AMD3100 plus G-CSF can successfully mobilize CD34+ cells from non-Hodgkin's lymphoma, Hodgkin's disease and multiple myeloma patients previously failing mobilization with chemotherapy and/or cytokine treatment: compassionate use data.

AMD3100 given with G-CSF has been shown to mobilize CD34+ cells in non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), and Hodgkin's disease (HD) patients who could not collect sufficient cells for autologous transplant following other mobilization regimens. These poor mobilizers are usually excluded from company-sponsored trials, but have been included in an AMD3100 Single Patient Use protocol, referred to as a Compassionate Use Protocol (CUP). A cohort of 115 data-audited poor mobilizers in CUP was assessed, with the objective being to collect > or =2 x 10(6) CD34+ cells per kg following AMD3100 plus G-CSF mobilization. The rates of successful CD34+ cell collection were similar for patients who previously failed chemotherapy mobilization or cytokine-only mobilization: NHL -- 60.3%, MM -- 71.4% and HD -- 76.5%. Following transplant, median times to neutrophil and PLT engraftment were 11 days and 18 days, respectively. Engraftment was durable. There were no drug-related serious adverse events. Of the adverse events considered related to AMD3100, two (1.6%) were severe (one patient -- headache, one patient -- nightmares). Other AMD3100-related adverse events were mild (84.8%) or moderate (13.6%). The most common AMD3100-related adverse events were gastrointestinal reactions, injection site reactions and paresthesias. AMD3100 plus G-CSF offers a new treatment to collect CD34+ cells for autologous transplant from poor mobilizers, with a high success rate.

Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo.

BACKGROUND: Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis. AIMS: To determine the effects of relaxin on activated HSC. METHODS: Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor beta1 (TGF-beta1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively. RESULTS: Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-beta1 mRNA expression. CONCLUSION: These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation.

Rab7 regulates phagosome maturation in Dictyostelium.

A Dictyostelium Rab7 homolog has been demonstrated to regulate fluid-phase influx, efflux, retention of lysosomal hydrolases and phagocytosis. Since Rab7 function appeared to be required for efficient phagocytosis, we sought to further characterize the role of Rab7 in phagosomal maturation. Expression of GFP-Rab7 resulted in labeling of both early and late phagosomes containing yeast, but not forming phagocytic cups. In order to determine if Rab7 played a role in regulating membrane traffic between the endo/lysosomal system and maturing phagosomes, latex bead containing (LBC) phagosomes were purified from wild-type cells at various times after internalization. Glycosidases, cysteine proteinases, Rab7 and lysosomally associated membrane proteins were delivered rapidly to nascent phagosomes in control cells. LBC phagosomes isolated from cells overexpressing dominant negative (DN) Rab7 contained very low levels of LmpA (lysosomal integral membrane protein) and alpha-mannosidase was not detectable. Interestingly, cysteine proteinases were delivered to phagosomes as apparent pro-forms in cells overexpressing DN Rab7. Despite these defects, phagosomes in cells overexpressing DN Rab7 matured to form multi-particle spacious phagosomes, except that these phagosomes remained significantly more acidic than control phagosomes. These results suggested that Rab7 regulates both an early and late steps of phagosomal maturation, similar to its role in the endo/lysosomal system.

Intracellular distribution of gravin, a PKA and PKC binding protein, in vascular endothelial cells.

Gravin, a high-molecular-weight protein expressed widely in tissues and cells, is upregulated in cultured endothelial cells under conditions which suggest that it may play a role in wound repair and vascular development. In the current study, we examined the intracellular distribution of gravin to determine if it is associated with the cytoskeleton or with another intracellular compartment. Immunofluorescence microscopy of human umbilical vein endothelial cells (HUVEC) revealed that gravin had a punctate staining distribution that extended to the cell margin and did not appear to colocalize with stress fibers, microtubules, and intermediate filaments. Moreover, disruption of the cytoskeletal structures with either cytochalasin D or colchicine did not alter gravin distribution. However, confocal and immunoelectron microscopy clearly revealed that gravin was concentrated at the cell margin in close association with the plasma membrane. Immunoprecipitation of gravin from endothelial cell lysates resulted in coprecipitation of protein kinase activity that could be eluted from the immunoprecipitates with cAMP and that was inhibitable with a PKA-specific inhibitor. An anti-PKA catalytic subunit antibody reacted with a 40-kD band on immunoblots of the cAMP eluate. Immunoblots of the immunoprecipitates further revealed that PKCalpha coprecipitated with gravin from endothelial cell lysates. This study indicates that gravin is associated with either the plasma membrane or the membrane skeleton and may play a role in endothelial wound healing by targeting PKA and PKC to specific membrane-associated sites and regulating PKA/PKC-dependent cellular activities associated with endothelial wound healing.

Relaxin decreases renal interstitial fibrosis and slows progression of renal disease.

BACKGROUND: Relaxin, a hormone of the insulin-growth factor family, promotes collagen remodeling. In rodent models of pulmonary and dermal fibrosis, relaxin reduced interstitial fibrosis. To study relaxin's effect in renal disease, we used the experimental bromoethylamine (BEA) model that leads to severe renal interstitial fibrosis, a decrease in glomerular filtration rate, and albuminuria at one month. METHODS: Rats were injected with BEA one week prior to implantation of an osmotic pump delivering relaxin (2 microg/hour) or vehicle continuously for 28 days. RESULTS: BEA caused a significant decrease in creatinine clearance, which was partially prevented by relaxin. In the relaxin-treated BEA rats, serum creatinine was normal, and albumin excretion was slightly decreased. By morphometric measurement, relaxin administration was associated with a significant decrease in interstitial fibrosis at the corticomedullary junction. This was accompanied by a decrease in the number of ED-1 positive cells (an index of macrophage infiltration) and in the intensity of immunohistochemical staining for transforming growth factor-beta. This antifibrotic effect of relaxin did not appear to be mediated by systemic hemodynamic changes since the mean arterial pressure was not significantly different among the groups. CONCLUSIONS: Relaxin may have a useful application in decreasing interstitial fibrosis and thereby slowing the progression of renal disease.

p110-related PI 3-kinases regulate phagosome-phagosome fusion and phagosomal pH through a PKB/Akt dependent pathway in Dictyostelium.

The Dictyostelium p110-related PI 3-kinases, PIK1 and PIK2, regulate the endosomal pathway and the actin cytoskeleton, but do not significantly regulate internalization of particles in D. discoideum. Bacteria internalized into delta ddpik1/ddpik2 cells or cells treated with PI 3-kinase inhibitors remained intact as single particles in phagosomes with closely associated membranes after 2 hours of internalization, while in control cells, bacteria appeared degraded in multi-particle spacious phagosomes. Addition of LY294002 to control cells, after 60 minutes of chase, blocked formation of spacious phagosomes, suggesting PI 3-kinases acted late to regulate spacious phagosome formation. Phagosomes purified from control and drug treated cells contained equivalent levels of lysosomal proteins, including the proton pump complex, and were acidic, but in drug treated cells and delta ddpik1/ddpik2 cells phagosomal pH was significantly more acidic during maturation than the pH of control phagosomes. Inhibition of phagosomal maturation by LY294002 was overcome by increasing phagosomal pH with NH(4)Cl, suggesting that an increase in pH might trigger homotypic phagosome fusion. A pkbA null cell line (PKB/Akt) reproduced the phenotype described for cells treated with PI 3-kinase inhibitors and delta ddpik1/ddpik2 cells. We propose that PI 3-kinases, through a PKB/Akt dependent pathway, directly regulate homotypic fusion of single particle containing phagosomes to form multi-particle, spacious phagosomes, possibly through the regulation of phagosomal pH.

Relaxin induces vascular endothelial growth factor expression and angiogenesis selectively at wound sites.

Relaxin is a reproductive hormone that has historically been characterized as being responsible for pubic ligament loosening and cervical ripening. Recently, relaxin has been associated with neovascularization of the endometrial lining of the uterus, potentially via specific induction of vascular endothelial growth factor. Previously conducted clinical studies using partially purified porcine relaxin have described relaxin's ability to stimulate the healing of ischemic wounds, suggesting that relaxin may also have angiogenic effects at sites of ischemic wound healing. In the present study, relaxin's angiogenic effects in the context of wound repair were tested in rodent models of angiogenesis and wound healing. Relaxin showed an ability to stimulate new blood vessel formation, particularly at ischemic wound sites, and to induce both vascular endothelial growth factor and basic fibroblast growth factor specifically in cells, presumably including macrophages, collected from wound sites. Resident macrophages collected from nonwound sites, such as the lung, did not show altered expression of these cytokines following relaxin administration. Because angiogenic wound cells are frequently macrophages, THP-1 cells, a cell line of monocyte lineage that binds relaxin specifically, were tested for and shown to induce vascular endothelial growth factor and basic fibroblast growth factor in response to relaxin. In conclusion, relaxin may be useful in the treatment of ischemic wounds by stimulating angiogenesis via the induction of vascular endothelial growth factor and basic fibroblast growth factor in wound macrophages.

E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium.

Ovarian carcinomas are thought to arise in the ovarian surface epithelium (OSE). Although this tissue forms a simple epithelial covering on the ovarian surface, OSE cells exhibit some mesenchymal characteristics and contain little or no E-cadherin. However, E-cadherin is present in metaplastic OSE cells that resemble the more complex epithelia of the oviduct, endometrium and endocervix, and in primary epithelial ovarian carcinomas. To determine whether E-cadherin was a cause or consequence of OSE metaplasia, we expressed this cell-adhesion molecule in simian virus 40-immortalized OSE cells. In these cells the exogenous E-cadherin, all three catenins, and F-actin localized at sites of cell-cell contact, indicating the formation of functional adherens junctions. Unlike the parent OSE cell line, which had undergone a typical mesenchymal transformation in culture, E-cadherin-expressing cells contained cytokeratins and the tight-junction protein occludin. They also formed cobblestone monolayers in two-dimensional culture and simple epithelia in three-dimensional culture that produced CA125 and shed it into the culture medium. CA125 is a normal epithelial-differentiation product of the oviduct, endometrium, and endocervix, but not of normal OSE. It is also a tumor antigen that is produced by ovarian neoplasms and by metaplastic OSE. Thus, E-cadherin restored some normal characteristics of OSE, such as keratin, and it also induced epithelial-differentiation markers associated with weakly preneoplastic, metaplastic OSE and OSE-derived primary carcinomas. The results suggest an unexpected role for E-cadherin in ovarian neoplastic progression.

Relaxin stimulates expression of vascular endothelial growth factor in normal human endometrial cells in vitro and is associated with menometrorrhagia in women.

Although the role of the reproductive hormone, relaxin, in rodents is well documented, its potential contribution to human reproduction is less well defined. In this study, we examine the effects of relaxin on human endometrial cells in vitro and describe the clinical effects of relaxin on menstrual flow in women. In cultured endometrial cells, relaxin specifically induces the expression of an angiogenic agent, vascular endothelial growth factor (VEGF). cAMP is implicated as a second messenger involved in VEGF stimulation. VEGF expression is temporally regulated in the endometrium, and our results suggest that relaxin, which is secreted by the corpus luteum and is present in the endometrium during the menstrual cycle and pregnancy, may be involved in regulating endometrial VEGF expression. Relaxin was recently tested in a clinical trial for efficacy in the treatment of progressive systemic sclerosis, and was administered at levels up to 10 times higher than that measured during pregnancy. The most frequent relaxin-related adverse event reported during the course of the study was the onset of menometrorrhagia, defined in this study as heavier-than-usual or irregular menstrual bleeding. The intensification of menstrual flow observed in these patients is consistent with the hypothesis that relaxin mediates neovascularization of the endometrial lining.

A Philadelphia-negative chronic myeloid leukemia with a BCR/ABL fusion gene on chromosome 9.

A 40-year-old man had chronic myeloid leukemia (CML) and an apparently normal karyotype. Fluorescence in situ hybridization with a BCR/ABL1-S probe, which is formatted to display a BCR/ABL fusion signal on chromosome 22, gave a positive fusion signal on a chromosome 9. Therefore this patient has a BCR/ABL fusion gene on chromosome 9. The BCR/ABL1-D probe, formatted to display a fluorescent signal for both the reciprocal products of a 9/22 rearrangement, gave a positive fusion signal on the derivatives 9 and 22. These findings favor either a cryptic reciprocal exchange between BCR and ABL loci or the reversal of a Philadelphia translocation. An insertion of BCR next to ABL is ruled out. The reverse-transcriptase polymerase chain reaction provided molecular evidence that a typical CML chimeric product resulting from a fusion of BCR exon 2 with C-ABL exon II, a2b2, is present.

Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization.

Phosphatidylinositide 3-kinases (PI3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles. There are at least three genes in Dictyostelium discoideum. DdPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mammalian 110-kD PI-3 kinase in amino acid sequence within the kinase domain. A mutant disrupted in DdPIK1 and DdPIK2 (delta ddpik1/ddpik2) grows slowly in liquid medium. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic postlysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase. Mutant cells were also almost completely devoid of large postlysosomal vacuoles as determined by transmission EM. However, delta ddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme alpha-mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologically normal in mutant cells. Light microscopy revealed that delta ddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1-3% of the mutant cells were also connected by a thin cytoplasmic bridge. Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a cortical pattern in control cells. Finally, delta ddpik1/ddpik2 cells responded and moved more rapidly towards cAMP. Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization.

Relaxin induces an extracellular matrix-degrading phenotype in human lung fibroblasts in vitro and inhibits lung fibrosis in a murine model in vivo.

Pulmonary fibrosis is the common end stage of a number of pneumopathies. In this study, we examined the ability of the human cytokine, relaxin, to block extracellular matrix deposition by human lung fibroblasts in vitro, and to inhibit lung fibrosis in a bleomycin-induced murine model. In vitro, relaxin (1-100 ng/ml) inhibited the transforming growth factor-beta-mediated over-expression of interstitial collagen types I and III by human lung fibroblasts by up to 45% in a dose-dependent manner. Relaxin did not affect basal levels of collagen expression in the absence of TGF-beta-induced stimulation. Relaxin also blocked transforming growth factor-beta-induced upregulation of fibronectin by 80% at the highest relaxin dose tested (100 ng/ml). The expression of matrix metalloproteinase-1, or procollagenase, was stimulated in a biphasic, dose-dependent manner by relaxin. In vivo, relaxin, at a steady state circulating concentration of approximately 50 ng/ml, inhibited bleomycin-mediated alveolar thickening compared with the vehicle only control group (P < 0.05). Relaxin also restored bleomycin-induced collagen accumulation, as measured by lung hydroxyproline content, to normal levels (P < 0.05). In summary, relaxin induced a matrix degradative phenotype in human lung fibroblasts in vitro and inhibited bleomycin-induced fibrosis in a murine model in vivo. These data indicate that relaxin may be efficacious in the treatment of pathologies characterized by lung fibrosis.

Restricted endothelial cell expression of gravin in vivo.

BACKGROUND: Gravin, a novel, high molecular weight, intracellular protein, is expressed in endothelial cells and several other adherent cell types in vitro. To gain insights into its function, we examined the distribution of gravin in tissues. METHODS: Affinity-purified polyclonal and monoclonal antibodies were raised against a bacterial fusion protein corresponding to the carboxyl terminus of gravin and against affinity-isolated gravin. The specificity of the antibodies was characterized by immunoblotting bacterial, cell, and tissue extracts. The characterized antibodies were used to localize gravin in baboon tissue sections by immunocytochemistry and immunofluorescence microscopy. RESULTS: The antibodies specifically immunoblotted the fusion protein and recognized either a band at 250 kDa or a doublet at 300 kDa on immunoblots of MG63 cells, HEL cells stimulated with phorbol ester, and several baboon tissues. In tissue sections, cell types that express gravin included fibroblasts, components of the peripheral and central nervous system, the adrenal medulla, the somatic layer of Bowman's capsule, cells associated with the glomerulus, and smooth muscle of certain organs. In contrast, most epithelia and all endothelia, with the exception of endothelia of the hepatic sinusoids and intestinal lacteals, lacked gravin. Levels of gravin mRNA expression in stimulated HEL cells increased dramatically when cells were stimulated in the presence of cycloheximide, suggesting that gravin expression may be partly regulated by protein-dependent mRNA catabolism. CONCLUSIONS: These data indicate that gravin expression is regulated in endothelial cells, possibly through protein-dependent mRNA catabolism. The strong expression of gravin in fibroblasts, neurons, and cells derived from neural crest in vivo and in adherent cells in vitro further suggests that this protein may play role in the modulation of cell motility and adhesion.

Molecular cloning and preliminary characterization of a novel cytoplasmic antigen recognized by myasthenia gravis sera.

A cDNA clone was isolated by screening of a lambda gt11 endothelial expression library with serum from a patient with myasthenia gravis (MG). Rabbit antisera raised against the recombinant protein and human MG sera reactive with the clone immunoblotted an M(r) integral of 250,000 polypeptide (gravin) present in endothelial cells and several adherent cells. Gravin was not detected in platelets, leukocytes, U937, or human erythroleukemic (HEL) cell lines, but was expressed in HEL cells after induction with phorbol myristate acetate. Northern blot analysis showed two transcripts of approximately 6.7 and 8.4 kb in endothelial cells but not U937 or HEL cells. Indirect immunofluorescence of permeabilized cells revealed a trabecular network of gravin staining with a distinct linear component. Antibodies to gravin, were present in sera from 22:72 (31%) of MG patients. In contrast 0:50 normal sera and 1:72 sera from patients with other autoimmune diseases contained antigravin antibodies. Gravin is not likely to be a nonerythroid spectrin, talin, myosin, or actin-binding protein based on the lack of reactivity of antigravin with these polypeptides in immunoblots. The nucleotide sequence of the immunoreactive clone indicated that it encodes a highly acidic polypeptide fragment that contains the carboxyl terminus of the protein. Neither amino acid nor nucleotide sequences were present in Genbank, EMBL, or Swissprot databases as of March, 1992. These data indicate that gravin is an inducible, cell type-specific cytoplasmic protein and that auto-antibodies to gravin may be highly specific for MG.

Dominant rearrangements among human tumor-infiltrating lymphocytes. Analysis of T-cells derived from 32 patients with melanoma, lung, and renal cell carcinoma.

Dominant rearrangements of T-cell receptor (TCR) beta-chain genes are reported among tumor-infiltrating lymphocytes (TIL). After interleukin-2 expansion of TIL from renal and lung carcinoma and melanoma biopsy tissues, rearrangements of TCR beta-chain genes were analyzed by Southern blotting. Nongermline restriction fragments, indicating dominant rearrangements, were detected among the TIL from all 6 patients with renal cell carcinoma, 17 of 20 patients with melanoma, and 3 of 6 patients with lung tumors. The restriction-fragment sizes of these dominant rearrangements were heterogeneous among the various patients. Rearrangements into C beta 1 were more common than C beta 2 rearrangements. Phenotypic analyses indicated that dominant rearrangements occurred in both CD4 and CD8 predominant TIL populations. The TIL populations that were extracted were expanded to derive large cell numbers suitable for in vivo transfer in an interleukin-2 and TIL immunotherapy program. The data indicated that the cells delivered to these patients usually were characterized by dominant populations of T-cells with selective TCR gene rearrangements. The significance of selective TCR use requires evaluation of the function and specificity of the TIL comprising these dominant populations both in their native in vivo setting and in the context of therapeutic transfer.

Brain-associated glycoproteins expressed in bovine heart Purkinje fibres.

The Purkinje fibres of bovine heart were investigated immunohistochemically by use of monoclonal antibodies with specificity against the glycoproteins Thy-1 and Gp120, expressed in human brain. The existence and expression in bovine tissues (brain and thymus) of antigens displaying similar properties and immunochemical crossreactivity with monoclonal antibodies against the human antigens were confirmed. Both these antigens, as identified by use of anti Thy-1 and anti-Gp120 monoclonal antibodies were found to be associated with the membranes of the impulse conduction system. The presence of the antigens was seen in areas facing other conduction cells. No parts of the cells facing the basal membrane of the fibres were stained. The continuous staining between the cells was different from that of desmosome related proteins. These findings may have physiological and functional implications and are interesting in relation to recent evidences suggesting that the conduction tissue might be a derivative of the neural crest.

Immunofluorescence localization of vinculin in ectoplasmic ("junctional") specializations of rat Sertoli cells.

We have investigated, using indirect immunofluorescence techniques, the possibility that vinculin is a component of Sertoli cell ectoplasmic specializations. Affinity-purified polyclonal antibodies produced against human platelet vinculin were used to probe fixed frozen sections of rat testis. Specific fluorescence occurs in Sertoli cell regions adjacent to spermatids and to basally situated junctional complexes, sites at which ectoplasmic specializations are known to occur. Staining also occurs in Sertoli cell regions associated with tubulobulbar complexes. The antibody also labels focal contacts in cultured human dermal fibroblasts, apical junctional sites of rat epididymal epithelium, and dense plaques of smooth muscle. Our results are consistent with the prediction that vinculin is likely a component of ectoplasmic specializations and are also consistent with the hypothesis that these structures are a form of actin-associated adhesion complex.

Myofibrillar M-band proteins in rat skeletal muscles during development.

The distribution of three myofibrillar M-band proteins, myomesin, M-protein and the muscle isoform of creatine kinase, was investigated with immunocytochemical techniques in skeletal muscles of embryonic, fetal, newborn and four-week-old rats. Furthermore, muscles of newborn rats were denervated and examined at four weeks of age. In embryos, myomesin was present in all myotome muscle fibres of the somites, whereas M-protein was detected only in a small proportion of the myotome muscle fibres and muscle creatine kinase was not detected at all. In fetal and newborn muscles, all fibres contained all three M-band proteins. At four weeks of age, when fibre types (type 1 or slow twitch fibres and type 2 or fast twitch fibres) were clearly discernable, the pattern was changed. Myomesin and muscle creatine kinase were still observed in all fibres, whereas M-protein was present only in type 2 fibres. On the other hand, in muscle fibres denervated at birth all three M-band proteins were still detected. Our results suggest 1) that during the initial stages of myofibrillogenesis expression and incorporation of myomesin into the M-band precede that of M-protein and muscle creatine kinase; 2) that expression and incorporation of all three M-band proteins during fetal development is nerve independent and non coordinated to the expression of different forms of myosin heavy chains, and 3) that the suppression of M-protein synthesis during postnatal development is nerve dependent and reflects the maturation of slow twitch motor units.

Sertoli cell ectoplasmic specializations: a type of actin-associated adhesion junction?

In this paper we provide evidence that ectoplasmic specializations are a form of intercellular adhesion junction. Ectoplasmic specializations, found at basal junctions between adjacent Sertoli cells and at sites of adhesion between Sertoli cells and germ cells, consist of actin filament bundles sandwiched between the plasma membrane and a cistern of endoplasmic reticulum. The actin filaments in each bundle are unipolar and are hexagonally packed. The bundles are coupled to the adjacent membranes and to each other. Because ectoplasmic specializations are associated with junctional sites, they may play a role in intercellular adhesion. In this study, we report a procedure for obtaining samples enriched for ectoplasmic specializations and identify polypeptides that may be associated with ectoplasmic specializations. On SDS-polyacrylamide gels, an 83K (K = 10(3) Mr) polypeptide is specific to the ectoplasmic specialization-enriched sample, suggesting that it may be a component of ectoplasmic specializations. Other polypeptides at 38, 53, 56 and 69K also may be associated with ectoplasmic specializations. Immunoblots further indicate that fimbrin and vinculin are present in the ectoplasmic specialization-enriched fraction. In addition, immunofluorescence indicates that vinculin is associated with spermatid-Sertoli cell and Sertoli-Sertoli cell junctions. We suspect that fimbrin, an actin-bundling protein, may be involved in cross-linking the hexagonally packed actin filaments in ectoplasmic specializations while vinculin may be associated with actin-membrane linkages. If so, ectoplasmic specializations may be a new class of actin-associated junctional site. Moreover, the presence of vinculin in testicular fractions enriched for ectoplasmic specializations and at junctional sites supports the view that these structures may play a role in intercellular adhesion, possibly by stabilizing an adhesive membrane domain.