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PubMed1993

Overexpression of acidic and basic fibroblast growth factors in human pancreatic cancer correlates with advanced tumor stage.

Acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) are mitogenic polypeptides that may contribute to cancer cell proliferation. In the present study we examined aFGF and bFGF expression in human pancreatic cancer. Northern blot analysis of total RNA isolated from 12 pancreatic cancers revealed elevated aFGF and bFGF mRNA levels in 12 and 10 samples, respectively, by comparison with the normal human pancreas. Immunostaining demonstrated the presence of aFGF and bFGF in many cancer cells and in the atrophic acini and ducts adjacent to the cancer cells, but to a much lesser extent in the surrounding fibroblasts. By in situ hybridization, both mRNA moieties colocalized with their respective proteins and were abundant in many cancer cells. Immunoblotting confirmed that cancer tissues with increased aFGF and bFGF immunoreactivity contained elevated levels of both proteins. To determine the significance of aFGF and bFGF expression in the pancreatic cancer cells, immunohistochemical analysis of 78 human pancreatic carcinomas was performed. aFGF and bFGF immunoreactivity was present in the cancer cells in 47 (60%) and 44 (56%) of the tumors, respectively. There was a significant correlation between the presence of either aFGF or bFGF in the cancer cells and advanced tumor stage, and the presence of bFGF and shorter patient survival. These data suggest that aFGF and bFGF are overexpressed in a significant proportion of human pancreatic carcinoma cells and that this overexpression may contribute to disease progression.

Adult↗
PubMed1986

Comparative distribution of 3H-GABA uptake and GAD immunoreactivity in goldfish retinal amacrine cells: a double-label analysis.

The comparative distribution of 3H-GABA uptake and glutamic acid decarboxylase immunoreactivity (GAD-IR) in amacrine cells of goldfish retina was studied simultaneously by using a combined autoradiographic/immunocytochemical technique in order to determine the degree of colocalization of these two markers of GABAergic neurons; 3H-GABA was taken up most intensely by large pyriform Ab amacrine cells (3% of inner nuclear layer (INL) somata), and less intensely by smaller, polyform amacrine cells (12% of INL somata), cell bodies in the ganglion cell layer (one-half as common as Ab cells), and cell bodies in the inner plexiform layer (very rare). GAD-IR was observed in 25% of amacrine cells in the INL, the vast majority of which were polyform in shape, and cell bodies in the ganglion cell layer. Twice as many cells were labeled for GAD-IR as for 3H-GABA uptake. Of the cells that took up 3H-GABA, colocalization of 3H-GABA uptake and GAD-IR was observed in 90% of the polyform amacrine cells, 80% of the cells in the ganglion cell layer, and none of the pyriform Ab amacrine cells or cells in the inner plexiform layer. We suggest that the polyform cells compose the major population of GABAergic amacrine cells in the goldfish retina, rather than the pyriform Ab cells. GABAergic displaced amacrine cells or ganglion cells are also indicated by our data. The implications of these data with regard to the physiology of the goldfish retina are discussed as well.

Animals↗
PubMed1990

Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor.

We have identified a region within the steroid binding domain of the mouse estrogen receptor that is required for both receptor dimerization and high affinity DNA binding. Analysis of sequences in this region revealed that a heptad repeat of hydrophobic residues was conserved in all members of the nuclear receptor superfamily. Single amino acid substitutions of residues in the N-terminal half, but not the C-terminal half, of the repeat prevented receptor dimerization. Steroid binding was abolished by point mutations in the center of the conserved region, implying that the steroid binding and dimerization domains overlap. The role of this region in steroid receptor function is discussed in relation to other models of protein dimerization and DNA binding.

Amino Acid Sequence↗
PubMed1993

Tropomodulin is highly concentrated at the postsynaptic domain of human and rat neuromuscular junctions.

Tropomodulin is expressed in skeletal muscle and recent studies suggest that tropomodulin is associated with synaptic membranes. Therefore, we have examined neuromuscular junctions by immunofluorescence analysis for tropomodulin localization. Anti-tropomodulin antibodies generated against either the whole protein or a 15-amino acid peptide fragment label human neuromuscular junctions as demonstrated by colocalization with alpha-bungarotoxin label. Tropomodulin labeling also colocalizes with desmin and beta-amyloid proteins which are concentrated in the postsynaptic region. Comparable results are found with immunostaining of neuromuscular junctions in rat diaphragm muscle. These immunofluorescence results suggest that tropomodulin is an integral component of the cytoskeletal lattice associated with the postsynaptic region of neuromuscular junctions.

Animals↗
PubMed1995

B-50 (GAP-43) immunoreactivity is rarely detected within intact catecholaminergic and serotonergic axons innervating the brain and spinal cord of the adult rat, but is associated with these axons following lesion.

The persistence of high levels of B-50 (GAP-43) in fibers innervating various regions of the adult central nervous system is generally thought to characterize neuronal systems capable of undergoing morphological plasticity. In a recent series of in situ hybridization studies, it has been shown that most catecholaminergic and serotonergic neurons of the adult rat brain express high levels of B-50 mRNA. The present study addresses the question whether high expression of B-50 mRNA in the catecholaminergic and serotonergic perikarya corresponds with detectable high levels of the B-50 protein in the efferent axonal fibers that innervate various regions of the adult rat brain and spinal cord. For this purpose, vibratome sections were doubly immunostained for B-50 and for tyrosine hydroxylase or serotonin and were analyzed by laser scanning confocal microscope. Colocalizations were investigated either (1) in regions of intact rat brain and spinal cord in which particular concentrations of B-50 immunoreactive fibers appeared codistributed with catecholaminergic or serotonergic fibers or (2) in intrahypothalamic portions of the medial forebrain bundle in which a surgical lesion was made. In the intact brain, frequent colocalizations of B-50 and tyrosine hydroxylase were detected in fibers innervating both the mediobasal hypothalamus and the neurointermediate hypophysial lobe. In all the other regions examined, the analysis of thin optical sections demonstrated that immunoreactivity to B-50 was only rarely associated with axonal profiles immunoreactive to tyrosine hydroxylase or to serotonin. By contrast, in the lesioned medial forebrain bundle B-50 immunoreactivity was found to be associated with numerous catecholaminergic and serotonergic axonal sprouts that regenerate around the surgical lesion. These data indicate that the majority of intact catecholaminergic and serotonergic axons innervating the adult rat brain and spinal cord contains low levels of B-50. However, following axotomy, B-50 is immunocytochemically detectable in the regenerating sprouts produced by both types of axonal fibers. This suggests that under basal conditions the relatively high content of B-50 mRNA in monoaminergic perikarya does not lead to appreciable accumulation of B-50 within corresponding axonal fibers and terminals, whereas conditions of morphological reorganization induce increased production of B-50 that accumulates within monoaminergic axonal sprouts.

Animals↗
PubMed1987

[Conformation analysis of modified tetrasaccharide sequences of the N-glycoprotein type--problem of the alpha-(1 to 6)-glycosidic bond].

The conformational analysis of the recently synthesized tetrasaccharides alpha-D-Manp (1----3)-[alpha-D-Manp-(1----6)]-4-deoxy-beta-D-lyx-hexp+ ++-(1----4)-D-GlcNAc (2) and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)-D-GlcNAc (3) will be described. The preferred solution conformation of 2 and 3 is a gt-conformation, which is nearly identical with the preferred conformation of the naturally occurring tetrasaccharide alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-D-GlcNAc (1). The main structural feature is the backfolding of the alpha-(1----6)-linked D-Man to the reducing D-GlcNAc unit. Conformational analysis of the tetrasaccharides alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-1,6- anhydro-beta-D-GlcNAc (4), alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)]-4-deoxy-beta-D- lyx-hexp-(1----4)- 1,6-anhydro-beta-D-GlcNAc (5), and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)- 1,6-anhydro-beta-D-GlcNAc (6) gave additional proof for this backfolding. The substitution of the reducing unit leads to a smaller amount of gt- and a greater amount of gg-conformers. The method used for conformational analysis of 2-6 is a combination of n.m.r.-experiments and HSEA-calculations with the program GESA. Concerning the application of new 2D-techniques, the COLOC-experiment turned out to be extremely useful in sequencing oligosaccharides.

Carbohydrate Conformation↗
PubMedDate not supplied

Distribution and co-localization of calbindin D28k with VIP and neuropeptide Y but not somatostatin, galanin and substance P in the enteric nervous system of the rat.

Calbindin D28k, previously demonstrated in the mammalian central nervous system, has been localized to discrete neurons in the enteric nervous system of the rat. Calbindin D28k is present in cell bodies in both the myenteric and submucous plexi and in interganglionic nerve fibers in all regions of the gastrointestinal tract. Immunoreactive nerve fibers were also detected in the mucosal region, although none were observed in the pyloric sphincter, circular or longitudinal muscle layers. The highest concentration of immunoreactivity was present in the submucosal plexus and mucosa of the colon. Western blot analysis of the protein detected by the antiserum confirmed that it comigrated with purified calbindin D28k and the single immunoreactive band seen in extracts from rat brain. The colocalization of calbindin D28k with components of the peptidergic innervation was also investigated. Of the peptides studied the neurons containing both vasoactive intestinal polypeptide and neuropeptide Y in the submucous plexus were seen to exhibit calbindin D28k immunoreactivity. The neurons containing somatostatin, galanin and substance P did not demonstrate co-localization. In the stomach, calbindin D28k was detected within a small number of epithelial cells which were found to correspond to a sub-population of the somatostatin-immunoreactive endocrine cells.

Animals↗
PubMed1994

Immunohistochemical localization of collagen VI in diabetic glomeruli.

Late stage diabetic nephropathy is histologically characterized by either diffuse or nodular expansion of the glomerular matrix. This is presumed to represent the morphological correlate for the functional impairment of the kidney. The exact matrix composition of the nodular glomerulosclerosis lesion of end-stage diabetic nephropathy is not known. Biochemical studies have provided evidence that the microfibrillar collagen type VI is increased in diabetic nephropathy. Consequently, this immunohistochemical study was designed to evaluate the extent and exact morphologic location of increased collagen VI deposition at various stages of diabetic glomerulosclerosis (GS). An irregular, sometimes spot-like staining of collagen VI was observed in diffuse GS in the mesangial portion. The uninterrupted staining which was evident along the glomerular basement membrane in normal glomeruli was discontinuous in diffusely sclerotic glomeruli. In nodular GS, the markedly increased deposition of collagen VI appeared to be evenly distributed throughout the entire nodular lesion. At the same time, mesangial staining for collagen IV was reduced in nodular GS, suggesting that in the expanded mesangial matrix collagen IV is progressively substituted by collagen VI during the transition from diffuse to nodular GS. The colocalization of PAS staining with collagen VI deposition in nodular GS suggests that the typical Kimmelstiel-Wilson lesions at least in part consist of collagen VI. Biochemical analysis confirmed the increased collagen VI deposition in glomeruli extracted from diabetic patients with nodular GS. Application of two antisera, recognizing primarily the alpha 1(VI)- and alpha 2(VI)-chains and the N-terminal part of alpha 3(VI)-chain, respectively, revealed no difference in staining pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Release of Ca2+ from individual plant vacuoles by both InsP3 and cyclic ADP-ribose.

Calcium mobilization from intracellular pools couples many stimuli to responses in plant cells. Cyclic adenosine 5'-diphosphoribose (cADPR), which interacts with a ryanodine receptor in certain animal cells, was shown to elicit calcium release at the vacuolar membrane of beet storage root. The vacuolar calcium release pathway showed similarities to cADPR-gated calcium release in animal cells, including inhibition by ruthenium red, ryanodine activation, and high affinity for cADPR [Michaelis constant (Km) = 24 +/- 7 nanomolar]. Analysis by patch-clamping demonstrated that the cADPR-gated pathway in beet is voltage-dependent over the physiological range, does not spontaneously desensitize, and is colocalized with an inositol 1,4,5-trisphosphate (InsP3)-gated calcium release pathway in individual vacuoles.

Adenosine Diphosphate Ribose↗
PubMed2026

FANCI promotes esophageal squamous cell carcinoma progression and cell cycle regulation and interacts with FANCD2.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with poor clinical outcomes, and reliable molecular biomarkers and therapeutic targets remain limited. Fanconi anemia group I protein (FANCI) is a core component of the Fanconi anemia (FA) pathway, but its expression pattern, clinical significance, and functional role in ESCC have not been comprehensively defined. This study aimed to investigate FANCI expression and prognostic value in ESCC, assess its effects on malignant cellular phenotypes and tumor growth, and explore its potential mechanistic relationship with Fanconi anemia group D2 protein (FANCD2) and cell-cycle regulation. METHODS: Multi-cohort analyses were performed using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets, together with ESCC single-cell RNA sequencing (RNA-seq) data. FANCI functions were assessed by bidirectional gain- and loss-of-function experiments in vitro (proliferation, colony formation, migration, invasion, apoptosis, and cell-cycle assays) and by xenograft models in vivo. Mechanistic studies included protein-protein interaction (PPI) analyses, co-immunoprecipitation (Co-IP), and immunofluorescence (IF) colocalization. RESULTS: FANCI was consistently upregulated in ESCC across bulk transcriptomic datasets and was further supported by quantitative polymerase chain reaction (qPCR), Western blotting, and immunohistochemistry (IHC). FANCI discriminated ESCC from normal tissues in TCGA-ESCC and was independently validated in GSE53624 [area under the curve (AUC) =0.940 and 0.975, respectively]. FANCI was associated with poorer overall survival (OS) and shorter disease-free interval (DFI), and these findings were validated in an independent GEO cohort. Functionally, FANCI promoted ESCC cell proliferation, migration, and invasion, while inhibiting apoptosis; FANCI knockdown suppressed tumor growth in vivo and induced G2/M cell-cycle arrest. Mechanistically, FANCI physically interacted with FANCD2, colocalized with FANCD2 in the nucleus, and was associated with altered FANCD2 protein abundance, consistent with cell-cycle and DNA repair-related programs. Single-cell analysis indicated that FANCI was enriched in epithelial cells and associated with higher activity of malignant functional programs. In TCGA-ESCC, FANCI-high tumors showed distinct mutation profiles, a trend toward increased tumor mutation burden (TMB), and altered immune-associated signatures. CONCLUSIONS: FANCI is upregulated in ESCC and is associated with diagnostic and prognostic value. It promotes malignant phenotypes and tumor growth, potentially through a FANCI-FANCD2-linked cell-cycle/DNA repair program, supporting FANCI as a candidate biomarker and therapeutic target in ESCC.

Esophageal squamous cell carcinoma (ESCC)↗
PubMed1990

Use of enhanced silver staining combined with electron microscopical immunolabelling to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in cerebrovascular nerves.

The combination of immunolabelling at the electron microscope level and enhanced silver staining has been used to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in perivascular nerves supplying cerebral arteries of the rat. This has been shown in control tissue, but it is easier to demonstrate after long-term sympathectomy since that leads to an enhancement of neuropeptide Y in vasoactive intestinal polypeptide-containing parasympathetic nerves supplying these vessels. Immunolabelling of the antigens for these peptides was performed sequentially with the biotin streptavidin diaminobenzidine method, and the end product to the first antiserum was gold-silver intensified before the visualization of the second antigen. Using this technique, it was shown that all the neuropeptide Y immunoreactivity present in the rat cerebral vessels after long-term sympathectomy with guanethidine was localized in vasoactive intestinal polypeptide-containing nerves. Furthermore, an immunohistochemical analysis of the parasympathetic pterygopalatine ganglia in guanethidine-treated rats showed an increase in the percentage of neurons displaying neuropeptide Y immunoreactivity. In order to clarify if the pterygopalatine ganglion was the origin of those neuropeptide Y/vasoactive intestinal polypeptide-immunoreactive cerebrovascular nerves, which had increased in number after sympathectomy, a fluorescent neuronal tracer (Fast Blue) was applied to the right middle cerebral artery of rats which had undergone guanethidine treatment for six weeks. Immunohistochemical analysis of the ipsilateral ganglion 72 h after application of the tracer revealed the presence of immunoreactivity to both these peptides in retrogradely labelled neurons. It is concluded that neuropeptide Y and vasoactive intestinal polypeptide are colocalized in perivascular parasympathetic nerves supplying the middle cerebral artery of the rat, which have their origin in the pterygopalatine ganglion. Furthermore, long-term sympathectomy with guanethidine leads to an increase in the expression of neuropeptide Y in these vasoactive intestinal polypeptide-immunoreactive neurons.

Animals↗
PubMed1993

Colocalization of GABA and glycine in the rabbit oculomotor nucleus.

In the present study we examined the possible colocalization of the inhibitory neurotransmitters glycine and GABA in the oculomotor nucleus of the rabbit. Serial sections were processed alternately for glycine and GABA postembedding immuno-cytochemistry. Ultrastructural analysis revealed that all terminals that showed glycine-positive immunoreactivity were also GABA positive; up to 5% of the GABA-positive terminals were also glycine positive.

Animals↗
PubMed1993

Receptor-mediated endocytosis of alpha 2 macroglobulin by a glioma cell line.

Previous experiments have shown that human neoplastic and embryonic glial cell lines synthesize and secrete in culture, alpha 2 macroglobulin (alpha 2M), a broad spectrum proteinase inhibitor present in serum and extracellular fluids. The present study was aimed to investigate the presence of alpha 2M receptors on glial cell membrane, since several non-neural cell types producing alpha 2M also express alpha 2M receptors. By flow cytometric analysis, immunofluorescence and immunoelectronmicroscopy techniques we demonstrate an alpha 2M receptor-related immunoreactivity on the plasma membrane of a human glioma cell line. Ultrastructural experiments reveal a close colocalization of immunoreactivities for alpha 2M and its receptor in clathrin-coated pits and vesicles, structures typically involved in receptor-mediated endocytic pathways.

Endocytosis↗
PubMed1995

The Vif protein of human and simian immunodeficiency viruses is packaged into virions and associates with viral core structures.

The vif gene of human and simian immunodeficiency viruses (HIV and SIV) encodes a late gene product that is essential for viral infectivity in natural target cells. Virions produced in the absence of Vif are abnormal in their ultrastructural morphology and are severely impaired in the ability to complete proviral DNA synthesis upon entry into new target cells. Because previous studies failed to detect Vif protein in virus particles, Vif is believed to influence virus infectivity indirectly, by affecting virion assembly, release, and/or maturation. In this report, we reexamined the possibility that Vif is a virion-associated protein. Utilizing high-titer Vif-specific antibodies, a sensitive immunoblot technique, and highly concentrated virus preparations, we detected a 23-kDa Vif-reactive protein in wild-type HIV type 1 (HIV-1) and a 27-kDa Vif-reactive protein in wild-type SIVSM virions. Neither protein was present in virions derived from vif-deficient HIV-1 and SIVSM proviral constructs. Vif protein content was similar among different strains of HIV-1 and was independent of the cell type (permissive or nonpermissive) used to produce the virus. To determine the subvirion localization of Vif, HIV-1 virions were treated with proteinase K or Triton X-100 to remove virion surface proteins and the viral membrane, respectively, purified through sucrose, and analyzed by immunoblot analysis. Vif protein content was not affected by the removal of external surface proteins or by the removal of the viral membrane and submembrane p17Gag matrix protein. Instead, Vif colocalized with viral core structures which sedimented at a density of 1.25 g/ml on linear sucrose gradients (enveloped HIV-1 particles sediment at a density of 1.17 g/ml). Finally, the amount of Vif protein packaged into virions was estimated to be on the order of 1 molecule of Vif for every 20 to 30 molecules of p24Gag, or between 60 and 100 molecules of Vif per particle. These results indicate that Vif represents an integral component of HIV and SIV particles and raise the possibility that it plays a direct role in early replication events.

Amino Acid Sequence↗
PubMed1994

cDNA cloning and characterization of a vitamin D3 hydroxylase-associated protein.

We previously reported the generation of monoclonal antibodies which immunoprecipitate a fraction of the total chick kidney 1,25-dihydroxyvitamin D3-24R-hydroxylase activity. These antibodies were used to screen a chick kidney lambda gt11 cDNA library resulting in the isolation of a full-length cDNA encoding a protein that is not the 1,25-dihydroxyvitamin D3-24R-hydroxylase but another protein we term the vitamin D3 hydroxylase-associated protein (VDHAP). The deduced amino acid sequence agreed with an NH2-terminal amino acid sequence from the isolated VDHAP. Gene and protein bank search did not identify homology to known sequences or functional domains in the VDHAP cDNA. VDHAP mRNA levels are not altered by conditions which either induce 1,25-dihydroxyvitamin D3-24R-hydroxylase activity (78-fold) or 25-hydroxyvitamin D3-1 alpha-hydroxylase activity (30-fold). Northern analysis of poly(A)+ RNA from chick tissues revealed VDHAP only in kidney. Cellular fractionation experiments demonstrated that VDHAP and the 25-hydroxyvitamin D3-1 alpha-hydroxylase are colocalized in the inner membrane of mitochondria. The VDHAP antibody immunoprecipitates 14% of the total 1,25-dihydroxyvitamin D3-24R-hydroxylase activity (7-fold over background) and immunoprecipitates 21% of the total 25-hydroxyvitamin D3-1 alpha-hydroxylase activity (2-fold over background). VDHAP is a novel chick kidney-specific inner membrane protein of mitochondria, which associates with a fraction of the 1,25-dihydroxyvitamin D3-24R-hydroxylase and 25-hydroxyvitamin D3-1 alpha-hydroxylase.

Amidohydrolases↗
PubMed1994

Mutational analysis of human papillomavirus E4 proteins: identification of structural features important in the formation of cytoplasmic E4/cytokeratin networks in epithelial cells.

We have previously demonstrated that human papillomavirus type 1 (HPV 1) and 16 (HPV 16) E4 proteins form cytoplasmic filamentous networks which specifically colocalize with cytokeratin intermediate-filament (IF) networks when expressed in simian virus 40-transformed keratinocytes. The HPV 16 (but not the HPV 1) E4 protein induced the collapse of the cytokeratin networks. (S. Roberts, I. Ashmole, G. D. Johnson, J. W. Kreider, and P. H. Gallimore, Virology 197:176-187, 1993). The mode of interaction of E4 with the cytokeratin IFs is unknown. To identify E4 sequences important in mediating this interaction, we have constructed a large panel of mutant HPV (primarily HPV 1) E4 proteins and expressed them by using the same simian virus 40-epithelial expression system. Mutation of HPV 1 E4 residues 10 to 14 (LLGLL) abrogated the formation of cytoplasmic filamentous networks. This sequence corresponds to a conserved motif, LLXLL, found at the N terminus of other E4 proteins, and similar results were obtained on deletion of the HPV 16 motif, LLKLL (residues 12 to 16). Our findings indicate that this conserved motif is likely to play a central role in the association between E4 and the cytokeratins. An HPV 1 E4 mutant protein containing a deletion of residues 110 to 115 induced the collapse of the cytokeratin IFs in a manner analogous to the HPV 16 E4 protein. The sequence deleted, DLDDFC, is highly conserved between cutaneous E4 proteins. HPV 1 E4 residues 42 to 80, which are rich in charged amino acids, appeared to be important in the cytoplasmic localization of E4. In addition, we have mapped the N-terminal residues of HPV 1 E4 16-kDa and 10/11-kDa polypeptides expressed by using the baculovirus system and shown that they begin at tyrosine 16 and alanine 59, respectively. Similar-sized E4 proteins are also found in vivo. N-terminal deletion proteins, which closely resemble the 16-kDa and 10/11-kDa species, expressed in keratinocytes were both cytoplasmic and nuclear but did not form cytoplasmic filamentous networks. These findings support the postulate that N-terminal proteolytic processing of the E1-- E4 protein may modulate its function in vivo.

Adult↗
PubMed1994

Utrophin localization in normal and dystrophin-deficient heart.

BACKGROUND: The localization of dystrophin at the sarcolemma of cardiac skeletal fibers and cardiac Purkinje fibers has been described. Dystrophin deficiency produces clinical manifestations of disease in skeletal muscles and hearts of patients with Duchenne and Becker muscular dystrophy. Utrophin (or dystrophin-related protein), a dystrophin homologous protein, was found to be expressed in fetal muscles and reexpressed in dystrophin-deficient skeletal muscle fibers. We therefore examined utrophin expression in normal and in dystrophin-deficient hearts. METHODS AND RESULTS: The expression and subcellular distribution of utrophin was examined in cardiac muscle by immunoblot and immunofluorescence analysis in normal bovine heart compared with dystrophin. Utrophin expression was also examined in normal and dystrophin-deficient hearts of MDX mice. Three monoclonal antibodies reacting with dystrophin and utrophin solely or reacting with both proteins along with two polyclonal antibodies reacting with either utrophin or dystrophin and utrophin were tested. In normal bovine heart, utrophin was not expressed at the periphery of fibers but was strongly expressed in intercalated disks and in the cytoplasm of cardiac Purkinje fibers. In cardiocytes, utrophin was colocalized along transverse T tubules with dystrophin. Dystrophin was present at the periphery of cardiocytes and cardiac Purkinje fibers as well as in transverse T tubules but was absent or faintly expressed in intercalated disks. The results with monoclonal and polyclonal antibodies were identical. Western blot analysis revealed that the detected molecules corresponded only to a 400-kD protein band and not to possible shorter transcripts of utrophin or dystrophin (apo-utrophin or apo-dystrophin). In dystrophin-deficient hearts of MDX mice, utrophin alone was abundant but not organized in the same networklike distribution. CONCLUSIONS: This first localization of utrophin in normal heart (in Purkinje fibers, transverse tubules, and intercalated disks) showed a distinct subcellular localization of this protein with dystrophin, suggesting an important function of this protein in intercellular communication. In dystrophin-deficient hearts of MDX mice, utrophin alone is overexpressed as in skeletal muscle sarcolemma, an area normally occupied by dystrophin but not organized in the same networklike distribution.

Animals↗
PubMed1989

Direct evidence for nuclear and cytoplasmic colocalization of proteasomes (multiprotease complexes) in liver.

Subcellular localization of the large multicatalytic protease complexes called proteasomes, which have been found in soluble fractions of various cells, was examined by biochemical, immunological, and immunohistological methods. Rat liver nuclei, purified by two different procedures, showed high activities for degrading [3H]methylcasein and various fluorogenic oligopeptides with neutral and weakly alkaline pH optima. On gel filtration, all of these peptidase activities were recovered in a single peak with the unusually large molecular weight of about 600,000. Properties of the proteolytic activity in crude extracts of the nucleus and the cytoplasm were very similar. Immunoelectrophoretic blot analysis showed the presence of appreciable concentrations of proteasomes with similar immunoreactivity in isolated nuclear and cytosolic fractions. Moreover, immunohistochemical staining of human liver showed that proteasomes were predominantly localized in the nuclear matrix but also were present diffusely in the cytoplasm of hepatocytes. These findings indicate the nuclear and cytoplasmic colocalization of proteasomes.

Animals↗