The human T-cell leukemia/lymphotropic virus type I p12I protein cooperates with the E5 oncoprotein of bovine papillomavirus in cell transformation and binds the 16-kilodalton subunit of the vacuolar H+ ATPase.

The human T-cell leukemia/lymphotropic virus type I (HTLV-I) induces T-cell leukemia and transforms human T cells in vitro. A recently identified protein with a molecular weight of 12,000 (12K) (p12I), encoded by single- and double-spliced mRNAs transcribed from the 3' end of the HTLV-I genome, has been shown to localize in the perinuclear compartment and in the cellular endomembranes. The p12I protein exhibits significant amino acid sequence similarity to the E5 oncoprotein of bovine papillomavirus type 1 (BPV-1). Both proteins are very hydrophobic, contain a glutamine residue in the middle of a potential transmembrane region(s), and are localized in similar cellular compartments. Because of these observations, we investigated whether the p12I resemblance to E5 correlated with a similarity in their biological behavior. We expressed the p12I protein to evaluate its ability to functionally cooperate with the BPV-1 E5 oncoprotein and to bind to a cellular target of the E5 protein, the 16K component of the vacuolar H+ ATPase. Cotransfection of the mouse C127 cell line with the p12I and E5 cDNAs showed that although p12I alone could not induce focus formation, it strongly potentiated the transforming activity of E5. In addition, the p12I protein bound to the 16K protein as efficiently as the E5 protein. These findings might provide new insight for potential mechanisms of HTLV-I transformation and suggest that p12I and E5 represent an example of convergent evolution between RNA and DNA viruses.     

The E5 oncoprotein of bovine papillomavirus binds to a 16 kd cellular protein.

The E5 oncoprotein of bovine papillomavirus type 1 is the smallest known viral transforming protein. It is a 44 amino acid polypeptide asymmetrically oriented in Golgi and plasma membranes which appears to modify (either directly or indirectly) the internalization and phosphorylation of at least two growth factor receptors: EGF and CSF-1. To identify cellular proteins associated with E5, we have constructed two E5 fusion proteins, each of which contains a well-characterized epitope at the E5 amino terminus. These E5-epitope fusion proteins are biologically active, localize normally to cellular membranes and form dimers. Both monoclonal and polyclonal antibodies against the inserted epitopes specifically co-precipitate E5 and an associated 16 kd cellular protein. A transformation-defective E5 mutant containing a substitution within the hydrophobic portion of E5 is defective in its ability to bind the 16 kd protein. These findings suggest a role for E5/16 kd binding in the process of cellular transformation.     

Low-dose adenovirus vaccine encoding chimeric hepatitis B virus surface antigen-human papillomavirus type 16 E7 proteins induces enhanced E7-specific antibody and cytotoxic T-cell responses

Induction of effective immune responses may help prevent cancer progression. Tumor-specific antigens, such as those of human papillomaviruses involved in cervical cancer, are targets with limited intrinsic immunogenicity. Here we show that immunization with low doses (10(6) infectious units/dose) of a recombinant human adenovirus type 5 encoding a fusion of the E7 oncoprotein of human papillomavirus type 16 to the carboxyl terminus of the surface antigen of hepatitis B virus (HBsAg) induces remarkable E7-specific humoral and cellular immune responses. The HBsAg/E7 fusion protein assembled efficiently into virus-like particles, which stimulated antibody responses against both carrier and foreign antigens, and evoked antigen-specific kill of an indicator cell population in vivo. Antibody and T-cell responses were significantly higher than those induced by a control adenovirus vector expressing wild-type E7. Such responses were not affected by preexisting immunity against either HBsAg or adenovirus. These data demonstrate that the presence of E7 on HBsAg particles does not interfere with particle secretion, as it occurs with bigger proteins fused to the C terminus of HBsAg, and results in enhancement of CD8(+)-mediated T-cell responses to E7. Thus, fusion to HBsAg is a convenient strategy for developing cervical cancer therapeutic vaccines, since it enhances the immunogenicity of E7 while turning it into an innocuous secreted fusion protein.     

The v-sis protein retains biological activity as a type II membrane protein when anchored by various signal-anchor domains, including the hydrophobic domain of the bovine papilloma virus E5 oncoprotein

Membrane-anchored forms of the v-sis oncoprotein have been previously described which are oriented as type I transmembrane proteins and which efficiently induce autocrine transformation. Several examples of naturally occurring membrane-anchored growth factors have been identified, but all exhibit a type I orientation. In this work, we wished to construct and characterize membrane-anchored growth factors with a type II orientation. These experiments were designed to determine whether type II membrane-anchored growth factors would in fact exhibit biological activity. Additionally, we wished to determine whether the hydrophobic domain of the E5 oncoprotein of bovine papilloma virus (BPV) can function as a signal-anchor domain to direct type II membrane insertion. Type II derivatives of the v-sis oncoprotein were constructed, with the NH2 terminus intracellular and the COOH terminus extracellular, by substituting the NH2 terminal signal sequence with the signal-anchor domain of a known type II membrane protein. The signal-anchor domains of neuraminidase (NA), asialoglycoprotein receptor (ASGPR) and transferrin receptor (TR) all yielded biologically active type II derivatives of the v-sis oncoprotein. Although transforming all of the type II signal/anchor-sis proteins exhibited a very short half-life. The short half-life exhibited by the signal/anchor-sis constructs suggests that, in some cases, cellular transformation may result from the synthesis of growth factors so labile that they activate undetectable autocrine loops. The E5 oncoprotein encoded by BPV exhibits amino acid sequence similarity with PDGF, activates the PDGF beta-receptor, and thus resembles a miniature membrane-anchored growth factor with a putative type II orientation. The hydrophobic domain of the E5 oncoprotein, when substituted in place of the signal sequence of v-sis, was indistinguishable compared with the signal-anchor domains of NA, TR, and ASGPR, demonstrating its ability to function as a signal-anchor domain. NIH 3T3 cells transformed by the signal/anchor-sis constructs exhibited morphological reversion upon treatment with suramin, indicating a requirement for ligand/receptor interactions in a suramin- sensitive compartment, most likely the cell surface. In contrast, NIH 3T3 cells transformed by the E5 oncoprotein did not exhibit morphological reversion in response to suramin.     

A carboxy-terminal region required by the adenovirus type 9 E4 ORF1 oncoprotein for transformation mediates direct binding to cellular polypeptides.

Human adenovirus type 9 (Ad9) is unique among oncogenic adenoviruses in that it elicits exclusively mammary tumors in rats and requires the viral E4 region open reading frame 1 (9ORF1) gene for tumorigenicity. The 9ORF1 oncogenic determinant codes for a 14-kDa transforming protein, and three separate regions of this polypeptide, including one at the extreme C terminus, are necessary for transforming activity. In this study, we investigated whether the 9ORF1 transforming protein interacts with cellular factors. Following incubation with cell extracts, a glutathione S-transferase (GST)-9ORF1 fusion protein associated with several cellular phosphoproteins (p220, p180, p160, p155), whereas GST fusion proteins of transformation-defective 9ORF1 C-terminal mutants did not. Similar interactions requiring the 9ORF1 C terminus were revealed with protein-blotting assays, in which a GST-9ORF1 protein probe reacted specifically with cellular polypeptides having gel mobilities resembling those of the 9ORF1-associated cellular phosphoproteins, as well as with additional cellular polypeptides designated p140/p130. In addition, GST fusion proteins containing 9ORF1 C-terminal fragments associated with some of the 9ORF1-associated cellular polypeptides, as did GST fusion proteins of full-length wild-type Ad5 and Ad12 E4 ORF1 transforming proteins. Significantly, the results of coimmunoprecipitation analyses suggested that the same cellular polypeptides also associate with wild-type but not C-terminal-mutant 9ORF1 proteins in vivo. Together, these findings suggest that the 9ORF1 C terminus, which is essential for transformation, participates in specific and direct binding of the 9ORF1 oncoprotein to multiple cellular polypeptides. We propose that interactions with these cellular factors may be responsible, at least in part, for the transforming activity of the 9ORF1 viral oncoprotein.     

Combined immunization with fusion genes of mutated E7 gene of human papillomavirus type 16 did not enhance antitumor effect

BACKGROUND: The E7 oncoprotein of human papillomavirus type 16 (HPV16) is frequently used as a model tumor-associated antigen. Its immunogenicity has been substantially enhanced by fusion with several proteins of various origins and functions. Different mechanisms have been responsible for increased vaccination efficacy of fusion proteins. METHODS AND RESULTS: We linked E7 and its mutated form (E7GGG) with the mouse heat-shock protein 70.1 (HSP70.1). Enhanced immunogenicity of both fusion genes administered via a gene gun was demonstrated by protection of C57BL/6 mice against oncogenic MHC class I positive TC-1 cells producing the HPV16 E7 oncoprotein but not against the MHC class I negative TC-1/A9 subline. To assess if the efficacy of E7-based DNA vaccines could be increased by combination of various fusion genes, we combined the HSP70.1 fusion genes (i.e. E7HSP or E7GGGHSP) with the fusion construct linking E7GGG with targeting signals of lysosome-associated membrane protein 1 (Sig/E7GGG/LAMP-1). Treatment of mice 4 days after TC-1 cell inoculation showed moderately higher immunization potency of HSP70.1 fusion genes in comparison with the Sig/E7GGG/LAMP-1 gene. Any combination of two fusion genes given in the same gene gun shot neither was more effective compared with single genes nor protected mice against TC-1/A9 cells. As fusion of E7GGG with E. coli glucuronidase (E7GGG.GUS) had been previously proven to provide partial protection from TC-1/A9-induced tumors, we also combined E7GGGHSP with E7GGG.GUS. The genes were inoculated either in mix in two gene gun shots or separately each gene in one shot into opposite sides of the abdomen. Neither mode of combined immunization induced higher protection than E7GGG.GUS alone. However, doubling the DNA dose considerably enhanced the antitumor efficacy of E7GGG.GUS. CONCLUSIONS: We constructed highly immunogenic fusions of HPV16 E7 and E7GGG with mouse HSP70.1. Furthermore, we substantially enhanced protection against TC-1/A9 cells with downregulated MHC class I expression by doubling the pBSC/E7GGG.GUS dose, but we failed to demonstrate a beneficial effect of any combination of two fusion genes with different mechanisms causing enhancement of HPV16 E7 immunogenicity.     

Purification of annexin I and annexin II from human placental membranes by high-performance liquid chromatography.

Annexin I and annexin II were extracted from human placental membranes with ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) and purified by high-performance liquid chromatography by measuring their ability to inhibit phospholipase A2 activity in vitro. Neither protein was capable of binding to a DEAE-5PW HPLC column at neutral pH; however, they were resolved through binding to a Mono S column and passage through size-exclusion HPLC columns. Annexin I and its covalently linked dimer (36 and 66 kDa, respectively, by sodium dodecyl sulfate (SDS)-gel electrophoresis) reacted in one-dimensional immunoblots with monoclonal antibodies to annexin I and calpactin II, and with monoclonal and polyclonal antibodies to lipocortin I, confirming that annexin I, calpactin II, and lipocortin I are the same or closely related proteins. Milligram amounts of monomeric annexin I containing negligible amounts of the cross-linked dimeric annexin I were selectively isolated from placental membranes by using buffers containing the sulfhydryl reagent iodoacetic acid. Milligram amounts of cross-linked annexin I were selectively isolated when placental membranes were initially treated with buffers that did not contain iodoacetic acid and then extracted with Triton X-100, suggesting that sulfhydryl-dependent transglutaminase activity contributes to the selective isolation of this protein. A third phospholipase A2-inhibitory protein (35 kDa by SDS-gel electrophoresis) that reacted in immunoblots with monoclonal antibodies to calpactin I and annexin II, indicating their similar identity, was isolated. The procedure employed allows the rapid purification of annexins I and II in milligram amounts from placental membranes within 2 days.     

Differential Subcellular Distribution of p36 (the Heavy Chain of Calpactin I) and Other Annexins in the Adrenal Medulla

The annexins are a group of highly related Ca2(+)-dependent membrane-binding proteins that are present in a wide variety of cells and tissues. We have examined the subcellular distribution of five members of the annexin family in the adrenal medulla. Bovine adrenal medullary tissue was homogenized in buffers containing EGTA and fractionated on sucrose gradients. p36 (the large subunit of calpactin I) was found to be predominantly membrane associated, with approximately 20% present in fractions enriched in chromaffin granules. In contrast, lipocortin I was localized primarily to the cytosol, with only a small proportion found in plasma membrane-containing fractions. Like lipocortin I, endonexin I was found to be present almost entirely in the soluble fractions. The 67-kDa calelectrin was localized primarily to the plasma membrane fractions, with a small amount present in the chromaffin granule and cytoplasmic fractions. Synexin was present in both membranous and cytoplasmic fractions. p36 appeared to be a peripherally associated granule membrane protein in that it was dissociated from the membrane by addition of base and it partitioned with the aqueous phase when granule membranes were treated with Triton X-114. Antiserum against p10 (the small subunit of calpactin I) reacted with a protein of 19 kDa that is specifically localized in chromaffin granule membrane fractions. The differences in subcellular distributions of the annexins suggest that these proteins have distinct cellular functions. The finding that p36 is associated with chromaffin granule and plasma membrane fractions provides further support for a possible role of calpactin in exocytosis.     

Annexin-mediated membrane fusion of human neutrophil plasma membranes and phospholipid vesicles.

Membrane fusion was studied using human neutrophil plasma membrane preparations and phospholipid vesicles approximately 0.15 microns in diameter and composed of phosphatidylserine and phosphatidylethanolamine in a ratio of 1 to 3. Liposomes were labeled with N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD) and lissamine rhodamine B derivatives of phospholipids. Apparent fusion was detected as an increase in fluorescence of the resonance energy transfer donor, NBD, after dilution of the probes into unlabeled membranes. 0.5 mM Ca2+ alone was sufficient to cause substantial fusion of liposomes with a plasma membrane preparation but not with other liposomes. Both annexin I and des(1-9)annexin I caused a substantial increase in the rate of fusion under these conditions while annexin V inhibited fusion. Fusion mediated by des(1-9)annexin I was observed at Ca2+ concentrations as low as approximately 5 microM, suggesting that the truncated form of this protein may be active at physiologically low Ca2+ concentrations. Trypsin treated plasma membranes were incapable of fusion with liposomes, suggesting that plasma membrane proteins may mediate fusion. Liposomes did not fuse with whole cells at any Ca2+ concentration, indicating that the cytoplasmic side of the membrane is involved. These results suggest that annexin I and unidentified plasma membrane proteins may play a role in Ca(2+)-dependent degranulation of human neutrophils.     

Structural determinant of the vesicle aggregation activity of annexin I.

Some annexins, including annexins I, II, IV, and VII, can promote membrane aggregation. To identify amino acids involved in annexin I-mediated membrane aggregation, we generated truncated mutants of human annexin I lacking various parts of the amino terminus. The in vitro vesicle binding and aggregation activities of these mutants indicated that both the amino-terminal region of annexin I spanning residues 26-29 and the carboxy-terminal core are involved in membrane aggregation. This notion was further supported by the finding that a chimera composed of residues 24-35 of annexin I and the core of annexin V has vesicle aggregation activity that is significantly higher than that of annexin V but lower than that of annexin I. Further site-specific mutations in the amino-terminal region of annexin I indicated that Lys-26 and Lys-29 are essential for its membrane aggregation activity. The comparison of tryptic digest patterns of free and vesicle-bound wild type and K29E mutant suggests that a primary role of Lys-26 and Lys-29 is to induce and stabilize an active conformation of annexin I for vesicle aggregation.     

A new class of receptor for herpes simplex virus has heptad repeat motifs that are common to membrane fusion proteins

We isolated a human cDNA by expression cloning and characterized its gene product as a new human protein that enables entry and infection of herpes simplex virus (HSV). The gene, designated hfl-B5, encodes a type II cell surface membrane protein, B5, that is broadly expressed in human primary tissue and cell lines. It contains a high-scoring heptad repeat at the extracellular C terminus that is predicted to form an alpha-helix for coiled coils like those in cellular SNAREs or in some viral fusion proteins. A synthetic 30-mer peptide that has the same sequence as the heptad repeat alpha-helix blocks HSV infection of B5-expressing porcine cells and human HEp-2 cells. Transient expression of human B5 in HEp-2 cells results in increased polykarocyte formation even in the absence of viral proteins. The B5 protein fulfills all criteria as a receptor or coreceptor for HSV entry. Use by HSV of a human cellular receptor, such as B5, that contains putative membrane fusion domains provides an example where a pathogenic virus with broad tropism has usurped a widely expressed cellular protein to function in infection at events that lead to membrane fusion.     

The hydrophobic domain of cytochrome b5 is capable of anchoring beta-galactosidase in Escherichia coli membranes.

Cytochrome b5 is inserted posttranslationally into membranes in vivo and spontaneously into liposomes in vitro by a short carboxyl-terminal hydrophobic membrane-anchoring sequence. DNA corresponding to this hydrophobic sequence has been synthesized, and two gene fusions with the Escherichia coli enzyme beta-galactosidase have been constructed by locating the hydrophobic domain in one case at the EcoRI site near the C terminus and in the other at the normal C terminus of the enzyme. The latter fusion protein was enzymatically active, having approximately 50% of the specific activity of beta-galactosidase, and cells expressing this protein grew normally with lactose as the sole carbon source. Both fusion proteins were localized to the E. coli inner membrane, converting beta-galactosidase from a cytoplasmic enzyme to a membrane-associated enzyme. The hydrophobic domain of cytochrome b5 therefore contains the information required to target polypeptides containing this domain to the membrane. Use of the cytochrome b5 hydrophobic peptide, either alone or in conjunction with other localizing sequences such as signal sequences, provides a general procedure for associating proteins with membranes. Polypeptides bearing this hydrophobic peptide may have considerable use as pharmaceuticals when associated with liposomes or cellular membranes.     

Clustering and redistribution of late endocytic compartments in response to Helicobacter pylori vacuolating toxin

Helicobacter pylori VacA is a secreted protein toxin that may contribute to the pathogenesis of peptic ulcer disease and gastric adenocarcinoma. When added to cultured mammalian cells in the presence of weak bases (e.g., ammonium chloride), VacA induces the formation of large cytoplasmic vacuoles. Here, we report a previously unrecognized capacity of VacA to induce clustering and perinuclear redistribution of late endocytic compartments. In contrast to VacA-induced cell vacuolation, VacA-induced clustering and redistribution of late endocytic compartments are not dependent on the presence of weak bases and are not inhibited by bafilomycin A1. VacA mutant toxins defective in the capacity to form anion-selective membrane channels fail to cause clustering and redistribution. VacA-induced clusters of late endocytic compartments undergo transformation into vacuoles after the addition of ammonium chloride. VacA-induced clustering and redistribution of late endocytic compartments occur in cells expressing wild-type or constitutively active Rab7, but not in cells expressing dominant-negative mutant Rab7. In VacA-treated cells containing clustered late endocytic compartments, overexpression of dominant-negative Rab7 causes reversion to a nonclustered distribution. Redistribution of late endocytic compartments to the perinuclear region requires a functional microtubule cytoskeleton, whereas clustering of these compartments and vacuole formation do not. These data provide evidence that clustering of late endocytic compartments is a critical mechanistic step in the process of VacA-induced cell vacuolation. We speculate that VacA-induced alterations in late endocytic membrane traffic contribute to the capacity of H. pylori to persistently colonize the human gastric mucosa.     

Enhancement of immunogenicity of HPV16 E7 oncogene by fusion with E. coli beta-glucuronidase

BACKGROUND: Human papillomavirus type 16 (HPV16) E7 is an unstable oncoprotein with low immunogenicity. In previous work, we prepared the E7GGG gene containing point mutations resulting in substitution of three amino acids in the pRb-binding site of the HPV16 E7 protein. METHODS AND RESULTS: To increase E7GGG immunogenicity we constructed fusion genes of E. coli beta-glucuronidase (GUS) with one or three copies of E7GGG. Furthermore, a similar construct was prepared with partial E7GGG (E7GGGp, 41 amino acids from the N-terminus). The expression of the fusion genes was examined in human 293T cells. Quantification of GUS activity and the amount of E7 antigen showed substantially reduced GUS activity of fusion proteins with complete E7GGG that was mainly caused by decrease of their steady-state level in comparison with GUS or E7GGGpGUS. Still, the steady-state level of E7GGG.GUS was about 20-fold higher than that of the E7GGG protein. The immunogenicity of the fusion genes with complete E7GGG was tested by DNA immunisation of C57BL/6 mice with a gene gun. TC-1 cells and their clone TC-1/A9 with down-regulated MHC class I expression were subcutaneously (s.c.) inoculated to induce tumour formation. All mice were protected against challenge with TC-1 cells and most animals remained tumour-free in therapeutic-immunisation experiments with these cells, in contrast to immunisation with unfused E7GGG and the fusion with the lysosome-associated membrane protein 1 (Sig/E7GGG/LAMP-1). Significant protection was also recorded against TC-1/A9 cells. Both tetramer staining and ELISPOT assay showed substantially higher activation of E7-specific CD8+ lymphocytes in comparison with E7GGG and Sig/E7GGG/LAMP-1. Deletion of 231 bp in the GUS gene eliminated enzymatic activity, but did not influence the immunogenicity of the E7GGG.GUS gene. CONCLUSIONS: The findings demonstrate the superior immunisation efficacy of the fusion genes of E7GGG with GUS when compared with E7GGG and Sig/E7GGG/LAMP-1. The E7GGG.GUS-based DNA vaccine might also be efficient against human tumour cells with reduced MHC class I expression.     

The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C

Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.     

Epidermal growth factor induces the accumulation of calpactin II on the cell surface during membrane ruffling

Confluent and proliferatively quiescent T51B rat liver epithelial cells provide a cellular model for the study of epidermal growth factor (EGF) effects in non-neoplastic cells. Immunoreactive calpactin II, a well-known substrate for EGF-receptor kinase, was found predominantly in the cytosol, although a second immunoreactive pool was found in a Triton X-100-extractable membrane fraction. Stimulation with EGF resulted in a rapid and transient (2-5 min) formation of ruffles at the cell surface and at the cell-cell contacts. Both calpactin II and filamentous actin were found co-localized at the membrane ruffles. Immunoprecipitations of membrane-bound calpactin II from 32P-labeled cells indicate a transient EGF-dependent phosphorylation of calpactin II correlating with membrane ruffling. These results suggest a temporal (2-5 min) function for calpactin II at the plasma membrane during the EGF-induced mitogenesis of T51B cells.     

S100-annexin complexes: some insights from structural studies

Several annexins have been shown to bind proteins that belong to the S100 calcium-binding protein family. The two best-characterized complexes are annexin II with p11 and annexin I with S100C, the former of which has been implicated in membrane fusion processes. We have solved the crystal structures of the complexes of p11 with annexin II N-terminus and of S100C with annexin I N-terminus. Using these structural results, as well as electron microscopy observations of liposome junctions formed in the presence of such complexes (Lambert et al., 1997 J Mol Biol 272, 42-55), we propose a computer generated model for the entire annexin II/p11 complex.     

Interaction of endocytotic vacuoles with the inner nuclear membrane in simian virus 40 entry into CV-1 cell nucleus.

The transfer of endocytosed simian virus 40 (SV40) to the nuclear position was investigated ultrastructurally using cationized ferritin (CF), ferritin labelled concanavalin A (Fer-Con A) and Con A as cell membrane markers. In the cells incubated with these markers and SV40 at 4 degrees C, and then chased for 2 h at 37 degrees C in serum-free medium, ferritin particles representing CF and/or Fer-Con A binding sites were found in vacuoles with SV40. The membrane of some vacuoles seemed to be in contact with the outer nuclear membrane. Several ferritin particles were located in the perinuclear cisterna and within the nucleoplasm, but not within the nuclear pores. In addition, there were vacuoles with ferritin particles and SV40 near the nuclear membrane, which looked like a single diaphragm with heterochromatins inside it. The outer nuclear and vacuole membranes were often obscure in the areas where the vacuole was very close to the diaphragm. In the case of cells incubated with CF, SV40 and Con A at 4 degrees C, chased for 2 h at 37 degrees C, and then reacted with horseradish peroxidase (HRP), HRP activity showing Con A-binding sites was also observed along the nuclear side of the inner nuclear membrane as well as in the perinuclear cisterna along the outer membrane. These results confirm that SV40-induced endocytotic vacuoles fuse with the outer nuclear membrane, and further indicate that some endocytotic vacuoles may well interact directly with the diaphragm, suggesting another path for migration of SV40 into CV-1 cell nuclei besides the path going through the process of fusion of the vacuole membrane with the outer nuclear membrane.     

Annexin II (calpactin I) in the mouse mammary gland: immunolocalisation by light- and electron microscopy

Summary To elucidate the putative role of annexin II (calpactin I) in the secretory function of mammary tissue its immunolocalisation in the mammary gland of pregnant and lactating mice was investigated by light- and electron microscopy using the immunoperoxidase technique. A low level of fairly uniform annexin II staining was evident throughout the gland despite its mixed composition during pregnancy. In lactating tissue it was revealed that apparently mature alveoli contained a concentration of annexin II staining outlining their epithelium. The staining was localised by immuno-electron microscopy to the apical membrane of these alveolar epithelial cells and their microvillar extentions. There was also an apparent association of annexin II with vesicles of a range of sizes located near, or actually fused with, the apical membrane. Many of the small, stained vasicles could clearly be identified as casein-containing vesicle while the large vesicles were apparently associated with either casein granules or possibly lipid. The appearance of a selective concentration of annexin II in apparently actively secreting mammary epithelial cells, as revealed in this study, is consistent with a possible structural and/or functional role for this protein at the membranes participating in the secretion of protein and possibly lipid from these secretory cells.