Perinuclear Cytoplasm Research Articles

Selection and identification of a specific binding peptide targeting to blood vessel of hmnan colon cancer

Objective To select, identify and analyze a peptide binding specifically to blood vessels of human colon cancer by phage displayed peptide library in vivo. Methods Animal models were established using sub-renal capsular assay （SRCA） in immunosuppressive mice implanted with human colon cancer xenografts. The phage displayed peptide library was injected intravenously into mice. After 4 rounds of selection, 20 clones were picked up randomly and sequenced individually. The homing ability to human colon cancer xenografts and co-cultured human umbilical vein endothelial cells （ Co-HUVECs with human colon cancer Lovo cells） of the positive phage clones were determined by in vivo binding assay and in vitro cell enzyme linked immunosorbent assay （ ELISA ）. The binding ability to Co-HUVECs of peptide-displayed phage clone was identified by immunocytoehemieal stain. Peptide displayed on the phage was syn- thesized and competitive binding assays were performed to observe the competitive inhibition effect of the peptide with their phage counterpart, hnmunofluoreseence microscopy was used to study the binding of synthesized peptides to Co-HUVECs and vascular vessels in colon cancer. Results Two peptide sequences were obtained finally and named CV1 and CV2. In vivo binding assay showed that the homing ability to human colon cancer xenografts of peptides of CV1 or CV2 was higher than that of control organs. In vitro cell ELISA suggested that CV2 phage preferably binded to Co-HUVECs rather than control HUVECs. Then CV2 phage clone was identified further. Immunocytoehemical staining revealed that CV2 phage preferably binded to Co-HUVECs rather than the control. Competition binding assays demonstrated a significant competition between the synthesized peptide CV2 and the phage displaying CV2 while binding to Co-HUVECs or human colon cancer xenografts. Under the immunofluorescence microscopy, fluorescence-labeled CV2 peptide was seen on the membrane and in the perinuclear cytoplasm of Co-HUVECs, and bound to colon cancer xenografts rather than control organs. Conclusion Two phage clones displaying CV1 and CV2 peptide could target to human colon cancer xenografts. The peptide CV2 and its displayed phage were identified binding preferably with Co-HUVECs. And the cyclic nonapeptide （CV2） was binding site of the CV2 phage with Co-HUVECs. Synthesized nonapeptide CV2 had specificity to Co-HUVECs and colon cancer vascular endothelial ceils. The peptide CV2 could be used in target therapy of tumor angiogenesis. Key words: Colon carcinoma; Phage displayed peptide library; Angiogenesis; Peptide

Abstract P4-06-23: KLF8 Is Regulated by Its Novel Interacting Protein PARP-1

Abstract Krüppel-like factor 8 (KLF8) plays an important role in the progression of breast cancer invasion and metastasis. Using mass spectrometry and co-immunoprecipitation, we identified PARP-1, a chromatin-associated enzyme that catalyzes protein poly(ADP-ribosyl)ation (PARylation), as a novel KLF8 interacting protein. Co-IP and western blotting indicated that KLF8 is a PARylation substrate. Mutation of the cystein residues in the first and second zinc-finger motifs of KLF8 abolishes its interaction with PARP-1. Luciferase assays showed KLF8 activation on the cyclin D1 promoter was significantly reduced when PARP-1 was physically deleted or functionally inhibited or when KLF8-PARP-1 interaction was disrupted. Surprisingly, immunofluorescent staining demonstrated that KLF8 was localized in the nuclei of wide type MEFs, but was mislocalized in the perinuclear cytoplasm of the PARP-1-/- cells, which could be rescued by re-expression of PARP-1. The detection of γH2A.X, comet assay and clonogenic assay indicated KLF8 play a DNA repair role in a PARP-1- dependent matter. Cychloheximade-chase assay revealed the stability of KLF8 dramatically reduced by loss of PARP-1 or when KLF8-PARP-1 interaction was disrupted, meanwhile ubiquitination assay showed heavier ubiquitin chains of KLF8. This work has identified PARP-1 as a novel binding partner and post-translational modifier of KLF8 and a critical regulator of KLF8 localization, functions and stability in the cells and suggested a critical role of the interaction between KLF8 and PARP1 during metastatic progression of breast cancer. Support by: NIH, ACS and Komen grants to J.Z. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-23.

39 REGULATION OF THE SEMINIFEROUS TUBULES MOTILITY IN PATIENTS WITH SECONDARY SCO: PRESENCE OF C-KIT + CELLS

Introduction: Sertoli-cell-only (SCO) syndrome, is also called germ cell aplasia. The histological diagnosis of Sertoli-cell-only (SCO) syndrome is confirmed when the examined tissue reveals that all seminiferous tubules contain only Sertoli cells. Theoretically the SCO is caused by a prenatal defect in migration of germ cells into the seminiferous tubules, resulting in sterility. Conversely, secondary SCO is a result of postnatal damage to healthy testicular tissue that may result only in a focal histological SCO pattern. Primary and secondary SCO differ substantially in tubular wall histology, in morphology and function of Sertoli cells, and in the appearance of the interstitial tissue. However, it is possible that they also differ in the motility of the seminiferous tubules. Objective: In this research we aim to identify and quantify the c-kit + cells in human testes with histological confirmation of SCO secondary. Material and Methods: Seven samples with confirmed diagnosis of secondary SCO were taken from the Laboratory of Reproduction, Faculty of Medicine of the University of Chile were obtained, and the appropriate authorizations for bioethics. Paraffin sections were mounted on silanized slides to conduct immunohistochemistry protocols HRP / DAB to identify cells expressing c-kit protein with the primary antibody anti c-kit (CD177, clone YR145, BSB 5322). Optical microscopy to quantify the number of positive cells per unit area present in the interstitial compartment of the testis (using Micrometrics SE Premium software, 2009). Then the means and standard deviations between patients were compared by t test with p 0.05. Results: The biopsies with a diagnosis of secondary SCO is possible to recognize the presence of c-kit + cells through immunohistochemistry. Phenotypically these cells in the interstitium were observed with a core comatina rounded and dense, with abundant perinuclear cytoplasm in an approximate ratio of 3/1 to the core. Equally distributed in the interstitium itself also in the vicinity of the seminiferous tubules. Discussion: C-kit cells are involved in contractile activity in a wide variety of organs, and usually associated with contractile cells function properly. These cells operate independently of the general nervous system. Normally in a healthy testicle cells are scarce and rarely seen their cytoplasm, which is occupying the proprietary extensions that contact with the effector cells. Therefore, the diagnosis of secondary SCO observed changes in the quantity (more, p 0.05) and distribution of cytoplasm (mainly perinuclear) taking lead to modifications in the motility of the seminiferous tubules and runoff of its content, which together lead to changes in male fertility (Grant Fondecyt No. 1101046, 2010). 40 THE EFFECTS OF PROGESTERONE AND EXOGENOUS GONADOTROPIN ON THE INCIDENCE OF APOPTOSIS IN MOUSE BLASTOCYSTS M. Salehnia1, F. Panahi1, M.F. Moghadam2. 1Department of Anatomy, Tarbiat Modares University, Tehran, Iran, 2Department of Biotechnology, Tarbiat Modares University, Tehran, Iran

Inter- and intrachromosomal asynchrony of cell division cycle events in root meristem cells of Allium cepa: possible connection with gradient of cyclin B-like proteins

Alternate treatments of Allium cepa root meristems with hydroxyurea (HU) and caffeine give rise to extremely large and highly elongated cells with atypical images of mitotic divisions, including internuclear asynchrony and an unknown type of interchromosomal asynchrony observed during metaphase-to-anaphase transition. Another type of asynchrony that cannot depend solely on the increased length of cells was observed following long-term incubation of roots with HU. This kind of treatment revealed both cell nuclei entering premature mitosis and, for the first time, an uncommon form of mitotic abnormality manifested in a gradual condensation of chromatin (spanning from interphase to prometaphase). Immunocytochemical study of polykaryotic cells using anti-β tubulin antibodies revealed severe perturbations in the microtubular organization of preprophase bands. Quantitative immunofluorescence measurements of the control cells indicate that the level of cyclin B-like proteins reaches the maximum at the G2 to metaphase transition and then becomes reduced during later stages of mitosis. After long-term incubation with low doses of HU, the amount of cyclin B-like proteins considerably increases, and a significant number of elongated cells show gradients of these proteins spread along successive regions of the perinuclear cytoplasm. It is suggested that there may be a direct link between the effects of HU-mediated deceleration of S- and G2-phases and an enhanced concentration of cyclin B-like proteins. In consequence, the activation of cyclin B-CDK complexes gives rise to an abnormal pattern of premature mitotic chromosome condensation with biphasic nuclear structures having one part of chromatin decondensed, and the other part condensed.

Abstract 4985: Identification of PARP-1 as a novel KLF8 interacting and regulating protein

Abstract KLF8 transcription factor regulates important genes including cyclin D1 and E-cadherin and plays a critical role in cell cycle progression, oncogenic transformation and EMT. Transcription factors need to interact with both DNA and other proteins in order to regulate transcription. However, little is known about KLF8-protein interaction. Using mass spectrometry and co-immunoprecipitation (co-IP), we identified PARP-1, a chromatin-associated enzyme that catalyzes protein poly(ADP-ribosyl)ation (PARylation), as one of novel proteins that interact with KLF8. Co-IP and western blotting indicated that KLF8 is a PARylation substrate. Mutation of the cystein residues in the first and second zinc-finger motifs of KLF8 abolishes its interation with PARP1. Luciferase assays showed KLF8 activation on the cyclin D1 promoter was significantly reduced when PARP1 was physically deleted or functionally inhibited or when KLF8-PARP-1 interation was disrupted. Surprisingly, immunofluorescent staining demonstrated that KLF8 was localized in the nuclei of the wild-type mouse embryonic fibroblasts (MEF), but was mislocalized in the perinuclear cytoplasm of the PARP-1−/− MEFs, which could be rescued by re-expression of PARP1. Interestingly, while the PARP1-interation deficient KLF8 mutant was also mislocalized in the cytoplasm of the wild-type MEFs, inhibition of PARP1 activity in the MEFs did not affect the nuclear localization of wild-type KLF8. Cychloheximade (CHX)-chase assay revealed the stability of KLF8 dramatically reduced by loss of PARP-1 or when KLF8-PARP-1 interaction was disrupted. Ubiquitination assay showed heavier ubiquitin chains in PARP-1−/− MEFs or when KLF8-PARP-1 interaction was disrupted. Furthermore, DNA damage assay under Doxorubin treatment indicated KLF8 involved in PARP-1-dependent DNA damage repair pathway. Those results suggest that PARP-1 work as a co-activator in KLF8's transcriptional activity on Cyclin D1 promoter, keep KLF8's nuclear localization, inhibit KLF8's ubiquitination and contribute to KLF8's stability. This work has identified PARP1 as a novel binding partner and post-translational modifier of KLF8 and a critical regulator of KLF8 localization, function and stability in the cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4985.

The identification of intestinal M cells in the sacculus rotundus and appendix of the Angora rabbit

The present study was aimed at the immunohistochemical demonstration of M cells, found in the follicle-associated epithelium (FAE) of the sacculus rotundus (SR) and appendix of the Angora rabbit, using anti-vimentin primary antibodies, and at the determination of certain fine structural characteristics. Ten adult Angora rabbits constituted the material of the study. Immunohistochemical staining revealed that many cells composing the FAE, which covered the dome regions of the SR and appendix, reacted positively with vimentin. FAE contained two different types of vimentin-positive cells. The first type surrounded intraepithelial lymphocytes (IEL) with a basolateral invagination in the apex and periphery of the dome epithelium, whilst the second type consisted of columnar cells found in the FAE near crypts. The immunoreactivity of the cells found in the FAE covering the apex and periphery of the domes was observed particularly in the perinuclear cytoplasm and the cytoplasm surrounding the IEL. Electron microscopic examination demonstrated that the M cells found in the FAE covering the apex and periphery of the dome regions of the SR and appendix did not exhibit any microvilli on their apical surface. The FAE near crypts contained columnar cells, which resembled enterocytes. The apical membrane of these cells exhibited shorter and irregular microvilli, in contrast to neighbouring enterocytes. It was determined that M cells, found in the FAE of the SR and appendix in the Angora rabbit, displayed similarities in terms of localization and fine structure. This situation may be indicative of the two lymphoid structures with different localization having similar functional properties.

Transmission electron microscopic observation on ultrastructural alterations in Schistosoma japonicum caused by mefloquine

The purpose of the study was to explore the ultrastructural alterations of adult Schistosoma japonicum induced by mefloquine. Eight out of ten mice infected with 60-80 S. japonuicum cercariae for 35 days were treated orally with mefloquine at a single dose of 400 mg/kg. Four groups of two mice were killed at 8 h, 24 h, 3 days, and 7 days post-treatment, and schistosomes were collected by perfusion technique, fixed, and examined under a transmission electron microscope. Schistosomes obtained from the remaining two mice served as control. Eight hours after mefloquine 400 mg/kg was administered to the infected mice, various alterations in the tegument and subtegument tissues of both male and female worms were seen, which included focal lysis of tegmental matrix resulted in vacuole formation, decrease in rod-like and discoid-like secretary bodies, light swelling or focal lysis of musculature, extensive lysis of internal structure of sensory organelles, and appearance of vacuole or myelin-like structure in perinuclear cytoplasm of syncytium and epithelial cells. In vitelline cells of female worms, the most significant alteration was extensive lysis or fusion of vitelline balls in vitelline droplets and decrease in granular endoplasmic reticulum Twenty-four hours post-treatment, damage to the tegument and subtegument tissues had increased in severity. In male worms, the most prominent alterations were emergence of large vacuoles in the tegument, detachment of cytoplasmic process from the tegumental surface, focal collapse of internal structure of sensory organelle, and loss of definition of syncytium and gut epithelial cell. In female worms, focal lysis in tegumental matrix, musculature, and parenchymal tissues resulted in emergence of vacuole or myelin-like structure, reduction of nucleoli, fusion of partial nuclear membrane together with cytoplasm in epithelial cell, and lysis of interstitial tissues among the vitelline cells which were universal. Three and 7 days post-treatment, besides the aforementioned alterations, the significant damage to the male worms were disrupted outer plasma membrane detached from the cytoplasmic process, swelling of individual cytoplasmic process, extensive swelling and focal lysis in the musculature, parenchymal tissues and perinuclear cytoplasm of syncytium, accompanied by emergence of swollen mitochondria, vacuoles, and myelin-like structure, and severe damage to gut epithelial cell. In female worms, apart from disruption of outer plasmic membrane in cytoplasmic process, severe swelling of tegumental matrix accompanied by emergence of vacuoles, swollen mitochondria and myelin-like structure, focal lysis of heterochromatin and nucleoli, disappearance of microvilli in gut epithelial cells, and emergence of myelin-like structures in vitelline cells were observed. The results indicate that administration of mefloquine to mice infected with adult S. japonicum exhibits an extensive damage to the ultrastructure in tegument and subtegument tissues including syncytium, gut epithelial cells, parenchymal tissues, and vitelline cells of schistosomes.

Force generated by actomyosin contraction builds bridges between adhesive contacts

Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)-coated stripes separated by non-adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non-adhesive regions in a treadmilling network. Inhibition of myosin-II (MII) or Rho-kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin-A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross-links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another.

Histochemical method for demonstrating quail mast cell types simultaneously

The development and application of selective staining methods for routine detection of mast cells are of considerable interest, because these cells play an important role in health and disease. The composition of cytoplasmic mast cell granules depends on the species and type of mast cell. The study reported here was conducted to investigate the combined use of aldehyde fuchsin (AF) and the Alcian blue-critical electrolyte concentration (AB-CEC) (pH 5.8, 0.3 M MgCl2) techniques for differentiating avian mast cell subtypes. Tissue samples from skin, intestines, and lungs of six healthy adult quail and two control rats were fixed in Carnoy's solution and 10% formolin for routine histological processing. To determine the staining properties of sulfated glycosaminoglycans (GAGs), a three-step staining technique was applied using berberine sulfate, AF, and AB-CEC. In quail, AF positivity following application of the AB-CEC technique was found only in the lungs, mostly in cells that gave a berberine sulfate-positive reaction, and this positivity was determined to be localized particularly in the nucleus and perinuclear cytoplasm. In other regions, the pale AF staining of cells that did not emit fluorescence when stained with berberine sulfate was determined to be replaced by a blue color after application of AB-CEC. The AF/AB-CEC (pH 5.8, 0.3 M MgCl2) technique demonstrated that rat and quail mast cells varied in both GAG types and their distribution within the cell. Especially in avian species, this technique can be applied to distinguish mast cells according to their GAG content. It can be used as an alternative to the AB/safranin O staining procedure for differentiating mast cells that contain and lack heparin.

PKA‐Mediated Golgi Remodeling During cAMP Signal Transmission

Cyclic AMP (cAMP)-dependent protein kinase A (PKA) is part of the set of signaling proteins that are stably associated to the cytosolic surface of Golgi membranes in mammalian cells. In principle, Golgi-associated PKA could participate in either signal transduction events and/or the coordination of Golgi transport activities. Here, we show data indicating that although Golgi-associated PKA is activated fast and efficiently during cell stimulation by an extracellular ligand it does not contribute significantly to cAMP signal transmission to the nucleus. Instead, most of the PKA catalytic subunits Calphaderived from the Golgi complex remain localized in the perinuclear cytoplasm where they induce changes in Golgi structural organization. Thus, in stimulated cells the Golgi complex appears collapsed, showing increased colocalization of previously segregated markers and exhibiting merging of different proximal cisternae within a single stack. In contrast, the trans-Golgi network remains as a separate compartment. Consequently, the rate of protein transport is increased whereas glycan processing is not severely affected. This remodeling process requires the presence of PKA activity associated to the Golgi membranes. Together these data indicate that Golgi-associated PKA activity is involved in the adaptation of Golgi dynamic organization to extracellular signaling events.

Spectroscopic and biological studies of a novel synthetic chlorin derivative with prospects for use in PDT

Photosensitizers with desirable combinations of chemical, photophysical and biological properties are essential for improving the efficacy of photodynamic therapy (PDT) against various cancers. Chlorins seem to be promising candidates for photodynamic therapy (PDT) owing to their photophysical properties. This paper reports spectroscopic and biological properties of a novel synthetic chlorin derivative. Cytotoxicity, phototoxicity as well as subcellular localization of the novel derivative was studied using Lewis lung carcinoma cultured cells (LLC). In the examined concentration range no significant cytotoxic effects were found but high phototoxicity was observed. Confocal laser scanning microscopy demonstrated that the compound, upon entering cells, was localized in the perinuclear cytoplasm of LLC cells. Using fluorescent microscopy we investigated the impact of PDT based on the novel compound upon cytoskeleton and DNA structure of LLC cells. Our results indicate that liposomes are effective in transferring the chlorin photosensitizer into the studied cells, leading to their high photosensitization, whereas the non-carrier delivery mode (i.e., DMSO) is rather useless for such purposes.

Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments

Extracellular deposits of amyloid fibrils in the form of parenchymal plaques and cerebrovascular lesions, as well as intracellular accumulation of paired-helical filaments in the form of neurofibrillary tangles (NFT) in selected neuronal populations are the main neuropathologic hallmarks of Alzheimer's disease. Amyloid fibrils composed of polymeric structures of the amyloid-beta (Abeta) concentrate at the center of senile plaques and accumulate in the walls of cerebral blood vessels, exhibiting extensive Congo red/thioflavin S staining. Intraneuronal NFT are composed of building blocks of aberrantly hyperphosphorylated species of the microtubule-associated protein tau, which accumulate in the perinuclear cytoplasm of vulnerable neurons in the form of paired helical filaments (PHF). This unit presents a variety of protocols for the isolation, biochemical analysis, and characterization of amyloid fibrils and neurofibrillary tangles.

Distribution of rat hepatic steroid 5 alpha-reductase 1 as shown by immunohistochemistry.

Peptide fragments of rat liver 5 alpha-reductase were synthesized according to the published primary sequence data. The peptide were used (i) in free form and (ii) linked to keyhole limpet hemocyanin (KLH), respectively, for immunization of rabbits. Resulting antisera showed positive reaction with the respective peptide-antigen in an ELISA. In Western blot experiments the antisera specifically recognized a 26,000 mol wt protein in extracts from female and male rat liver. All antisera, as well as isolated immunoglobulins, showed inhibition of 5 alpha-reductase activity in microsomes prepared from rat liver. Positive immunohistochemical reactions were observed in the perinuclear cytoplasm of hepatocytes. No reaction was seen in Kupffer- and connective tissue cells. Acinar heterogeneity of the immunoreaction was seen in the male rat liver with a gradient from the portal canal to the central vein. After androgen-deprivation a considerable increase in immunoreactivity was seen in male rat liver cells, indicating androgen-dependent suppression of the enzyme in male liver.

Ultrastructure of the early gravid uterus of Corallobothrium fimbriatum (Cestoda: Proteocephalidea)

The ultrastructure of the uterine epithelium and underlying extracellular matrices has been studied in several cyclophyllideans, but relatively little has been published on uterine fine structure in other cestode orders. To develop comparative data, we initiated a study focusing on a common and widespread genus of the order Proteocephalidea. Adult specimens of Corallobothrium fimbriatum were removed from the small intestine of brown bullhead catfish, Ameiurus nebulosus, from the St. Lawrence River in North America. Early gravid proglottids were examined by light microscopy as whole mounts or diced in buffered glutaraldehyde, processed by standard techniques and examined by as stained sections by light microscopy and by transmission electron microscopy. Voucher specimens were deposited in the Museum of Comparative Zoology at Harvard University. The uterus consisted of an epithelial wall supported basally by an extracellular matrix and parenchyma cells, which together formed epitheliomesenchymal villi at many points. The uterine epithelium consisted of a single thin syncytial layer, folded distally into long epithelial villi that protruded into the lumen at some points. The epithelium's numerous nuclei were very close to the lumen, but bulged along with the perinuclear cytoplasm proximally into the surrounding medullary parenchyma. Each nucleus contained small amounts of evenly dispersed heterochromatin and euchromatin, with a central nucleolus. Numerous mitochondria occurred in the cytoplasm, which was densely filled with free ribosomes but contained scant endomembrane components and showed little evidence of secretory activity. The uterine cells possessed few of the apical microvilli that are typical of some cyclophyllidean species, but the apical plasma membrane and cortical cytoplasm were extensively developed into long microlamellae similar to other species. The basic syncytial nature of the epithelium is similar to that of other cestodes studied previously, but the specific combination of features is unlike any previously described. Based on this one proteocephalidean species, we discerned no pattern that would distinguish between Proteocephalidea and Cyclophyllidea based on uterine structure.

W2068 Interstitial Cell of Cajalopathy (ICCpathy) of Human Stomach in Type 2 DM

Background & Aim: Patients with long-standing diabetes mellitus (DM) commonly suffer from gastropathy. However, the pathophysiology is not fully understood. Interstitial Cells of Cajal (ICC) have been identified as pacemakers and mediators of neurotransmission in GI tract. A deficiency and ultrastructural changes of ICC have been reported in diabetic animal models. The aim of this study was to evaluate ultrastructural changes of ICC in patients with type 2 DM. Methods: Gastric specimens were obtained from 13 diabetic patients (mean age: 65.8 yrs, range: 52-75 yrs) and 6 control subjects (mean age: 60.3yrs, range: 34-72 yrs) with gastric cancer who underwent gastrectomy at Samsung Medical Center between June and November, 2008. All the specimens were taken from the disease-free area. Samples were processed for electron microscopic examination and analyzed. Medical records of diabetic patients which included duration of DM, chronic complication and symptoms were reviewed. . Results: In normal subjects, ICC features were similar to animal models. The nucleus was ovoid and had peripherally condensed heterochromatin. The cytoplasm with numerous lateral branches had abundant mitochondria, intermediate filaments, Golgi apparatus and endoplasmic reticulum. In diabetic patients, several ultrastructural changes of ICC were observed. First, the changes of nucleus was observed. The heterochromatin of the nucleus was decreased and became euchromatic. The wrinking of nuclear envelope which was spreading in whole from part was observed. Some nuclei were shrunken and showed karyorrhectic changes and pyknosis. Second, the changes of cytoplasm was observed. The perinuclear cytoplasm and lateral branches were reduced. Endoplasmic reticulum was markedly dilated and cytoplasmic vacuoles were formed. These changes were more prominent in patients who had long duration of diabetes or autoneuropathy. In some specimens, normal appearing ICCs and degenerated ICCs were coexisted. Conclusion: In diabetes, preapoptotic changes of ICCwere observed.We are willing to reffer to these changes as an Interstitial Cell of Cajalopathy(ICCpathy). These results suggest that degeneration of ICCs may be associated with the pathogenesis of diabetic gastroparesis.

Connexin32 Mutations Cause Loss of Function in Schwann Cells and Oligodendrocytes Leading to PNS and CNS Myelination Defects

The gap junction (GJ) protein connexin32 (Cx32) is expressed by myelinating Schwann cells and oligodendrocytes and is mutated in X-linked Charcot-Marie-Tooth disease. In addition to a demyelinating peripheral neuropathy, some Cx32 mutants are associated with transient or chronic CNS phenotypes. To investigate the molecular basis of these phenotypes, we generated transgenic mice expressing the T55I or the R75W mutation and an IRES-EGFP, driven by the mouse Cnp promoter. The transgene was expressed in oligodendrocytes throughout the CNS and in Schwann cells. Both the T55I and the R75W mutants were localized in the perinuclear cytoplasm, did not form GJ plaques, and did not alter the expression or localization of two other oligodendrocytic GJ proteins, Cx47 and Cx29, or the expression of Cx29 in Schwann cells. On wild type background, the expression of endogenous mCx32 was unaffected by the T55I mutant, but was partly impaired by R75W. Transgenic mice with the R75W mutation and all mutant animals with Gjb1-null background developed a progressive demyelinating peripheral neuropathy along with CNS myelination defects. These findings suggest that Cx32 mutations result in loss of function in myelinated cells without trans-dominant effects on other GJ proteins. Loss of Cx32 function alone in the CNS causes myelination defects.

Neuronal and Glial Alterations Due to Focal Cortical Hypoxia Induced by Direct Cobalt Chloride (CoCl2) Brain Injection

Ischemic brain injury is a dynamic process that involves oxidative stress, inflammation, and cell death, as well as activation of endogenous adaptive and regenerative mechanisms depending on activation of transcription factors such as hypoxia inducible factor 1-alpha (HIF-1alpha). Because CoCl2 activates HIF-1alpha, we described a new focal-hypoxia model by direct intracerebral CoCl2 injection. Adult male Wistar rats were intracerebrally injected with CoCl2 (2 microl-50 mM), in frontoparietal cortex of right hemisphere, and saline (2 microl) in the contralateral hemisphere. In slides of fixed brains at 1, 6, 9, 24 h or 5 day after treatment, TTC, histochemistry (toluidine blue, Hoescht-33342, TUNEL), immunostaining (HIF-1alpha, GFAP), Lycopersicon esculentum lectin staining, and electron microscopy (EM) were performed. Immediately after 1 h post CoCl2 injection, HIF-1alpha stabilization and neuronal nuclear shrinkage and cromathin condensation were observed by immunostaining and EM, respectively. Neuronal apoptotic nuclear morphology and GFAP immunoreactivity and lectin maximal reactivity were detected during 6-9 h. Ultrastructural alterations of morphology included edematous perinuclear cytoplasm, organelles and endoplasmic reticulum (RE) enlargement, mitochondrial swelling with increased matrix density, and deposits of electron-dense material. Neurons showed particular nuclear indentations. Astrocytes and oligodendrocytes presented alterations in both nuclei and RE with dilated lumen and altered mitochondrias, and all these ultrastructural changes became detectable at day 5. CoCl2 cortical injection mimics focal brain ischemia, inducing neuronal death and glial activation. This model brings the opportunity to develop focal ischemia in selected brain areas to study their functional consequences and potential pharmacological therapies for in vivo models of stroke.

The Catalytic Subunit of Drosophila Glutamate-Cysteine Ligase Is a Nucleocytoplasmic Shuttling Protein

GSH concentration is considerably lower in the nucleus than in the cytoplasm; however, it is significantly elevated during active cell proliferation. The main purpose of this study was to understand the mechanism underlying these variations in nuclear/cytoplasmic distribution of GSH. The rate-limiting step in the de novo GSH biosynthesis pathway is catalyzed by glutamate cysteine ligase (GCL), a heterodimer, composed of a catalytic subunit (GCLc) and a modulatory subunit (GCLm). In Drosophila, GCLc, but not GCLm, contains a nuclear localization signal (NLS). Drosophila S2 cells, constitutively expressing regular GCLc protein or expressing GCLc protein with a mutated NLS motif, were generated by transfection. In quiescent S2 cells, GCLc is aggregated in the perinuclear cytosol and the nucleus, whereas GLCm resides solely in the cytosol. In actively proliferating S2 cells, expressing the normal NLS motif, GCLc migrates from the perinuclear cytoplasm into the nucleus, and the nuclear GSH level becomes elevated; in contrast, in proliferating cells, expressing the mutated NLS motif, neither does the GCLc migrate into the nucleus nor does the nuclear GSH amount rise. In S2 cells expressing wild type GCLc, perturbation of cellular redox state by exposure to cadmium resulted in the migration of GCLc into the nucleus but not in cells expressing GCLc with the mutated NLS motif. Overall, results indicated that GSH biosynthesis in the nucleus is associated with migration of only the GCLc subunit from the cytoplasm into the nucleus, and this migration requires the presence of an intact NLS.

The Familial Mediterranean Fever Protein Interacts and Colocalizes with a Putative Golgi Transporter

Abstract. The biological function of pyrin, the protein mutated in Familial Mediterranean Fever (FMF), has not been elucidated. Based on sequence homology, a transcription factor activity was proposed for this neutrophil-specific protein. In a yeast two-hybrid assay, neither transcription activation activity nor any self interaction was detected for pyrin. Screening of an expression cDNA library of peripheral blood leukocytes using as bait the carboxyl portion of pyrin (amino acids 557–781), which contains most of the FMF mutations, led to the identification of P/M-IP1 (pyrin/marenostrin interacting protein 1). A splice variant of P/M-IP1, GTC-90, had previously been described as a component of the 13S hetero-oligomeric protein complex that stimulates in vitro Golgi transport. We have now shown that P/M-IP1 colocalizes with pyrin in the perinuclear cytoplasm of Cos-7 cells and that the interaction between these two proteins is impaired by FMF causing mutations in pyrin. These data suggest that, at some stage of its functional pathway, pyrin resides in the cytoplasm and might be involved in, or impacted by, cellular protein sorting by the Golgi apparatus. The data also imply that P/M-IP1 may be involved in the abnormal inflammatory response that occurs in patients with FMF.

The phosphorylation state of serine 256 is dominant over that of serine 261 in the regulation of AQP2 trafficking in renal epithelial cells

Phosphorylation of serine 256 (S256) plays a critical role in vasopressin (VP)-mediated membrane accumulation of aquaporin-2 (AQP2). Recently, phosphorylation of serine 261 was also reported, raising the possibility that it has a role in AQP2 trafficking. We addressed this issue using transfected LLC-PK(1) cells that express point mutations of AQP2 S261 and S256, mimicking the phosphorylated (S to D) or dephosphorylated (S to A) states of these residues. Both AQP2 (S261A) and AQP2 (S261D) were located in the perinuclear cytoplasm without stimulation but, like wild-type AQP2, they both accumulated on the plasma membrane after 20-min exposure to VP or forskolin. Following membrane accumulation, S261A, S261D, and wild-type AQP2 reinternalization was complete over a similar time frame, between 30 and 60 min after VP washout. Using various combinations of point mutations, we showed that the phosphorylation state of S256 is dominant with respect to AQP2 behavior; AQP2 membrane accumulation and internalization were not detectably affected by the phosphorylation state of S261. Finally, blocking AQP2 endocytosis by methyl-beta-cyclodextrin caused membrane accumulation of AQP2 in cells expressing either a single S-A mutation or double mutations of S256 and S261, although as previously reported, the S256D mutation was always present at the cell surface. This suggests that constitutive recycling of AQP2 was not modified by the phosphorylation state of S261. Together, our data indicate that the phosphorylation state of AQP2 at S261 does not detectably affect regulated or constitutive trafficking of AQP2. The potential role of S261 phosphorylation/dephosphorylation in vasopressin action remains to be determined.