges. The bridges were extremely slender throughout most of their lengths and could not be properly resolved by phase-contrast microscopy. Staining with a DNA-specific fluorescent dye revealed, however, the presence of a thin DNA thread. This finding strongly suggests that the bridges arise during mitosis through faults in chromosome segregation. The bridges persist and most probably elongate, when the cells separate from one another after completion of mitosis. Some bridges showed also highly fluorescent DNA masses, which were detected by a CREST anti-centromere serum. Thus, a subset of the cytoplasmic bridges contained centromeres. The DNA-containing bridges between carcinoma cells in culture signal continuous rearrangements of the karyotype at a relatively high rate. The presence of extended cytoplasmic bridges between the cells could be a morphological marker for highly mobile tumor cell types and has, therefore, diagnostic value.

Adult↗
PubMed1995

The short DNA sequences in the cytoplasm of Ehrlich ascites tumor cells are tightly associated with proteins.

Mouse Ehrlich tumor cells harbor extrachromosomal DNA elements in a non-mitochondrial fraction of the cytoplasm (Abken et al., Proc. Natl. Acad. Sci USA 90: 6518-6522, 1993). The cytoplasmic DNA sequences constitute a distinct group of extrachromosomal genetic elements with common properties: (i) the DNA molecules are 50-500 bp in length and of linear configuration, (ii) most of the DNA sequences exhibit the potential to modulate the activity of a transcriptional promoter, and (iii) the DNA elements are preferentially found in tumor cells, not in cells with normal phenotype. Unexpectedly, the extrachromosomal DNA sequences are found to be tightly associated with at least three proteins (52 kD, 62 kD, 64 kD) forming DNA-protein (DNP) complexes. The DNA-protein interaction is stable during extraction with phenol and chloroform and during incubation with proteinase K and pronase P, in the presence of detergents (SDS, NP40), guanidine-HCl, high salt concentrations, and alkali. Hydrolysis of the DNA by DNAse I makes the proteins of the DNP complex accessible to proteolytic degradation. Western blot analyses imply that the proteins associated with the cytoplasmic DNA sequences are antigenically related to nucleomatrix proteins that are covalently bound to certain regions of chromosomal DNA. Covalent bonds between the cytoplasmic DNA and the polypeptides would explain the unexpected high stability of the cytoplasmic DNA-protein complexes in tumor cells.

Animals↗
PubMed1996

Changes in ultrastructure of cytoplasm and nucleus during spermiogenesis in Chara vulgaris.

In the present study much attention was paid to the first and middle stages of spermiogenesis in Chara vulgaris. The spermiogenesis was divided into 8 phases (A-H). In telophase leading to spermatid formation (A phase) there are no structural changes, indicating that cells pass into a new functional stage-spermiogenesis.B phase: After the formation of a posttelophase nucleus, the difference is striking in chromatin of newly formed spermatids and chromatin nuclei in cycling cells. The nuclei of spermatids form big clusters of dense chromatin and nucleoli remain as in telophase. However, in cycling cells condensed chromatin forms fine reticulum while nucleoli contain abundant granular component. C phase: The movement of the nucleus to the side wall, characteristic for early spermiogenesis, is not preceded by changes in the structure of the nucleus. D phase: In D phase and later free spaces between plasmalemma and the cell wall appear. In these spaces two flagella have a typical structure (9 + 2). They are surrounded by plasmalemma with rhombus-shaped scales on its surface. The structure of nucleus is characterised by a thick, dense layer of chromatin and gradual disappearance of nucleoli by extrusion to the cytoplasm. In electron-transparent ground cytoplasm, numerous ribosomes forming clusters, spirals and chains are present. Numerous rough ER cisternae with light content also appear. Bodies similar to secondary lysosomes and intensified activity of Golgi structures are observed. E phase: Large amounts of proteins seem to be synthesized and accumulated in rough ER cisternae which are filled with an electron dense substance. The same kind of substance can be seen within the perinuclear space. One may assume that proteins migrate through the nuclear envelope to the cell nucleus. Probably at that moment the main proteins taking part in the reorganisation of chromatin structure and in the exchange of somatic proteins into generative ones are formed. Simultaneously synthesis of other types of proteins continues. Active Golgi structures producing numerous light and coated vesicles complete the picture of intensive metabolism. Parallelly reduction of cytoplasm takes place. F phase: The nuclear chromatin becomes netlike after DNP staining, without a thick layer of condensed chromatin close to the nuclear envelope. Simultaneously, all types of structures with positive contrast after EDTA migrate polarly from nucleus to the cytoplasm. Further reduction of cytoplasm takes place and protein synthesis continues as suggested by the presence of numerous free polysomes. G phase: The structure of chromatin fibrils undergoes a complete transformation. They become much thicker, parallel to each other, changing the orientation along with the formation of nucleus coils. The nucleus is devoid of most nucleoplasm structures. It has significantly elongated shape and shows the presence of manchette microtubules on the outside of nuclear envelope and lamina attached to the inner membrane of nuclear envelope. Condensed mitochondria are located near flagella and nucleus while plastids with starch grains lined up on the opposite side of the spermatozoid are also surrounded by microtubules. H phase: Nuclear fibrils form lamellar interconnections and the nucleus has a form of dense reticular structure surrounded by the nuclear envelope without pores.

Animals↗
PubMed1997

Concentration of RB protein in nucleus vs. cytoplasm is stable as phosphorylation of RB changes during the cell cycle and differentiation.

Unphosphorylated RB (retinoblastoma tumor suppressor) protein is known to bind isolated nuclear matrix in vitro, whereas phosphorylated RB has a lower affinity, suggesting a mechanism which might contribute to differential nuclear/cytoplasmic localization as part of its regulatory activity. This motivates interest in the in vivo localization of the endogenous RB protein as its phosphorylation state changes during the cell cycle and cell differentiation. It is known that in proliferating HL-60 cells all the RB protein is phosphorylated, but the extent of phosphorylation increases with progression from G1 to S to G2 + M. It has also been previously shown that retinoic acid and 1,25-dihydroxy vitamin D3 shift the RB protein to the unphosphorylated state with cell differentiation (Yen, A., S. Varvayanis, Exp. Cell Res. 214, 250-257 (1994)). The dependence of cell cycle progression and differentiation on RB nuclear versus cytoplasmic localization, as well as the dependence of RB localization on phosphorylation state can thus be tested. Confocal image analysis of the RB protein in vivo shows that the ratio of the concentration of the RB tumor suppressor gene protein in the nucleus versus the cytoplasm remains stable as the RB protein undergoes either phosphorylation during cell cycle progression or dephosphorylation during cell differentiation induced by retinoic acid or 1,25-dihydroxy vitamin D3. For the cell cycle analysis, HL-60 human promyelocytic leukemia cells were fluorescently stained for DNA and for the RB protein. G1, S, and G2 + M subpopulations were isolated by fluorescence-activated cell sorting. For each subpopulation, the relative concentration of RB protein in the nucleus and the cytoplasm was measured by laser confocal image analysis. To determine the effect of retinoic acid-induced myeloid differentiation or 1,25-dihydroxy vitamin D3-induced monocytic differentiation, the same cell sorting and image analysis was performed on cells treated with these inducers. In all cases the concentration of the RB protein in the nucleus was approximately 2 times that in the cytoplasm. Thus, the ratio of nuclear versus cytoplasmic RB protein concentration is stable and independent of phosphorylation or dephosphorylation of RB during both the cell cycle and cell differentiation.

Cell Cycle↗
PubMed1997

Nonlethal gamma radiation displays similar cytoplasmic rearrangements as 12(S)-HETE.

Timar et al. (Int. J. Canc., 52:594-603, 1992) and Onoda et al. (Rad. Res., 130:281-288, 1992) implicated 12(S)-HETE and nonlethal gamma radiation respectively, in enhanced expression of the alpha IIb beta 3 integrin receptor in B16a tumor cells. 12(S)-HETE has been reported to induce rearrangement of cytoplasmic elements and movement of cytoplasm (Haddad and Taylor, Proc. Amer. Cancer Res., 34:51, 1993); Timar et al., Cell Motil. Cytoskel., 26:49-65, 1993). Here, we report similar results in B16a tumor cells by post 90 cGy gamma radiation. Control cells were circular without cytoplasmic processes with the majority of their organelles and elements in perinuclear regions both surrounded by a ring of actin microfilaments. The cell's extensive leading edge was organelle free with the exception of occasional endoplasmic reticular cisternae parallel to one or two microtubules, and short bundles of actin microfilaments perpendicular to the plasma membrane. As early as 5 sec, but better defined by 60-90 sec, one end of elongated microfilaments bundles became associated with the plasma membrane while the other end of the bundles became associated with perinuclear mass protrusions. By 5 min, prominent by 15 min, protrusions, now separated from the leading edge by mixture of bundle and ring microfilaments, reached the plasma membrane to form cellular processes. By 30 min, some cytoplasmic masses showed signs of retraction along with the disappearance of their associated mixed microfilaments. As well as numerous vesicles of unknown origin, primarily associated with microtubules perpendicular to the plasma membrane, mitochondria and endoplasmic reticular cisternae were found in the cytoplasmic masses.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1997

Single amino acid mutations in the murine MHC class II A beta cytoplasmic domain abrogate antigen presentation.

Class II MHC molecules are heterodimeric transmembrane glycoproteins that function in the presentation of Ag to CD4+ T cells. Deletion of the cytoplasmic domains of the murine class II A alpha- and A beta-chains has previously been shown to diminish Ag presentation and abrogate rejection of class II-transfected tumor cells. To examine the contributions of individual amino acid residues of the A beta cytoplasmic domain to Ag presentation and tumor rejection, we have produced a series of cell lines expressing A beta class II molecules with site-directed mutations. An A beta(k) cDNA was constructed with mutations in the five conserved amino acid residues, Q224, K225, L235, L236, and Q237 (delta5). In addition, cDNA were produced in which alanine was individually substituted for A beta(k) cytoplasmic domain residues 224 through 237 or doubly substituted at residues G226 and P227 or L235 and L236. These mutant cDNAs were individually cotransfected with wild-type A alpha cDNA into the class II-negative M12.C3 B lymphoma and Sal sarcoma cell lines. As was previously reported for transfectants lacking the entire A beta(k) cytoplasmic domain, the delta5 M12.C3 transfectant could not effectively present Ag to an autoreactive Ak-restricted T cell hybrid, and the delta5 Sal transfectant was not rejected when inoculated into syngeneic hosts. A finer analysis revealed that alteration of the individual residue Q224 or the two residues G226 and P227 abrogated Ag presentation in vitro, while mutation of G226 diminished tumor rejection in vivo. Thus, the function of the A beta cytoplasmic domain in Ag presentation both in vitro and in vivo can be disturbed by mutation of single amino acid residues.

Amino Acid Sequence↗
PubMed1996

Analysis of myeloid and lymphoid markers on the surface and in the cytoplasm of mononuclear bone marrow cells in patients with myelodysplastic syndrome.

Mononuclear cells of the bone marrow (BM) of patients in various subgroups of the myelodysplastic syndrome (MDS) were studied by flow cytometry for the expression of myeloid and lymphoid markers both on the surface and in the cytoplasm. A significantly higher percentage of the BM cells of MDS patients reacted with monoclonal antibodies (mAbs) to myeloid antigens (CD13, CD15 and CD33) by cytoplasmic staining as compared with cell surface staining. The percentage of BM cells expressing CD34 was markedly elevated in patients with RAEB-T. A distinct finding in MDS patients was the expression of myeloid antigens on mononuclear BM cells. The proportion of individuals whose mononuclear BM cells were positive for surface reactivity with anti-CD13 and anti-CD33 mAbs was highest among RAEB-T patients while none of the patients with RA expressed these surface antigens. Cytoplasmic staining significantly increased the percentage of CD13+ and CD33+ BM cells among RAEB and RAEB-T patients. The proportion of individuals whose BM cells possessed myeloid antigens was increased by cytoplasmic staining in all subgroups of MDS. The BM of a considerable proportion of RAEB-T and RAEB patients showed cells which coexpressed the CD7 and CD3 lymphoid markers along with the CD13 and CD33 myeloid antigens. The present study indicates the importance of comparative surface and cytoplasmic immunophenotyping with CD13 and CD33 mAbs for the diagnosis of subgroups of MDS. The coexpression of CD3 and CD7 with markers of the myeloid lineage may reflect derangement of the differentiation of pluripotent stem cells characteristic for MDS.

Acute Disease↗
PubMed2000

Dilated rough endoplasmic reticulum corresponding to septate cytoplasmic vacuoles in papillary thyroid carcinoma.

Septate cytoplasmic vacuoles, which are one of the cytologic findings characteristic of papillary thyroid carcinoma, are small, uniform, and well-defined vacuoles, with defined strands of cytoplasm separating them. We report on a case with histologic and ultrastructural findings corresponding to septate cytoplasmic vacuoles, which have not been previously described. The patient was a 51-yr-old man with a mass in the left anterior of the neck, measuring 4.0 x 3.5 cm in diameter. Aspiration cytology smears revealed findings typical of papillary thyroid carcinoma, including papillary tissue fragments, intranuclear inclusions, nuclear grooves, powdery chromatin, and psammoma bodies. The tumor cells which were located at the periphery of small three-dimensional clusters, and others which showed a sheet-like arrangement, demonstrated uniform, small vacuoles in relatively abundant, dense cytoplasm. Histologically, a small number of papillary thyroid carcinoma cells with multiple vacuoles were observed, which were limited to the hobnail-shaped cells located at papillary configurations or floating tumor cells within the papillary lumen. Ultrastructurally, the hobnail-shaped tumor cells demonstrated various-sized dilated rough endoplasmic reticulum and rich heterochromatin. We emphasize that septate cytoplasmic vacuoles correspond to dilated rough endoplasmic vacuoles and are probably related to degenerative changes.

Carcinoma, Papillary↗
PubMed1991

Cytoplasmic distribution of poly(A)-containing RNA in developing Necturus maculosus oocytes with reference to annulate lamellae.

The cytoplasmic distribution of poly(A)+ mRNA and its relationship to annulate lamellae were examined in developing Necturus maculosus oocytes by in situ hybridization with [3H]poly(U). The specificity of [3H]poly(U) binding was tested by incubating control ovarian sections with either KOH or RNase A before in situ hybridization. In both experiments, the silver grain densities were markedly reduced. Poly(A)+ RNA is uniformly distributed in the cytoplasm until the mid-growth phase and then later in vitellogenesis becomes localized in the subcortical ooplasm. The silver grain density in the cytoplasm varied during oogenesis and was greatest in previtellogenic oocytes. Annulate lamellae commonly are observed with the light microscope in oocytes prior to vitellogenesis. In such oocytes, the labeled mRNA probe is observed over cytoplasmic regions of annulate lamellae. The results suggests that a differential localization of messenger RNA occurs during oogenesis in Necturus maculosus. Furthermore, poly(A)+ RNA is present in cytoplasmic regions of annulate lamellae.

Animals↗
PubMed2005

Up-regulated expression of cytoplasmic clusterin in human ovarian carcinoma.

BACKGROUND: Recently, tumorigenic roles of the clusterin gene in several human malignancies have been suggested, but its potential role in the development and progression of ovarian carcinoma is unclear. METHODS: In the current study, immunohistochemistry was used to examine the expression status of clusterin in 10 normal ovaries, 20 ovarian cystadenomas, 15 borderline ovarian tumors, and 240 ovarian carcinomas (nonmetastatic and metastatic) by tissue microarray. In addition, the apoptotic index of each tumor was assessed with a terminal deoxyuridine triphosphate nick-end labeling assay. RESULTS: Positive staining for clusterin in different ovarian tissues was observed primarily a cytoplasmic pattern. Cytoplasmic overexpression of clusterin was detected in none of the normal ovaries, in 17% of cystadenomas, in 38% of borderline tumors, and in 58% of invasive ovarian carcinomas. A significant association was observed (P < 0.001) between the overexpression of clusterin and late clinical stage according to the International Federation of Gynecology and Obstetrics staging system. In addition, the overexpression of clusterin was detected more frequently in metastatic lesions than that in their matched primary tumors. The current results also provided evidence that the overexpression of cytoplasmic clusterin in carcinomas was correlated inversely with the tumors' apoptotic index, demonstrating an antiapoptotic function of cytoplasmic clusterin in ovarian carcinomas. CONCLUSIONS: The current results suggested that the overexpression of cytoplasmic clusterin may represent an acquired malignant phenotypic feature of ovarian carcinoma and may be one of the important factors in determining the aggressive nature of ovarian carcinoma.

Adult↗
PubMed1988

Cytoplasmic membrane elaborations in oligodendrocytes during myelination of spinal motoneuron axons.

The ultrastructure of paranodal oligodendroglial cytoplasm, which is located in proximity to the forming myelin sheath, was studied during maturation of spinal motoneuron axons in rat. At 8 days postnatal, the paranodal oligodendroglial loops contain a network of membrane-bound tubulovesicular elements. These membrane elaborations are most common in oligodendroglial loops attached to the outermost layers of the myelin sheath, i.e., paranodal loops closest to the nodal gap. The number of oligodendroglial cytoplasmic profiles per paranodal loop falls over the course of five to ten sequential paranodal loops, and these profiles are nearly absent in paranodal oligodendroglial cytoplasm located distant from the nodal gap. Oligodendrocytes in spinal cords of 14- and 20-day-old rats and of adult rats did not exhibit networks of tubulovesicular profiles. The appearance of these membrane organelles within oligodendroglial cytoplasm during myelin maturation suggests increased membrane turnover within paranodal cytoplasm located adjacent to the axon that is being myelinated. Membrane turnover within oligodendrocytes may reflect axonal modulation of glial function during myelination.

Aging↗
PubMed1983

Partial rescue of dorsal, a maternal effect mutation affecting the dorso-ventral pattern of the Drosophila embryo, by the injection of wild-type cytoplasm.

Mutant alleles at the maternal effect locus dorsal cause a dorsalization of the Drosophila embryo. In extreme mutants, the embryos develop exclusively structures which derive from the dorsal-most region in normal eggs, in less strong phenotypes in addition to dorsal structures, structures normally derived from a dorso-lateral to lateral egg region are formed. Injection of cytoplasm from wild-type embryos into mutant embryos partially restores the dorso-ventral pattern in that injected embryos develop additional structures never formed in uninjected control embryos or embryos injected with mutant cytoplasm. The phenotype of injected embryos resembles that of weaker alleles at the dorsal locus indicating that the wild-type cytoplasm partially rescues the mutant phenotype. The response of the mutant embryos is restricted to the site of injection and occurs only when cytoplasm is injected into the ventral and not into the dorsal side of mutant embryos. The rescuing activity appears to be equally distributed in cleavage stage wild-type embryos, whereas, in syncytial blastoderm embryos, cytoplasm from the ventral side is about twice as effective as that taken from the dorsal side.

Journal Article↗
PubMed1989

The cytoplasmic matrix of the adrenal chromaffin cells of rats under normal and stressed conditions.

In embedment-free electron microscopy with polyethylene glycol embedding and subsequent deembedding, the conventional cytoplasm of the chromaffin cells was revealed to consist of a three-dimensional lattice of microtrabeculae and gives the impression that the chromaffin granules are held in place by the lattice. After the restraint stress, a substantial number of chromaffin cells were almost free of granules, and the microtrabecular lattice was much more compact than that in cytoplasmic regions occupied with remaining granules or increased mitochondria. In immunocytochemistry, actin immunofluorescence was confined to the subplasmalemmal regions, while tubulin and tropomyosin immunofluorescence appeared throughout the entire cytoplasm of normal chromaffin cells. After the stress, the immunofluorescence for actin and tubulin increased in intensity, while that for tropomyosin decreased. Immunogold labelings for actin and tubulin were found mainly on the thinner subplasmalemmal microtrabeculae and the thicker perikaryal ones, respectively, while some were deposited in the form of small aggregates on portions of microtrabeculae. No specific association between the gold labelings for actin or tubulin and the chromaffin granules was found, even in the subplasmalemmal regions. A hypothetical interpretation was proposed in which a more compact lattice of the microtrabeculae in spatial association with a looser lattice represents a gelated state of the cytoplasm. The significance of the gel-sol transition of the cytoplasmic matrix in relation to the secretory mechanism was discussed.

Animals↗
PubMed2006

Autonomous cytoplasmic linear plasmid pPac1-1 of Pichia acaciae: molecular structure and expression studies.

The genome organization of the linear DNA-element pPac1-1 from Pichia acaciae was determined. It turned out to be the smallest autonomous cytoplasmic yeast plasmid known so far, consisting of only 12 646 bp, carrying the shortest terminal inverted repeats yet found (138 bp). As for other cytoplasmic linear yeast plasmids, it is characterized by a strikingly high A + T content (75.35%). Ten putative genes (open reading frames, ORFs) reside on the element, leaving only 2.9% of the sequence outside a coding region. Highest similarities of the predicted proteins were obtained for proteins encoded by the three hitherto known autonomous cytoplasmic linear yeast plasmids. Amino acid sequences correspond to predicted polypeptides encoded by ORFs 2-11 of the linear plasmids pGKL2 of Kluyveromyces lactis, pSKL of Saccharomyces kluyveri and pPE1B of Pichia etchellsii. As for the latter, ORF1 existing on the two other plasmids is lacking on pPac1-1. Consistent with cytoplasmic localization, a cytoplasmic promoter termed upstream conserved sequence (UCS) is located in front of each reading frame. RT-PCR transcript analyses for ORFs 8, 9 and 11 proved expression of such genes but functions could not be attributed. The genome organization of pPac1-1 and other autonomous linear elements was found to be almost congruent, irrespective of the accompanying smaller elements, which may or may not encode their own element-specific DNA polymerases.

Amino Acid Sequence↗
PubMed1999

Mapping regions of the beta1 integrin cytoplasmic domain involved in migration and survival in primary oligodendrocyte precursors using cell-permeable homeopeptides.

The mapping of regions within integrin cytoplasmic domains responsible for the different effects on cell behaviour is an important part of an analysis of integrin-mediated signalling. In order to facilitate this analysis in primary cells, we have used cell-permeable homeopeptides to deliver sequences mimicking parts of the integrin beta1 cytoplasmic domain into the cell. In a study using oligodendrocyte precursors, the cells that give rise to myelin-forming oligodendrocytes during CNS development, we show that these peptides can be used to manipulate beta1 integrin signalling and that the regions of the cytoplasmic domain involved in migration and survival are distinct. Peptides mimicking the N-terminal portion of the cytoplasmic domain previously implicated in binding to Focal Adhesion Kinase (FAK) induce apoptosis, while peptides mimicking more C-terminal sequences do not cause cell death. In contrast they show that the NPIY sequence, the N-terminal one of two NPXY motifs previously implicated in signalling, is involved in migration. Peptides containing this sequence promote migration while alteration of NPIY to NPIA makes the peptide inhibitory to migration. Our results show that these peptides represent a novel approach to integrin signalling that allow rapid definition of critical cytoplasmic sequences in primary cells.

Amino Acid Sequence↗
PubMed2000

Deencryption of cellular tissue factor is independent of its cytoplasmic domain.

Tissue factor (TF) is a transmembrane molecule that, when exposed to plasma, is the key initiator of coagulation. Cellular TF activity is normally "encrypted", but treating cells with calcium ionophore (i.e. , ionomycin or A23187) increases ("deencrypts") TF activity without increasing TF mRNA or antigen expression. Deencryption results from both plasma membrane phosphatidylserine (PS)-dependent and -independent mechanisms; however, the nature of the PS-independent component is unclear. Since deencryption has been suggested to result from release of TF dimers on the cell surface, and since TF's cytoplasmic domain binds to actin-binding protein 280 and interacts with the cytoskeleton, we hypothesized that interactions with the cytoskeleton, through the cytoplasmic domain, play a role in mediating encryption/deencryption. We examined TF deencryption and the role of the cytoplasmic domain in the PS-independent component using baby hamster kidney (BHK) cells expressing full length TF (BHK-TF) or TF lacking its cytoplasmic domain (BHK-descyt) (Sorensen et al. (1999) J. Biol. Chem. 274, 21349). Both BHK-TF and BHK-descyt cells exhibited a dose-dependent, 1.5- to 10-fold increase in TF activity upon treatment with calcium ionophore, and this increase in activity was only partially blocked by annexin V. These results indicate that deencryption is not restricted to cells which naturally express TF and that the PS-independent component of deencryption is intact on cells transfected with either full length or truncated TF. Our results clearly indicate that deencryption is not dependent on an intact cytoplasmic domain in transfected BHK cells.

Animals↗