Spatial and functional relationship between poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase in the brain

Poly(ADP-ribose) polymerases (PARPs) are members of a family of enzymes that utilize nicotinamide adenine dinucleotide (NAD(+)) as substrate to form large ADP-ribose polymers (PAR) in the nucleus. PAR has a very short half-life due to its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). PARP-1 mediates acute neuronal cell death induced by a variety of insults including cerebral ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism, and CNS trauma. While PARP-1 is localized to the nucleus, PARG resides in both the nucleus and cytoplasm. Surprisingly, there appears to be only one gene encoding PARG activity, which has been characterized in vitro to generate different splice variants, in contrast to the growing family of PARPs. Little is known regarding the spatial and functional relationships of PARG and PARP-1. Here we evaluate PARG expression in the brain and its cellular and subcellular distribution in relation to PARP-1. Anti-PARG (alpha-PARG) antibodies raised in rabbits using a purified 30 kDa C-terminal fragment of murine PARG recognize a single band at 111 kDa in the brain. Western blot analysis also shows that PARG and PARP-1 are evenly distributed throughout the brain. Immunohistochemical studies using alpha-PARG antibodies reveal punctate cytosolic staining, whereas anti-PARP-1 (alpha-PARP-1) antibodies demonstrate nuclear staining. PARG is enriched in the mitochondrial fraction together with manganese superoxide dismutase (MnSOD) and cytochrome C (Cyt C) following whole brain subcellular fractionation and Western blot analysis. Confocal microscopy confirms the co-localization of PARG and Cyt C. Finally, PARG translocation to the nucleus is triggered by NMDA-induced PARP-1 activation. Therefore, the subcellular segregation of PARG in the mitochondria and PARP-1 in the nucleus suggests that PARG translocation is necessary for their functional interaction. This translocation is PARP-1 dependent, further demonstrating a functional interaction of PARP-1 and PARG in the brain.     

Basic studies on a labeled anti-mucin antibody detectable by infrared-fluorescence endoscopy

Background. We developed a fluorescent dye, indocyanine green (ICG)-sulfo-OSu, which was excited by infrared rays and conjugated to various antibodies. We attempted to clarify the staining patterns of anti-sulfomucin and anti-MUC1 antibodies in gastrointestinal cancer. We then evaluated the potential of the dye as a fluorescent label for antibodies specific to cancer, to be used as a diagnostic method for microcancer, with infrared fluorescence endoscopy. Methods. Paraffin sections of samples collected from 10 patients with esophageal cancer, 30 patients with gastric cancer, and 20 patients with colorectal cancer were immunohistologically stained using an anti-sulfomucin antibody and an anti-MUC1 antibody, and the staining patterns were examined. If a section had a high staining intensity, it was reacted with the ICG-suflo-OSu-labeled antibody and evaluated with infrared fluorescence imaging. Results. The staining patterns with the antibodies varied depending on the organs and the histological types and depth of the cancers, but the staining was generally good and the staining on the mucosal surface of cancer tissues was retained. Good images of cancer cells could be obtained by infrared fluorescence observation using the ICG-sulfo-OSu-labeled anti-MUC1 antibody. Conclusions. The anti-MUC1 antibody stained gastrointestinal cancer cells well, and nearly specific infrared fluorescence in cancer tissues was observed using the labeled anti-MUC1 antibody. The ICG-sulfo-OSu-labeled anti-MUC1 antibody has possible usefulness for the screening of cancer via infrared fluorescence endoscopy. Received: December 8, 2000 / Accepted: September 28, 2001     

Identification of CD157-Positive Vascular Endothelial Stem Cells in Mouse Retinal and Choroidal Vessels: Fluorescence-Activated Cell Sorting Analysis

PurposeCD157 (also known as Bst1) positive vascular endothelial stem cells (VESCs), which contribute to vascular regeneration, have been recently identified in mouse organs, including the retinas, brain, liver, lungs, heart, and skin. However, VESCs have not been identified in the choroid. The purpose of this study was to identify VESCs in choroidal vessels and to establish the protocol to isolate retinal and choroidal VESCs.MethodsWe established an efficient protocol to create single-cell suspensions from freshly isolated mouse retina and choroid by enzymatic digestion using dispase, collagenase, and type II collagenase. CD157-positive VESCs, defined as CD31⁺CD45⁻CD157⁺ cells, were sorted using fluorescence-activated cell sorting (FACS).ResultsIn mouse retina, among CD31⁺CD45⁻ endothelial cells (ECs), 1.6 ± 0.2% were CD157-positive VESCs, based on FACS analysis. In mouse choroid, among CD31⁺CD45⁻ ECs, 4.5 ± 0.4% were VESCs. The CD157-positive VESCs generated a higher number of EC networks compared with CD157-negative non-VESCs under vascular endothelial growth factor (VEGF) in vitro cultures. The EC network area, defined as the ratio of the CD31-positive area to the total area in each field, was 4.21 ± 0.39% (retinal VESCs) and 0.27 ± 0.12% (retinal non-VESCs), respectively (P < 0.01). The EC network area was 8.59 ± 0.78% (choroidal VESCs) and 0.14 ± 0.04% (choroidal non-VESCs), respectively (P < 0.01). The VESCs were located in large blood vessels but not in the capillaries.ConclusionsWe confirmed distinct populations of CD157-positive VESCs in both mouse retina and choroid. VESCs are located in large vessels and have the proliferative potential. The current results may open new avenues for the research and treatment of ocular vascular diseases.     

Usefulness of endoscopic biopsy using immunostaining of p53 and Ki-67 in tumors of the ampulla of Vater

We investigated the diagnostic and prognostic value of p53 expression and proliferative activity, as indicated by the Ki-67, in endoscopic biopsy specimens. Specimens were immunologically stained with p53 and MIB-1 (Ki-67), and the MIB-1/Ki-67 labeling index (LI) was calculated. Classification of adenomas was based on findings of H&E-stained preparations into those with low- or high-grade atypia. Well-differentiated tubular and papillary adenocarcinomas were classified as carcinomas with low- or high-grade atypia. There were significant differences among the control and adenoma patients in MIB-1/Ki-67 LI (P < 0.05). No significant difference was identified between adenomas with high grade atypia and carcinomas with low grade atypia. The p53 expression was negative in all adenomas, but it was positive in 68.2% of carcinomas. The current study demonstrated that p53 protein expression in endoscopic biopsy specimens was of preoperative diagnostic value for carcinoma of the ampulla of Vater. The p53 protein positive tumors had a relatively higher malignant potential than p53 protein negative ones. The MIB-1/Ki-67 LI was useful in differentiating non-tumorous lesions from adenomas and adenomas with low- or high-grade atypia. The MIB-1/Ki-67 LI had a prognostic value because clinicopathological factors of carcinoma of ampulla of Vater correlated with MIB-1/Ki-67 LI.     

A comparison of the diagnostic efficacy in type 1 autoimmune pancreatitis based on biopsy specimens from various organs

BackgroundComprehensive immunostaining evaluation of the biopsy specimens from various organs with type 1 autoimmune pancreatitis (AIP) has not been elucidated. Our aim was to clarify which of these biopsy specimens and counting method could be a useful tool for supporting the diagnosis of AIP.MethodsWe retrospectively evaluated biopsy specimens from pancreas (n = 19), stomach (n = 28), duodenum (n = 27), duodenal papilla (n = 25), colon (n = 19), liver (n = 11), bile duct (n = 24), and minor salivary gland (n = 13) in 36 patients with AIP. Positive IgG4 immunostaining (>10 plasma cells/high-power field [HPF]) and positive IgG4/IgG ratio (>40%) of biopsy specimens from 8 sites of 6 organs in one HPF and an average from 3 HPFs were compared between AIP and controls.ResultsThe sensitivity of IgG4 immunostaining for AIP in one HPF were 16% in pancreas, 14% in stomach, 15% in duodenum, 52% in duodenal papilla, 11% in colon, 27% in liver, 21% in bile duct and 8% in minor salivary gland, respectively. The positive IgG4 immunostaining of the duodenal papilla in one HPF showed the highest sensitivity (52%) and accuracy (73%) among the 8 sites. It also showed the highest sensitivity among 4 different counting methods (IgG4 immunostaining in one HPF and 3 HPFs, both IgG4 immunostaining and IgG/IgG4 ratio in one HPF and 3 HPFs), but there were no significant differences with respect to specificity and accuracy.ConclusionsIgG4 immunostaining of swollen duodenal papilla with more than 10 IgG4-positive plasma cells in at least one HPF is useful for supporting the diagnosis of AIP.     

Four PGE2 EP receptors are up-regulated in injured nerve following partial sciatic nerve ligation

We previously reported that cyclooxygenase 2 (COX2) is up-regulated in macrophages in injured nerve of rats with partial sciatic nerve ligation (PSNL) and that local injection of the COX inhibitor ketorolac reversed tactile allodynia (Eur. J. Neurosci. 15: 1037-1047, 2002). These findings suggest that prostaglandins (PGs) are overproduced in injured nerve and are involved in the pathogenesis of neuropathic pain. In this study, we examined whether overproduced PGs alter the expression of PGE2 receptors, EP1-EP4, in injured nerve of PSNL rats. We found that cell profiles immunoreactive (IR) for four EP receptors, EP1, EP2, EP3, and EP4, are dramatically increased in injured nerve 2 and 4 weeks after PSNL. EP4-IR cells were the most abundant among these receptor-expressing cells. Immunoreactivities of all four EP receptors were localized to the cell nucleus. These EP-IR cells were never found in uninjured nerve. More than 80% EP1- and about 30% EP4-IR cells were identified as infiltrating macrophages since they coexpressed ED1. Only 3% EP2- and 6% EP3-IR cells coexpressed ED1. These findings suggest that majority of EP2-, EP3-, and EP4-IR cells are other types of inflammatory cells than macrophages. About 48% of macrophages expressed EP1 and 45% expressed EP4. Only 3 and 6% of macrophages, respectively, expressed EP2 and EP3. Perineural injection of ketorolac reversed tactile allodynia and suppressed the up-regulation of EP1 and EP4, but not the recruitment of ED1-IR marcrophages, in injured nerve. Our data suggest that following PSNL, PGE2 is one of the possible PGs overproduced in injured nerve and PG overproduction is involved in the up-regulation of EP receptors in injured nerve.     

Connexin immunoreactivity in glial cells of the rat retina

The rat retina contains two types of macroglial cells, Müller cells, radial glial cells that are the principal macroglial cells of vertebrate retinas, and astrocytes associated with the surface vasculature. In addition to the often-described gap-junctional coupling between astrocytes, coupling also occurs between astrocytes and Müller cells. Immunohistochemistry and confocal microscopy were used to identify connexins in the retinas of pigmented rats. Several antibodies directed against connexin43 stained astrocytes, identified using antibodies directed against glial fibrillary acidic protein (GFAP). In addition, two connexin43 antibodies stained Müller cells, identified with antibodies directed against S100 or glutamine synthetase. Connexin30-immunoreactive puncta were confined to the vitreal surface of the retina and colocalized with GFAP-immunoreactive astrocyte processes. Connexin45 immunoreactivity was associated with both astrocytes and Müller cells. We conclude that retinal glial cells express multiple connexins, and the patterns of immunostaining that we observe in this study are consistent with the expression of connexins30, -43, and possibly -45 by astrocytes and the expression of connexins43 and -45 by Müller cells. As gap-junction channels may be formed by both homotypic and heterotypic hemichannels, and the hemichannels may themselves be homomeric or heteromeric, there exists a multitude of possible gap-junction channels that could underlie the homotypic coupling between retinal astrocytes and the heterotypic coupling between astrocytes and Müller cells.     

Demonstration of competition between endogenous dopamine and [11C]raclopride binding in in vitro brain slices using a dynamic autoradiography technique

Although the basolateral nucleus (BL) of the amygdala is known to contain an abundance of gamma-aminobutyric acid (GABA)ergic neurons that regulate the amygdaloid projection neurons and influence storage and consolidation of memory, it remains to be determined what type of neuronal input controls GABAergic neurons in the BL. We examined the synapses that GABAergic neurons form with GABAergic and noradrenergic neurons and terminals with unknown transmitters by double-labeling immunoelectron microscopy using anti-GABA and dopamine-beta-hydroxylase (DBH) antisera. The medium and small dendrites of the GABAergic neurons were shown to receive symmetric, inhibitory-type synapses from GABAergic axon terminals and asymmetric, excitatory-type synapses from noradrenergic axon terminals. Each segment of the GABAergic neurons from perikarya to dendritic spines received both symmetric and asymmetric synapses from unlabeled axon terminals of various forms and sizes. The incidence rates of the two types of synapses were almost identical. Our results suggest that GABAergic neurons in the BL of the rat amygdala might be affected by the excitatory influence of the noradrenergic system and the inhibitory influence of the GABAergic system. Furthermore, these neurons are also strongly influenced by both excitatory and inhibitory-type synapses from neuronal systems other than the GABAergic and noradrenergic systems.