Immunoelectron Microscopic Study Research Articles

The Behavior of Calpain-Generated N- and C-Terminal Fragments of Talin in Integrin-Mediated Signaling Pathways

Our previous results showed that the binding of an adhesive ligand to integrin αIIbβ3 on the surface of platelets triggers the activation of calpain and the limited proteolysis of talin by calpain. To explore the physiological significance of the calpain-mediated cleavage of talin, we analyzed the behavior of the calpain-generated fragments of talin (N-terminal 47 kDa and C-terminal 190 kDa) during platelet activation by biochemical and immunoelectron microscopic studies. Intact talin and μ-calpain translocate from the Triton X-100-soluble fraction to the insoluble fraction upon platelet stimulation by thrombin, and the limited proteolysis of talin occurs in the Triton X-100-insoluble fraction, the cytoskeletal fraction. The fully autolyzed 76-kDa μ-calpain (active form) is found predominantly in the Triton X-100-insoluble fraction in stimulated platelets. While the N-terminal 47-kDa fragment remains in the Triton X-100-insoluble fraction, the C-terminal 190-kDa fragment is released into the Triton X-100-soluble fraction in a time-dependent manner. Immunoelectron microscopic observations revealed that the 47-kDa fragment locates on the submembrane zone just beneath the plasma membrane, including the open canalicular systems, while most of the 190-kDa fragment exists diffusely in the cytoplasm in thrombin-stimulated platelets. These findings suggest that calpain may contribute to the reorganization of the cytoskeleton in an integrin-mediated signaling pathway through the redistribution of the functional domain of talin.

Sodium channels in astrocytes of rat optic nerve in situ: immuno-electron microscopic studies.

Sodium channels in astrocytes of rat optic nerve in situ: immuno-electron microscopic studies.

Sodium channels in astrocytes of rat optic nerve in situ: immuno-electron microscopic studies.

This paper presents immunocytochemical studies using Antibody 7493. We interpreted immunostaining with Antibody 7493 as providing information about sodium channel localization, based on an immunological characterization of Antibody 7493 carried out in the laboratory of K.J. Angelides, a co-author of this paper. As reported in the Federal Register on March 12, 1999, based on an investigation by the Baylor College of Medicine into allegations of scientific misconduct by Dr. Angelides, the NIH Office of Research Integrity, on March 10, 1997, found that Dr. Angelides falsified the description of the data in the text and in the legend of Figure 1 of this paper and that his conduct constituted scientific misconduct. The Appeals Board of the Department of Health and Human Services issued a decision on February 5, 1999, in which it affirmed the findings of the Office of Research Integrity. More recent immunocytochemical studies, carried out in our laboratory using additional antibodies generated in other laboratories against conserved polypeptide sequences of sodium channels, have confirmed the presence of sodium channel immunoreactivity in astrocytes in vivo and in vitro, but we have not been able to determine whether perinodal astrocyte processes exhibit sodium channel immunoreactivity. Given the allegations of irregularity in the immunological characterization of Antibody 7493 and the findings that ORI and the DAB have made, we do not feel that we can stand behind the interpretation of results using this antibody. We therefore retract this paper.

Mercaptopyruvate sulfurtransferase as a defense against cyanide toxication: molecular properties and mode of detoxification.

In cyanide poisoning, metalloproteins and carbonyl groups containing proteins are the main target molecules of nucleophilic attack by cyanide. To defend against this attack, cyanide is metabolized to less toxic thiocyanate via transsulfuration. This reaction is catalyzed by rhodanese and mercaptopyruvate sulfurtransferase (MST). Rhodanese is a well characterized mitochondrial enzyme. On the other hand, little was known about MST because it was unstable and difficult to purify. We first purified MST to homogeneity and cloned MST cDNA from rat liver to characterize MST. We also found that MST was an evolutionarily related enzyme of rhodanese. MST and rhodanese are widely distributed in rat tissues, and the kidney and liver prominently contain these enzymes. Immunohistochemical study revealed that MST is mainly distributed in proximal tubular epithelial cells in the kidney, pericentral hepatocytes in the liver, the perinuclear area of myocardial cells in the heart, and glial cells in the brain, and immunoelectron microscopical study concluded that MST was distributed in both cytoplasm and mitochondria, so that MST first detoxifies cyanide in cytoplasm and the cyanide which escapes from catalysis due to MST enters mitochondria. MST then detoxifies cyanide again in cooperation with rhodanese in mitochondria. Tissues other than the liver and kidney are more susceptible to cyanide toxicity because they contain less MST and rhodanese. Even in the same tissue, sensitivity to cyanide toxicity may differ according to the kind of cell. It is determined by a balance between the amount of proteins to be attacked and that of enzymes to defend.

HRC (Histidine-Rich Ca2+ Binding Protein) Resides in the Lumen of Sarcoplasmic Reticulum as a Multimer

HRC (histidine-rich Ca2+ binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca2+ with low affinity and high capacity that is reminiscent of calsequestrin. The physiological role of HRC is largely unknown. In this study, we show that HRC exists as a multimeric complex (probably larger than a pentamer) under physiological conditions. At higher Ca2+ concentrations, the complex appeared to dissociate into dimers or trimers that form a more relaxed structure. This is in striking contrast to the characteristics of calsequestrin. An earlier immuno-electron microscopic study showed that HRC resides in the lumen of the sarcoplasmic reticulum (SR), but this conclusion has been challenged by other data. By tryptic digestion and biotinylation of SR vesicles, we provide compelling evidence showing that HRC is indeed present in the lumen of the SR.

Antigen retrieval of loricrin epitopes at desmosomal areas of cornified cell envelopes: an immunoelectron microscopic analysis.

Cell envelopes (CEs) are insoluble, chemically and mechanically tough structures formed during terminal differentiation of keratinocytes, providing skin with a protective barrier against the environment. They are 15 to 20 nm thick structures beneath the plasma membrane and continuous with desmosomal attachment plaques. Sequential deposition of several proteins including involucrin and loricrin leads to a gradual increase in envelope thickness and rigidity. Cross-linking of desmosomal components to other CE-proteins has been demonstrated and desmosomes in the cornified cells have been regarded as a part of CEs. Our previous immunoelectron microscopy studies showed that desmosomal areas of granular cells were loricrin-positive, but those in cornified cells were negative. We asked whether this is due to epitope masking and applied trypsin digestion of the electron microscopy sections to retrieve the possibly masked epitopes. Since this treatment made desmosomal structures obscure, one side of the sections was stained with anti-desmoglein antibody as an indicator of desmosomes. Trypsin was applied on the other side followed by immunolabeling with anti-loricrin antibody. Trypsin digestion indeed unmasked the loricrin epitopes in the desmoglein-positive desmosomal areas of CEs. It seems therefore that loricrin is first accumulated at the desmosomes before the CE-assembly and cross-linking of loricrin occurs at the desmosomal areas of CEs as well as at the non-desmosomal areas.

Nucleolar localization of murine nuclear DNA helicase II (RNA helicase A).

Nuclear DNA helicase II (NDH II) is a highly conserved member of the DEXH superfamily of eukaryotic helicases, whose physiological role is still unclear. To explore the function of NDH II, we studied the intracellular distribution of NDH II of different mammalian species by immunofluorescence and compared these findings with the known role of the Drosophila homologue MLE that is involved in sex-specific gene dosage compensation. NDH II displayed an apparent nucleolar localization in murine cells, whereas in cells from all other mammalian species examined so far the protein was confined to the nucleoplasm and apparently excluded from the nucleoli. The nucleolar localization of mouse NDH II strongly suggests a role in ribosomal RNA biosynthesis. Immunoelectron microscopic studies revealed that the mouse NDH II was found at the dense fibrillar components of the nucleoli, and a significant percentage of NDH II molecules colocalized with the RNA polymerase I (Pol I) transcription factor UBF (upstream binding factor). Additionally, the nucleolar localization of NDH II coincided with a preferential immunolabeling pattern of nascent transcripts with bromouridine (BrUMP). Furthermore, mouse NDH II redistributed in mitosis in a manner highly correlated with Pol I activity. Conditions leading to the inhibition of Pol I activity in the interphase decreased the amount of NDH II in the nucleoli that diffused into the nucleoplasm and the cytosol. Contrary to the effect of inhibiting rRNA synthesis, treatment of mouse cells with the translation inhibitor cycloheximide did not compromise the nucleolar localization of murine NDH II.

Identification of a novel structural protein of arteriviruses.

Arteriviruses are positive-stranded RNA viruses with an efficiently organized, polycistronic genome. A short region between the replicase gene and open reading frame (ORF) 2 of the equine arteritis virus (EAV) genome was previously assumed to be untranslated. However, here we report that this segment of the EAV genome contains the 5' part of a novel gene (ORF 2a) which is conserved in all arteriviruses. The 3' part of EAV ORF 2a overlaps with the 5' part of the former ORF 2 (now renamed ORF 2b), which encodes the GS glycoprotein. Both ORF 2a and ORF 2b appear to be expressed from mRNA 2, which thereby constitutes the first proven example of a bicistronic mRNA in arteriviruses. The 67-amino-acid protein encoded by EAV ORF 2a, which we have provisionally named the envelope (E) protein, is very hydrophobic and has a basic C terminus. An E protein-specific antiserum was raised and used to demonstrate the expression of the novel gene in EAV-infected cells. The EAV E protein proved to be very stable, did not form disulfide-linked oligomers, and was not N-glycosylated. Immunofluorescence and immunoelectron microscopy studies showed that the E protein associates with intracellular membranes both in EAV-infected cells and upon independent expression. An analysis of purified EAV particles revealed that the E protein is a structural protein. By using reverse genetics, we demonstrated that both the EAV E and GS proteins are essential for the production of infectious progeny virus.

Microglia in Alzheimer's Disease and Transgenic Models: How Close the Fit?

Research on microglia has grown nearly exponentially over the last two decades, 1 fueled in part by the recognition that inflammation plays a role in the pathogenesis of Alzheimer’s disease (AD). 2 Whereas their existence as a distinct central nervous system (CNS) cell type was once debated, it is now widely accepted that microglia are monocyte-derived cells that enter the CNS at early stages of development as highly mobile, ameboid cells that are distributed widely throughout the brain and spinal cord, developing a highly ramified phenotype that eventually forms a uniform reticular array in both gray and white matter. Being related to other cells of the mononuclear phagocytic system they are considered to be poised for activation, each cell policing a discrete and nonoverlapping domain and acting as a sentinel to disruption of the normal homeostasis of the neural tissue. 3 Microglia respond rapidly to subtle alterations not associated with any apparent tissue damage such as spreading electrical depression 4 as well as more severe insults such as anoxic-ischemic injury and neuronal loss associated with a host of degenerative disorders with a sequence of antigenic and morphological changes. The latter form is less ramified, with more abundant cytoplasm in soma and cell processes. It has cell markers, such as class II major histocompatibility antigen, that are barely detectable in the ramified form and is referred to as activated. The full repertoire of ramified microglia is not known, but they appear to be long-lived cells with a limited potential to divide, which contrasts with perivascular macrophages. 5 The latter have a more dynamic life cycle and enter and leave the CNS compartment more readily throughout the life of the individual. The perivascular macrophage, which constitutively express class II major histocompatibility antigen (HLA-DR) participates in the immune response and is fully capable of antigen presentation. 6 The phagocytic potential of the perivascular macrophage is not disputed, but whether ramified microglia are phagocytic cells is debated. Progressive transformation of ramified microglia to macrophages has been shown in a limited number of demyelinating conditions, where it appears that at least some microglia may differentiate into brain macrophages. 3,7

Aquaporin-6: An intracellular vesicle water channel protein in renal epithelia.

All characterized mammalian aquaporins (AQPs) are localized to plasma membranes where they function chiefly to mediate water transport across cells. Here we show that AQP6 is localized exclusively in intracellular membranes in renal epithelia. By using a polyclonal antibody to the C terminus of AQP6, immunoblots revealed a major 30-kDa band in membranes from rat renal cortex and medulla. Endoglycosidase treatment demonstrated presence of an intracellular high mannose glycan on each subunit. Sequential ultracentrifugation of rat kidney homogenates confirmed that AQP6 resides predominantly in vesicular fractions, and immunohistochemical and immunoelectron microscopic studies confirmed that >98% of AQP6 is located in intracellular membrane vesicles. In glomeruli, AQP6 is present in membrane vesicles within podocyte cell bodies and foot processes. In proximal tubules, AQP6 is also abundant in membrane vesicles within the subapical compartment of segment 2 and segment 3 cells, but was not detected in the brush border or basolateral membranes. In collecting duct, AQP6 resides in intracellular membrane vesicles in apical, mid, and basolateral cytoplasm of type A intercalated cells, but was not observed in the plasma membrane. Unlike other members of the AQP family, the unique distribution in intracellular membrane vesicles in multiple types of renal epithelia indicates that AQP6 is not simply involved in transcellular fluid absorption. Moreover, our studies predict that AQP6 participates in distinct physiological functions such as glomerular filtration, tubular endocytosis, and acid-base metabolism.

Localization of ladsin, a homolog of laminin-5, in normal adult human tissues: immunohistochemical and immunoelectron microscopic studies.

Ladsin, a homolog of laminin-5, is a large cell-adhesive protein with potent cell-scattering activity. In the present study, we investigated, by immunohistochemistry, the distribution of ladsin in a wide range of normal adult human epithelial tissues along with that of integrin subunit alpha3 as a marker of integrin alpha3beta1, which is the primary receptor of ladsin. Our results demonstrated that ladsin was localized in the basement membranes of almost all the epithelial tissues with coexisting integrin subunit alpha3 along the cell membranes, suggesting that their interaction is important in the maintenance of normal architecture and function of these membranes. Only cytoplasmic localization of ladsin was observed in the fundic glands, hepatocytes, renal tubuli, and acini of the mammary glands, and in these tissues, integrin subunit alpha3 was also localized in the cytoplasm. Both ladsin and integrin subunit alpha3 were absent in the acini of the pancreas, major salivary glands, and bronchial glands. These tissue- and cell type-specific localizations of ladsin and integrin subunit alpha3 likely reflect the differences in their role and function in each tissue.

Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus.

The authors previously reported the cloning of a lytic-enzyme-encoding gene, lytM, from an autolysis-defective mutant of Staphylococcus aureus. In the present work, the lytM gene was overexpressed in Escherichia coli and the product was purified to homogeneity by affinity chromatography and HPLC. Biochemical analysis of LytM-cleaved peptidoglycan fragments indicated that LytM is a glycylglycine endopeptidase. Immunoelectron microscopic studies with anti-LytM rabbit IgG showed that LytM is expressed during the early exponential phase and is overexpressed in an autolysis-defective mutant compared with the parent strain. Also, a uniform distribution of gold particles on the surface of actively growing bacterial cells indicates that LytM plays a role in cell growth. Northern blot analyses of lytM expression in two global regulatory mutants, agr and sar, showed that expression of lytM is increased about twofold in these mutants as compared with the parents. Protein homology searches revealed that LytM could be a member of the zinc protease family, as it contained a homologous 38-amino-acid motif, Tyr-X-His-X11-Val-X12/20-Gly-X5-6-His. Atomic absorption spectrometric analysis of LytM revealed the presence of 0.9 mol zinc (mol LytM)(-1).

Immunoelectron microscopic study of the luminal release of chromogranin A from rat enterochromaffin cells

Recently we found that raising the intraluminal pressure caused an increase in the luminal release of serotonin from enterochromaffin (EC) cells and serotonin immunoreactivity normally restricted within the secretory granules was diffusely scattered over the extragranular matrix. In the present study we investigated the intracellular localization of chromogranin A, a protein co-stored with serotonin in the EC cells, after stimulating the luminal release of serotonin. In situ vascularly and luminally perfused rat duodenum was exposed to intraluminal pressure and fixed for immunoelectron microscopic study. For immunoelectron microscopy, the pre-embedding DAB reaction for serotonin combined with the postembedding immunogold reaction for chromogranin A was used. Results showed that a number of secretory granules labeled with immunogold chromogranin A immunoreactivity located close to the apical plasma membrane. Some EC cells showed that one part of the apical cytoplasm was protruded into the lumen and a number of secretory granules with immunogold labeling were included in the protruded cytoplasm. These results suggest that EC cells may release chromogranin A into the intestinal lumen together with serotonin, by means of a different manner of secretion from that in serotonin.

PETA-3/CD151, a member of the transmembrane 4 superfamily, is localised to the plasma membrane and endocytic system of endothelial cells, associates with multiple integrins and modulates cell function.

The Transmembrane 4 Superfamily member, PETA-3/CD151, is ubiquitously expressed by endothelial cells in vivo. In cultured human umbilical vein endothelial cells PETA-3 is present on the plasma membrane and predominantly localises to regions of cell-cell contact. Additionally, this protein is abundant within an intracellular compartment which accounts for up to 66% of the total PETA-3 expressed. Intracellular PETA-3 showed colocalisation with transferrin receptor and CD63 suggesting an endosomal/lysosomal localisation which was supported by immuno-electronmicroscopy studies. Co-immunoprecipitation experiments investigating possible interactions of PETA-3 with other molecules demonstrated associations with several integrin chains including beta1, beta3, beta4, (alpha)2, (alpha)3, (alpha)5, (alpha)6 and provide the first report of Transmembrane 4 Superfamily association with the (alpha)6beta4 integrin. Using 2-colour confocal microscopy, we demonstrated similar localisation of PETA-3 and integrin chains within cytoplasmic vesicles and endothelial cell junctions. In order to assess the functional implications of PETA-3/integrin associations, the effect of anti-PETA-3 antibodies on endothelial function was examined. Anti-PETA-3 mAb inhibited endothelial cell migration and modulated in vitro angiogenesis, but had no detectable effect on neutrophil transendothelial migration. The broad range of integrin associations and the presence of PETA-3 with integrins both on the plasma membrane and within intracellular vesicles, suggests a primary role for PETA-3 in regulating integrin trafficking and/or function.

The multihormonal character of pituitary adenomas: Immuno-electron microscopic studies

This study investigates the multihormonal character of pituitary adenomas at the ultrastructural level. The growth hormone (GH)- and prolactin (PRL)-secreting adenomas under study consisted of many moderately or densely granulated cells and a few sparsely or slightly granulated cells. The GH-secreting adenomas showed well-developed cytoplasmic organelles and many large (250 nm or more) or medium-sized (200 nm) secretory granules, as well as a few small (70-150 nm) secretory granules. The PRL-secreting hormones exhibited poorly or slightly developed cyto-plasmic organelles and several small secretory granules. Morphologically and antigenically, these sparsely or slightly granulated cells were similar to those of clinically non-functioning (CN-F) adenomas, which appeared identical to cells expressing little or slight immunoreaction to pituitary hormones at the light microscopic level. As well as those of CN-F adenomas, the small secretory granules of both densely and sparsely granulated cells expressed little or only slight antigenicity of various hormones. Con-comitantly showing slight or moderate antigenicity of hormones biochemically unrelated to GH or PRL, the medium-sized or large secretory granules larger than 140 or 160 nm significantly exhibited intense PRL or GH antigenicity, respectively (Fisher's exact test; P < 0.05 or 0.01). Their GH or PRL antigenicity increased as they grew larger. Showing intermediate cells between sparsely and densely granulated cells, two CN-F adenomas, however, appeared to develop into growth hormone-secreting adenomas. This study led us to conclude that pituitary adenomas may originate from sparsely granulated cells with slight biochemically unrelated hormones, and that their hormonality may be selectively activated singly or multiply in the course of their development.

Expression of Transforming Growth Factor (TGF)-.BETA.1 and its Type I and Type II Receptors in Carcinoma and Leukoplakia of the Tongue: Immunohistochemical and immunoelectron microscopic studies.

TGF-β1, TGF-βRI and TGF-βRII were examined in 80 cases of squamous cell carcinoma (SCC), 26 cases of leukoplakia and 10 cases of early carcinoma in leukoplakia of the tongue by immunohistochemical methods. Nine cases of SCC were analyzed immunoelectron microscopically. Expression of TGF-β1 was enhanced in SCC compared with early carcinoma in leukoplakia and with leukoplakia (P

Immunoelectronmicroscopical Study on the Axonal Coexistence of Serotonin and Substance-P of Fetal Nerve Tissue Transplantation into the Transected Spinal Cord of Rats

Immunoelectronmicroscopical Study on the Axonal Coexistence of Serotonin and Substance-P of Fetal Nerve Tissue Transplantation into the Transected Spinal Cord of Rats

Immunoelectron microscopic study of c-Fos, c-Jun and heat shock protein after transient cerebral ischemia in gerbils.

The neuroprotective role of the expression of heat shock protein (HSP) and immediate early gene remains unclear. Using immunoelectron microscopy, we examined the ultrastructural integrity of the neurons with expression of c-Fos, c-Jun and HSP70 in gerbils after transient cerebral ischemia and reperfusion. Induction of c-Fos and c-Jun was observed in the CA3 region resistant to ischemia, while HSP70 was expressed not only in the CA3 but also in the vulnerable CAI region. With immunoelectron microscopy, the expression of c-Fos/c-Jun and HSP70 was observed in the neurons which retained neuronal integrity except for mitochondrial swelling and polyribosomal disaggregation. In contrast, the CAI neurons without immunoreaction for HSP70 showed cytoplasmic vacuoles and parallel stacking of rough endoplasmic reticulum, the features associated with the process of delayed neuronal death. These findings suggested that c-Fos and c-Jun were induced selectively in reversibly damaged neurons, whereas HSP70 was up-regulated even in neurons with irreversible damage, but was more preferentially and intensely expressed in neurons with reversible damage.

Presence of invertebrate dystrophin-like products in obliquely striated muscle of the leech, Pontobdella muricata (Annelida, Hirudinea).

Dystrophin is a 427-kDa cytoskeletal protein, which occurs in scant amounts in vertebrate muscle and nerve cells. No previous references to dystrophin or associated proteins in invertebrates at the protein level have been found, while two recent studies investigated the presence of genes encoding proteins homologous to dystrophin in sea urchin and other invertebrates such as Drosophila melanogaster. In this study, the possible presence and distribution of dystrophin-like proteins were studied in different invertebrate muscle cell types and species through Western blot analysis and light and electron microscope immunohistochemistry using a panel of antibodies whose specificities have been determined in vertebrates. Crude protein extracts of leech Pontobdella muricata were analysed by Western blotting. The revealed protein band, with 140 kDa molecular weight, was related to dystrophin, utrophin or dystrophin-related protein-2 (DRP2) according to the specificities of the antibodies used to detect them. The immunofluorescence study showed positive immunoreactions in obliquely striated muscle of this hyrudinean. The immunoelectron microscopy study confirmed specific immunogold labelling beneath the sarcolemma of muscle cells. We thus assume that this protein is an invertebrate dystrophin-like product that is referred to as IDLp140. The potential functions of this invertebrate dystrophin-like protein in invertebrate muscles are discussed relative to previous data in vertebrate tissues.

Down-regulation and redistribution of GPV/GPVf2, a subunit of von Willebrand factor receptor (GPIb/IX/V complex), on the surface membrane of thrombin-stimulated human platelets.

We have studied down-regulation and redistribution of glycoprotein V (GPV) and its fragment GPVf2, a subunit of a receptor for von Willebrand factor (VWF), on the surface membrane of thrombin and thrombin receptor activating peptide (TRAP) stimulated platelets by using a newly developed GPVf2-specific monoclonal antibody (1D9). Immunoelectron-microscopical studies revealed that about 50% each of total GPV and GPIX were expressed on the surface membrane of the resting human platelets, and about 83% of GPlbalpha was expressed on the surface. In thrombin-stimulated platelets, the surface GPIbalpha, GPIX and GPV, after hydrolysis by thrombin, was converted to GPVf2, translocated from the surface to the intraplatelet pool and then again redistributed to the surface. In TRAP-stimulated platelets, GPIbalpha, GPIX and GPV, without conversion to GPVf2, were translocated from the surface to the intraplatelet pool and then returned to the surface. Ristocetin-induced agglutinations of both the thrombin- and TRAP-stimulated platelets were lowered during the decreased surface expressions of GPIbalpha, GPIX and GPV/GPVf2 and then normalized when these GPs were again redistributed onto the surface, indicating that the redistributed GPIb/IX/Vf2 complex on the surface can act as a VWF receptor as efficiently as an intact GPIb/IX/V.