Significant Immunoreactivity Research Articles

<i>Wrinkled</i> 1 (WRI1) Homologs, AP2-Type Transcription Factors Involving Master Regulation of Seed Storage Oil Synthesis in Castor Bean (<i>Ricinus communis</i>L.)

Among APETALA2 (AP2)-type plant specific transcription factor family, WRINKLED1 (WRI1), has appeared to be a master gene transcriptionally regulating a set of carbon metabolism- and fatty acid synthesis (FAS)-related genes responsible for seed specific triacylglycerols (TAGs) storage in oil plants. B3 type transcription factors, such as ABI3 and FUS3, are known to be involved in seed development, such as seed storage protein synthesis and maturation. Based on the recent whole genome sequence data of castor bean (Ricinus communis L.), putative WRI1 homologs (RcWRI1, RcWRI2) specifically expressed in castor bean seed have been identified by comparing organ specific expression profiles among seed development-related transcription factors, seed storage specific genes (Ricin, RcOleosin) and a set of FAS genes including genes for sucrose synthase (RcSUS2), biotin carboxyl carrier protein (a subunit of acetyl-CoA carboxylase, RcBCCP2) and ketoacyl-acyl carrier protein synthase (RcKAS1). Immunoreactive signals with WRI1, FUS3 and ABI5-related polypeptides were also detected in seed specifically, consistent with the expression profiles of seed development-related genes. The WRI1 binding consensus sites, [CnTnG](n)(7)[CG], designated as the AW-box, were found at the promoter region of RcBCCP2 and RcKAS1. Thus, RcWRI1 possibly play a pivotal role in seed specific TAGs storage during seed development by directly activating FAS -related genes.

The Expression Patterns of Reg IV Gene in Normal Rat Reproduction System

Reg IV, the latest member of the regenerating gene family, has been documented in different tissues of human and rat, such as the colon, small intestine, stomach, and pancreas. Expression of Reg IV gene in distinct cell types has been correlated with its various functions in regeneration, cell growth and survival, proliferation and differentiation, cell adhesion, and resistance to apoptosis. However, there was no evidence to show whether the Reg IV protein is present in the reproductive system of normal rat. The aim of this study was to reveal the expression patterns of Reg IV in rat ovary and uterus. The expression of Reg IV was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot at mRNA and protein levels, respectively. The localization of Reg IV protein within rat ovary and uterus was investigated by immunohistochemistry (IHC). Our results showed that the expression of Reg IV in ovary was significantly higher than that in the uterus. The strong immunoreactive signals of Reg IV was observed in granulosa cells and oocytes of ovarian follicles, corpus luteum, and interstitial cells in rat ovary; only weak signals were detected in luminal and gland epithelium of rat endometrium. These findings first demonstrate the expression of Reg IV in ovary and uterus of the healthy rat at both mRNA and protein levels. It provides an evidence of Reg IV expression in rat reproductive system, which may help elucidate a potential role in cell growth and proliferation of reproductive system.

Corrigendum] Effects of rotigaptide (ZP123) on connexin43 remodeling in canine ventricular fibrillation.

Mol Med Rep 12: [Related article:] 5746–5752, 2015; DOI: 10.3892/mmr.2015.4193 Following the publication of this article on-line ahead of print, an interested reader drew to our attention some anomalies associated with the presentation of Fig. 1. In the lower panel, the fourth image from the left resembled a mirror image representation of the image in the first panel; the fifth image from the left bore a marked resemblance to a section of the third image, albeit displaced at an angle and with a different magnification; and an internal office investigation drew our attention to the fact that the sixth image in the upper panel resembled a section of the image in the third panel, although rotated through 180°.

Expression of FSH and its co-localization with FSH receptor and GnRH receptor in rat cerebellar cortex

The expression of follicle-stimulating hormone (FSH) and its receptor in extrapituitary and non-HPG axis tissues has been demonstrated and their non-reproductive functions in these tissues have been found. However, there have been no reports concerning the expression and function of FSH and its receptor in the cerebellum. In our study, immunofluorescence staining and in situ hybridization were used to detect the expression of FSH, double-labeled immunofluorescence staining was used to detect co-localization of FSH and its receptor and co-localization of FSH and gonadotropin-releasing hormone (GnRH) receptor in the rat cerebellar cortex. Results showed that some cells of the Purkinje cell layer, granular layer, and molecular layer of the cerebellar cortex showed both FSH immunoreactivity and FSH mRNA positive signals; not only for FSH and FSH receptor, but also for FSH and GnRH receptor co-localized in some cells throughout the Purkinje cell layer, granular layer, and molecular layer of the cerebellar cortex. These suggested that rat cerebellum could express FSH; cerebellum is a target tissue of FSH; FSH may exert certain functions through FSH receptor in a paracrine or autocrine manner; GnRH may regulate FSH positive cells through GnRH receptor in the cerebellum. Our study provides morphological evidence for further functional research on FSH and related hormones in the cerebellum.

Dynamics of GFRα1‐positive spermatogonia at the early stages of colonization in the recipient testes of W/Wν male mice

The spermatogonial transplantation experiment can be used as an unequivocal detection assay of spermatogenic stem cells (SSCs) in both a qualitative and quantitative manner, based on their regenerative capacity. In this study, the proliferative patterns and kinetics of donor-derived GFRα1-positive spermatogonia containing potential SSCs were examined during early colonization following spermatogonial transplantation. Donor-derived GFRα1-positive cells frequently formed several aggregates of A(al(aligned)) /morula-like structures in a single spermatogenic cell patch before and on day 14 post-transplant, indicating a possible involvement in the formation of a stable spermatogenic colony at 21 days post-transplant. The appearance of these A(al) /morula-like aggregates is positively correlated with regional, high-level expression of immunoreactive GDNF signals, a ligand for GFRα1, associated with colony expansion. These data raise the hypothesis that regional GDNF signals regulate the balance between donor-derived A(al) -like cell aggregates and their differentiation in each small patch, which subsequently leads to further selection of survival colonies at later stages.

Molecular Insights into Arrhythmogenic Right Ventricular Cardiomyopathy Caused by Plakophilin-2 Missense Mutations

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disorder mainly caused by dominant mutations in several components of the cardiac desmosome including plakophilin-2 (PKP2), the most prevalent disease gene. Little is known about the underlying genetic and molecular mechanisms of missense mutations located in the armadillo (ARM) domains of PKP2, as well as their consequences on human cardiac pathology. We focused on in vivo and in vitro studies of the PKP2 founder mutation c.2386T>C (p.C796R), and demonstrated in cardiac tissue from 2 related mutation carriers a patchy expression pattern ranging from unchanged to totally absent immunoreactive signals of PKP2 and other desmosomal proteins. In vitro expression analysis of mutant PKP2 in cardiac derived HL-1 cells revealed unstable proteins that fail to interact with desmoplakin and are targeted by degradation involving calpain proteases. Bacterial expression, crystallization, and structural modeling of mutated proteins impacting different ARM domains and helices of PKP2 confirmed their instability and degradation, resulting in the same remaining protein fragment that was crystallized and used to model the entire ARM domain of PKP2. The p.C796R and other ARVC-related PKP2 mutations indicate loss of function effects by intrinsic instability and calpain proteases mediated degradation in in vitro model systems, suggesting haploinsufficiency as the most likely cause for the genesis of dominant ARVC due to mutations in PKP2.

Growth differentiation factor 9 (Gdf9) was localized in the female as well as male germ cells in a protogynous hermaphroditic teleost fish, ricefield eel Monopterus albus

Growth differentiation factor 9 (GDF9) is a member of the transforming growth factor beta (TGFb) superfamily. As an oocyte-derived growth factor, GDF9 plays key roles in regulating follicle development. In the present study, we identified a gdf9 homologue from the ovary of ricefield eel, and analyzed its expression both at the mRNA and protein levels. Ricefield eel Gdf9 showed high homologies with those of other teleosts, especially perciformes fish. RT-PCR analysis revealed that ricefield eel gdf9 was expressed exclusively in the ovary and testis. The mRNA levels of gdf9 in the ovary were increased significantly at the pre-vitellogenic (PV) stage and then decreased significantly along with vitellogenesis. During the natural sex change, expression of ricefield eel gdf9 was peaked at the intersexual stages. The immunoreactivity for Gdf9 was localized exclusively in the cytoplasm of the oocytes in the ovary, particularly the oocytes at early stages, but not in the oogonia. Interestingly, strong immunoreactive signals were also detected in the degenerating oocytes in the intersexual gonad. Furthermore, the Gdf9 immunoreactivity was demonstrated for the first time to be localized in the cytoplasm of spermatogonia and spermatocytes of ricefield eel, a teleost fish. Taken together, the results of present study suggested that Gdf9 may play important roles in the folliculogenesis as well as spermatogenesis in ricefield eels.

Novel Survivin Inhibitor YM155 elicits Cytotoxicity in Glioblastoma Cell Lines with Normal or Deficiency DNA-Dependent Protein Kinase Activity

Pediatric glioblastoma is a malignant disease with an extremely poor clinical outcome. Patients usually suffer from resistance to radiation therapy, so targeted drug treatment may be a new possibility for glioblastoma therapy. Survivin is also overexpressed in glioblastoma. YM155, a novel small-molecule survivin inhibitor, has not been examined for its use in glioblastoma therapy. The human glioblastoma cell line M059K, which expresses normal DNA-dependent protein kinase (DNA-PK) activity and is radiation-resistant, and M059J, which is deficient in DNA-PK activity and radiation-sensitive, were used in the study. Cell viability, DNA fragmentation, and the expression of survivin and securin following YM155 treatment were examined using MTT (methylthiazolyldiphenyl-tetrazolium) assay, ELISA assay, and Western blot analysis, respectively. YM155 caused a concentration-dependent cytotoxic effect, inhibiting the cell viability of both M059K and M059J cells by 70% after 48 hours of treatment with 50 nM YM155. The half-maximal inhibitory concentration (IC50) was around 30-35 nM for both cell lines. Apoptosis was determined to have occurred in both cell lines because immunoreactive signals from the DNA fragments in the cytoplasm were increased 24 hours after treatment with 30 nM YM155. The expression of survivin and securin in the M059K cells was greater than that measured in the M059J cells. Treatment with 30 nM YM155, for both 24 and 48 hours, significantly suppressed the expression of survivin and securin in both cell lines. The novel survivin inhibitor YM155 elicits potent cytotoxicity in glioblastoma cells in vitro via DNA-PK-independent mechanisms. YM155 could be used as a new therapeutic agent for the treatment of human glioblastomas.

Gabapentin reduces CX3CL1 signaling and blocks spinal microglial activation in monoarthritic rats

BackgroundSpinal glia, particularly microglia and astrocytes, are of the utmost importance in the development and maintenance of chronic pain. A recent study from our laboratory revealed that gabapentin, a recommended first-line treatment for multiple neuropathic conditions, could also efficiently antagonize thermal hyperalgesia evoked by complete Freund's adjuvant (CFA)-induced monoarthritis (MA). In the present study, we investigated whether the spinal glia are involved in the anti-hyperalgesic effect of gabapentin and how this event occurs.ResultsUnilateral intra-articular injection of CFA produced a robust activation of microglia and astrocytes. These cells exhibited large cell bodies, thick processes and increases in the ionized calcium binding adapter molecule 1 (Iba-1, a microglial marker) or the glia fibrillary acidic protein (GFAP, an astrocytic marker). These cells also displayed immunoreactive signals, and an upregulation of the voltage-gated calcium channels (VGCCs) α2/δ-1 subunit, CX3CL1 and CX3CR1 expression levels in the spinal cord. These changes were associated with the development of thermal hyperalgesia. Immunofluorescence staining showed that VGCC α2/δ-1 subunit, a proposed gabapentin target of action, was widely distributed in primary afferent fibers terminals and dorsal horn neurons. CX3CL1, a potential trigger to activate microglia, colocalized with VGCC α2/δ-1 subunits in the spinal dorsal horn. However, its receptor CX3CR1 was mainly expressed in the spinal microglia. Multiple intraperitoneal (i.p.) gabapentin injections (100 mg/kg, once daily for 4 days with the first injection 60 min before intra-articular CFA) suppressed the activation of spinal microglia, downregulated spinal VGCC α2/δ-1 subunits decreased CX3CL1 levels and blocked the development of thermal hyperalgesia in MA rats.ConclusionsHere we provide the first evidence that gabapentin diminishes CX3CL1 signaling and spinal microglia activation induced by joint inflammation. We also show that the VGCC α2/δ-1 subunits might be involved in these events.

Ontogeny of Immunoreactive Lh and Fsh Cells in Relation to Early Ovarian Differentiation and Development in Protogynous Hermaphroditic Ricefield Eel Monopterus albus1

Luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) control many aspects of gonadal development and function in teleosts. In the present paper, the specific antisera against ricefield eel Lhb (Lh beta subunit), Fshb (Fsh beta subunit), and Cga (the common pituitary glycoprotein hormone alpha subunit) were generated, and the cellular localization, initial appearance, and subsequent development of gonadotrophs in relation to early ovarian differentiation and development in the ricefield eel, a protogynous sex-changing teleost, were examined with immunochemistry. Lhb- and Fshb-immunoreactive signals were identified in distinct pituitary cells that occupied primarily the peripheral regions of the adenohypophysis. During ontogeny, Lhb-immunoreactive signals were first detected in the pituitary around 40 days after hatching (dah) when the oogonia transitioned into early primary growth oocytes, and the intensity of immunoreactivity increased concomitantly with the growth of primary oocytes from 60 to 140 dah. During overwintering from 170 to 230 dah, Lhb-immunoreactive signals were significantly decreased when a large proportion of perinucleolus oocytes contained intense Balbiani bodies. In contrast, Fshb-immunoreactive signals were not detectable in the pituitary until around 230 dah (in the spring after hatching) and slightly increased from 285 dah when the late perinucleolus oocytes began to enter the secondary growth phase. Both Lhb- and Fshb-immunoreactive cells were increased when the early cortical alveoli oocytes emerged at 300 dah. The mRNA expression of lhb and fshb coincided with their immunoreactive signals. Taken together, these results suggest that only Lh is involved in primary oocyte growth in ricefield eels, but both Fsh and Lh are important for the secondary ooctye growth.

Thr649Ala-AS160 knock-in mutation does not impair contraction/AICAR-induced glucose transport in mouse muscle

AS160 and its closely related protein TBC1D1 have emerged as key mediators for both insulin- and contraction-stimulated muscle glucose uptake through regulating GLUT4 trafficking. Insulin increases AS160 phosphorylation at multiple Akt/PKB consensus sites, including Thr(649), and promotes its binding to 14-3-3 proteins through phospho-Thr(649). We recently provided genetic evidence that AS160-Thr(649) phosphorylation/14-3-3 binding plays a key role in mediating insulin-stimulated glucose uptake in muscle. Contraction has also been proposed to increase phosphorylation of AS160 and TBC1D1 via AMPK, which could be detected by a generic phospho-Akt substrate (PAS) antibody. Here, analysis of AS160 immunoprecipitates from muscle extracts with site-specific phospho-antibodies revealed that contraction and AICAR caused no increase but rather a slight decrease in phosphorylation of the major PAS recognition site AS160-Thr(649). In line with this, contraction failed to enhance 14-3-3 binding to AS160. Consistent with previous reports, we also observed that in situ contraction stimulated the signal intensity of PAS antibody immunoreactive protein of ∼150-160 kDa in muscle extracts. Using a TBC1D1 deletion mutant mouse, we showed that TBC1D1 protein accounted for the majority of the PAS antibody immunoreactive signals of ∼150-160 kDa in extracts of contracted muscles. Consistent with the proposed role of AS160-Thr(649) phosphorylation/14-3-3 binding in mediating glucose uptake, AS160-Thr(649)Ala knock-in mice displayed normal glucose uptake upon contraction and AICAR in isolated muscles. We conclude that the previously reported PAS antibody immunoreactive band ∼150-160 kDa, which were increased upon contraction, does not represent AS160 but TBC1D1, and that AS160-Thr(649)Ala substitution impairs insulin- but neither contraction- nor AICAR-stimulated glucose uptake in mouse skeletal muscle.

The Role of CD147 in the Invasiveness of Follicular Thyroid Carcinoma Cells

In patients without metastases, capsular and vascular invasion must be noted to make the diagnosis of follicular thyroid carcinoma (FTC). Some patients are initially diagnosed as follicular adenoma (FA) but develop metastases, indicating the original lesion was FTC. A diagnostic marker for FTCs that appear to be FAs by conventional histopathology is urgently needed. CD147 is a transmembrane glycoprotein that induces matrix metalloproteinases (MMPs) and participates in carcinoma invasion. The objective of this study was to determine whether CD147 is upregulated in FTC and if measures directed against it could reduce the invasive activity of FTC cells. The expression levels of CD147, MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9 in surgical specimens of normal thyroid (n=8), FA (n=20), and FTC (n=9) was determined using immunoblot and immunohistochemical techniques. CD147 protein expression levels of epithelial growth factor stimulated FTC-133 cell lines was measured by immunoblotting with and without cell signaling inhibitors such as wortmannin, PD98059, SP600125, and SB203580. This was also done after exposure to short-hairpin interference RNA directed against CD147. Immunoblot analysis of thyroid tissues revealed significant increases in signals for CD147, MMP-3, MMP-7, and MMP-9 in FTC compared with FA or normal tissue, or both. Immunohistochemical analysis revealed colocalization of determinants of CD147 with those of all of MMPs studied, mainly in follicular cells in normal and neoplastic cells in FA and FTC; their immunoreactivities were to some extent more intense in the FTC than FA or normals. In FTC-133 cells, immunoreactive signals for CD147 were upregulated by epidermal growth factor (EGF), and the EGF-driven increases in CD147 were prevented by inhibitors against phosphoinositol-3 kinase (PI3K), extracellular signal-regulated protein kinase (ERK), or c-Jun N-terminal kinase (JNK) but not p38. RNA interference targeted against CD147 reduced the invasive activity of FTC-133 cells and was associated with downregulation of MMP-2, MMP-3, MMP-7, and MMP-9. These results provide in vivo evidence for CD147 upregulation in FTC and in vitro evidence for EGF-stimulated CD147 induction via the PI3K, ERK, and JNK pathways. They suggest the involvement of CD147 in the invasiveness of FTC cells via regulation of MMPs.

Immunochemical Analysis of Mutant CFTR in Lung Explants

Background/Aims: Knowledge about the abundance and distribution of CFTR protein glycoforms in native lung tissue is scarce. For upcoming studies with correctors and potentiators for CFTR it is important to get more information about mutant CFTR protein biochemistry. Target for novel treatment is the most afflicted organ in cystic fibrosis (CF), the lung. Methods: Lung tissue sampled from patients with CF and non-CF donors prior to lung transplantation was examined for CFTR-immunoreactive signals by immunoblot. Quantitation of the immunoreactive signals was carried out by densitometry. Results: The complexglycosylated and mannose-rich CFTR isoforms were present in all non-CF specimens, whereas no or only the immature CFTR isoform was visible in CF samples. Whereas some complexglycosylated CFTR was often present in rectal biopsies of F508del homozygous subjects, no mature CFTR was detectable in CF lungs at the stage of terminal respiratory insufficiency. Conclusion: Immunoblot analysis of CFTR in lung tissue is feasible, but in context of the upcoming studies of CFTR correctors and potentiators rectal biopsies seem to be a more appropriate choice because of their safe and repeatable excision.

Cyclical and Patch-Like GDNF Distribution along the Basal Surface of Sertoli Cells in Mouse and Hamster Testes

Background and AimsIn mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs) through GDNF family receptor α1 (GFRα1). It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFRα1-positive spermatogonia in vivo.Methodology and Principal FindingsHere we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules.Conclusion/SignificanceOur data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules.

Aquaporin-4 expression is not elevated in mild hydrocephalus

Aquaporin-4 (aqp-4) is a member of water channel family proteins primarily expressed in the central nervous system. Physiologically it is the main channel providing water transport into the nervous system water compartments and across the blood-brain barrier. Several studies demonstrated its compensatory role in severe hydrocephalus. However, its role is not clear during the initial stages of hydrocephalus. This study was designed to investigate aqp-4 expression in less severe forms of hydrocephalus and to determine its role in disease progression. Twenty-five male Wistar-Hannover rats, were distributed into experimental (n = 20) and control (n = 5) groups. Hydrocephalus was induced in the experimental group by injection of 5 μl 25% kaolin suspension into the cisterna magna. Control animals received an injection of 5 μl normal saline. Eight weeks later, the animals were killed by the perfusion-fixation method. Immunohistochemical and Western blot analysis were performed. Ventricular dilatations were noted in all experimental animals. Both groups demonstrated positive immunoreactive signals to aqp-4. Immunohistochemically there were no changes in aqp-4 pattern and expression intensity between experimental and control animals. Similarly, Western blot analysis revealed mean aqp-4 values in experimental and control groups as 0.3436 and 0.3917, respectively, and the difference did not reach statistical significance (p > 0.05). Our results indicate that aqp-4 is not up-regulated during the initial stages of hydrocephalus. This implies that aqp-4 may not play a significant role in hydrocephalus compensation until severe ventricular dilatation occurs.

Molecular changes in the heart of a severe case of arrhythmogenic right ventricular cardiomyopathy caused by a desmoglein-2 null allele

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder caused by mutations in desmosomal genes. It is often associated with life-threatening arrhythmias. Some affected individuals develop progressive heart failure and may require cardiac transplantation. The explanted heart of a young adult with end-stage heart failure due to a null allele in desmoglein-2 was studied at macroscopic, microscopic, and molecular level. Myocardial samples were probed for junctional localization of desmosomal components and the gap junction protein connexin43 by immunohistochemical staining. In addition, the protein content of desmosomal and adherens junction markers as well as connexin43 was assessed by Western blotting. Histological analysis confirmed ARVC. Despite the loss of specific immunoreactive signal for desmosomal components at the cardiac intercalated disks (shown for plakoglobin, desmoplakin, and plakophilin-2), these proteins could be detected by Western blotting. Only for desmoglein-2, desmocollin-2, and plakoglobin were reduced protein levels observed. Adherens junction proteins were not affected. Lower phosphorylation levels were observed for connexin43; however, localization of the gap junction protein displayed regional differences. At the molecular level, disease progression was more severe in the right ventricle compared to the left ventricle. Our data suggest that, in the ARVC heart, plakoglobin is mainly redistributed from the junctions to other cellular pools and that protein degradation only plays a secondary role. Homogenous changes in the phosphorylation status of connexin43 were observed in multiple ARVC samples, suggesting that this might be a general feature of the disease.

Altered Desmosomal Proteins in Granulomatous Myocarditis and Potential Pathogenic Links to Arrhythmogenic Right Ventricular Cardiomyopathy

Immunoreactive signal for the desmosomal protein plakoglobin (γ-catenin) is reduced at cardiac intercalated disks in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), a highly arrhythmogenic condition caused by mutations in genes encoding desmosomal proteins. Previously, we observed a false-positive case in which plakoglobin signal was reduced in a patient initially believed to have ARVC but who actually had cardiac sarcoidosis. Sarcoidosis can masquerade clinically as ARVC but has not been previously associated with altered desmosomal proteins. We observed marked reduction in immunoreactive signal for plakoglobin at cardiac myocyte junctions in patients with sarcoidosis and giant cell myocarditis, both highly arrhythmogenic forms of myocarditis associated with granulomatous inflammation. In contrast, plakoglobin signal was not depressed in lymphocytic (nongranulomatous) myocarditis. To determine whether cytokines might promote dislocation of plakoglobin from desmosomes, we incubated cultures of neonatal rat ventricular myocytes with selected inflammatory mediators. Brief exposure to low concentrations of interleukin (IL)-17, tumor necrosis factor-α (TNF-α), and IL-6 (cytokines implicated in granulomatous myocarditis) caused translocation of plakoglobin from cell-cell junctions to intracellular sites, whereas other potent cytokines implicated in nongranulomatous myocarditis had no effect, even at much higher concentrations. We also observed myocardial expression of IL-17 and TNF-α and elevated levels of serum inflammatory mediators, including IL-6R, IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1β, in patients with ARVC (all P<0.0001 compared with controls). The results suggest novel disease mechanisms involving desmosomal proteins in granulomatous myocarditis and implicate cytokines, perhaps derived in part from the myocardium, in disruption of desmosomal proteins and arrhythmogenesis in ARVC.

Expression of water channel aquaporin-4 during experimental syringomyelia

Aquaporins (aqp) are protein channels providing water transport across cell membranes. The main member of this family expressed in the CNS is aqp-4. The pattern and amount of expression of this channel suggest a dominant role in bulk water movement into the nervous tissue. It has also been shown to play a role in several water balance disorders in the CNS. In this study, the authors investigated the possible role of aqp-4 in syringomyelia. Twenty-five male Wistar-Hannover rats were divided into experimental (20 rats) and control (5 rats) groups. Syringomyelia was induced in the experimental group by kaolin injection into the cisterna magna. Eight weeks later, the animals were killed, and their spinal cords were removed. Central canal dilations were noted in all experimental animals. Immunohistochemistry and Western blot analysis were performed to evaluate aqp-4 expression. Both groups demonstrated positive immunoreactive signals to aqp-4. Western blot analysis revealed a slight decrease in the mean aqp-4 value in the experimental group; however, the difference did not reach statistical significance (p > 0.05). Immunohistochemical analysis showed a similar pattern and intensity of aqp-4 staining in both groups. The results of this study indicate that aqp-4 most likely does not play a major role in chronic syringomyelia. Its slight downregulation during the initial stage of syrinx formation is possibly a compensatory mechanism. This effect is not present during the late stage of syringomyelia, and aqp-4 is most likely not involved in the pathophysiology of syrinx cavity formation.

Estrogen regulates DNA synthesis in human gingival epithelial cells displaying strong estrogen receptor β immunoreactivity

Estrogen acts via estrogen receptor (ER) α and β. The expression pattern of ERs and their importance in gingival tissues are not fully understood. In this study, we investigate gingival ER expression and effects of estrogen on gingival epithelial cell proliferation. Gingival biopsies were obtained from both healthy and diseased sites in three male and three female subjects. Expression of ERα and β was determined by immunohistochemistry. Effects of 17β-estradiol (E(2) ) on cell proliferation, monitored by measuring DNA synthesis, were studied in cultured human gingival epithelial HGEPp.05 cells. Estrogen receptor β, but not ERα, immunoreactivity was demonstrated in nuclei of epithelial cells in all layers of the gingival epithelium, but also in cells of the lamina propria. No differences were observed between male and female subjects. The same pattern, i.e. high ERβ expression but no ERα expression, was observed in both healthy and diseased sites within each individual. No differences in the intensity of the ERβ immunoreactive signal and the number of ERβ-positive nuclei were observed between healthy and diseased gingiva. Treatment with a physiological concentration of E(2) (10 nm) had no effect on DNA synthesis in ERβ- and ERα-expressing HGEPp.05 cells. In contrast, E(2) at high concentrations (500 nm and 10 μm) reduced DNA synthesis by 60-70%. Human gingival epithelial cells display strong ERβ but low ERα immunoreactivity both in vivo and in culture. Estrogen attenuates gingival epithelial cell DNA synthesis at high but not low concentrations, suggesting a concentration-dependent mechanism.

Population-prevalent desmosomal mutations predisposing to arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a progressive myocardial disorder caused by mutations of desmosomal cell adhesion proteins. The prevalence of these variants in the general population is unknown. This study examined the spectrum and population prevalence of desmosomal mutations predisposing to ARVC in Finland. We screened 29 Finnish ARVC probands for mutations in the DSP, DSG2, and DSC2 genes. All Finnish-type ARVC-associated mutations, including those 3 previously identified in PKP2 in the same patient group, were analyzed in the population-based Health 2000 cohort of 6,334 individuals and tested for association with electrocardiographic variables. We detected 2 novel mutations: DSG2 3059_3062delAGAG and DSP T1373A. DSG2 3059_3062delAGAG was present in a family with 5 mutation carriers. The endomyocardial samples of the DSG2 deletion carrier showed reduced immunoreactive signal for desmoglein-2, plakophilin-2, plakoglobin, and desmoplakin. DSP T1373A was found in 1 proband with typical right ventricular disease and exercise-related ventricular tachycardia. In the population sample, the collective prevalence of all 5 mutations identified in the 29 ARVC patients (PKP2 Q62K, Q59L, N613K, DSG2 3059_3062delAGAG, and DSP T1373A) was 31 of 6,334 individuals, or 0.5%. The apparent founder mutation PKP2 Q59L is present in 0.3% of Finns and was previously shown to have an approximately 20% disease penetrance. One of 200 Finns carries a desmosomal mutation that may predispose to ARVC and its clinical sequelae. ARVC-associated mutations may thus be more prevalent in the population than expected based on the published ARVC prevalence data.