Fluorescent Antibody Technique

Melanosomes are melanocyte-specific lysosome-related organelles that are transferred to keratinocytes of the epidermis and anagen hair bulb. Transferred melanin forms supra-nuclear caps that protect epidermal keratinocytes against UV irradiation. The mechanism(s) responsible for melanosome transfer into keratinocytes and their subsequent intra-keratinocyte distribution has long remained one of the most enigmatic of heterotypic cell interactions. Although there have been many attempts to study this process, significant progress has been hindered by the absence of an adequate in vitro model. During our ongoing study of melanocyte-keratinocyte interactions in skin and hair follicle, we have developed a novel in vitro assay that exploits the specificity of Silv ⁄ Pmel17 ⁄ gp100 expression for melanosome ⁄ melanin granules. Using matched cultures of keratinocytes and melanocytes isolated from normal healthy epidermis together with double immunofluorescence, we have determined that gp100 is a surprisingly useful tracker of transferred melanin. Moreover, transferred gp100 stained melanin granules emit a bright fluorescence signal, facilitating ready quantification of melanin transfer levels between melanocytes and keratinocytes. This quantitative approach was validated using known inducers and inhibitors of the melanocyte phenotype. This assay further confirmed that cytophagocytosis of melanocyte components (e.g. dendrite tips) by keratinocytes is one route for melanin incorporation into keratinocytes. Lastly, a role for the recently proposed filopodium as a direct conduit for melanin transfer was substantiated using this novel approach. In conclusion, this assay promises to significantly aid our investigations of the molecular basis of melanosome transfer and offers a new tool for the clinical evaluation of melanocyte modulators.

Microtubules (MTs) are cytoskeletal components whose structural integrity is mandatory for the execution of many basic cell functions. Utilizing parental and drug-resistant ovarian carcinoma cell lines that have acquired point mutations in ␤-tubulin and p53, we studied the level of expression and modification of proteins involved in apoptosis and MT integrity. Extending previous results, we demonstrated phosphorylation of pro-survival Bcl-x L in an epothilone-A resistant cell line, correlating it with drug sensitivity to tubulin-active compounds. Furthermore, Mcl-1 protein turned over more rapidly following exposure to tubulin-modifying agents, the stability of Mcl-1 protein paralleling the drug sensitivity profile of the paclitaxel or epothilone-A resistant cell lines. The observed decreases in Mcl-1 were not a consequence of G 2 M arrest, as determined by flow cytometry analysis, which showed prominent levels of Mcl-1 in the absence of any drug treatment in populations enriched in mitotic cells. We also observed that a paclitaxel-resistant cell line expressed Bax at a much lower level than the sensitive parental line [A2780(1A9)], consistent with its mutant p53 status. MT-associated protein-4 (MAP4), whose phosphorylation during specific phases of the cell cycle reduces its MT-polymerizing and -stabilizing capabilities, was phosphorylated in response to drug challenge without a change in expression. Phosphorylation of MAP4 correlated with sensitivity to tubulin-binding drugs and with a dissociation from MTs. We propose that the tubulin mutations, which result in a compromised paclitaxel:tubulin or epothilone:tubulin interaction and paclitaxel or epothilone resistance, indirectly inhibit downstream events that lead to cell death, and this, in turn, may contribute to the drug-resistance phenotype.

Mammalian cells acquire cholesterol, a major membrane constituent, via low-density lipoprotein (LDL) uptake. However, the mechanisms by which LDL cholesterol reaches the plasma membrane (PM) have remained obscure. Here, we applied LDL labeled with BODIPY cholesteryl linoleate to identify this pathway in living cells. The egress of BODIPY cholesterol (BC) from late endosomal (LE) organelles was dependent on acid lipase and Niemann-Pick C1 (NPC1) protein, as for natural cholesterol. We show that NPC1 was needed to recruit Rab8a to BC-containing LEs, and Rab8a enhanced the motility and segregation of BC-and CD63-positive organelles from lysosomes. The BC carriers docked to the cortical actin by a Rab8a-and Myosin5b (Myo5b)dependent mechanism, typically in the proximity of focal adhesions (FAs). LDL increased the number and dynamics of FAs and stimulated cell migration in an acid lipase, NPC1, and Rab8a-dependent fashion, providing evidence that this cholesterol delivery route to the PM is important for cell movement. We labeled human LDL with BC LN (Figures and). A431 cells were preloaded with dextran to visualize LE/LY, and then

Normal lung epithelium and 52 lung carcinomas obtained at surgical resection were examined by immunofluorescence for their expression levels and patterns of the calcium-dependent intercellular adhesion molecule E-cadherin. In dysplastic lung tissue and in well-differentiated squamous cell and adenocarcinomas, expression of E-cadherin was confined to the lateral cell border, similar to the expression level and pattern of normal lung tissue. The E-cadherin level was reduced and expression pattern was spotty or diffuse in moderately and poorly differentiated squamous cell and in small cell carcinomas of the lung. Most metastases resected also had a reduced level and an altered pattern of E-cadherin expression. In contrast, no such correlation was found in adenocarcinomas of the lung. This indicates that different cellular mechanisms are responsible in the progression of squamous cell carcinomas and adenocarcinomas of the lung.

Oxidative stress mediated by nitric oxide (NO) and its toxic metabolite peroxynitrite has previously been associated with motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Degenerating spinal motor neurons in familial and sporadic ALS are typically surrounded by reactive astrocytes expressing the inducible form of NO synthase (iNOS), suggesting that astroglia may have a pathogenic role in ALS. We report here that a brief exposure of spinal cord astrocyte monolayers to peroxynitrite (0.25–1 mM) provoked long‐lasting reactive morphological changes characterized by process‐bearing cells displaying intense glial fibrillary acidic protein and iNOS immunoreactivity. Furthermore, peroxynitrite caused astrocytes to promote apoptosis of embryonic motor neurons subsequently plated on the monolayers. Neuronal death occurred within 24 hr after plating, as evidenced by the presence of degenerating motor neurons positively stained for activated caspase‐3 and nitrotyrosine. Motor n...

A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK- lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin- specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti- bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acineto- bacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK- sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK- lysin exhibits 33% identity with a putative human pre- proprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells.

Neuronal death caused by excessive excitatory signaling, excitotoxicity, plays a central role in neurodegenerative disorders. The mechanisms regulating this process, however, are still incompletely understood. Here we show that the coated vesicle-associated kinase SCYL2/CVAK104 plays a critical role for the normal functioning of the nervous system and for suppressing excitotoxicity in the developing hippocampus. Targeted disruption of Scyl2 in mice caused perinatal lethality in the vast majority of newborn mice and severe sensory-motor deficits in mice that survived to adulthood. Consistent with a neurogenic origin of these phenotypes, neuron-specific deletion of Scyl2 also caused perinatal lethality in the majority of newborn mice and severe neurological defects in adult mice. The neurological deficits in these mice were associated with the degeneration of several neuronal populations, most notably CA3 pyramidal neurons of the hippocampus, which we analyzed in more detail. The loss...

The effects of retinol (vitamin A) and retinoic acid on primary cultures of isolated chicken osteoclasts have been studied. The experiments were performed to establish the direct actions of these two agents on the organization of cytoskeletal structures, on the acid phosphatase contents, and on the bone resorption activities of these cells. The results showed that by treating the cultures with retinol or retinoic acid, from lo-" to M, there were dose-related responses of the osteoclasts. Adhesion to the substratum was stimulated by increasing the number of cells exhibiting the specialized dot-like adhesion structures, or podosomes, which represent the active part of the sealing zone. The treatments also induced rearrangement of the microtubular patterns with reversible depolymerization of microtubules. Acid phosphatase activity was significantly higher both in vitamin A-treated osteoclasts and in their media. When [3H]proline-labeled bone particles were added to the retinoid-treated osteoclasts, the release of [3H]proline was increased significantly compared to controls. These results suggest that the two vitamin A metabolites cause several modifications of the metabolic status of isolated osteoclasts that result in augmented rates of bone resorption.

Palisade endings are nerve specializations found in the extraocular muscles (EOMs) of mammals, including primates. They have long been postulated to be proprioceptors. It was recently demonstrated that palisade endings are cholinergic and that in monkeys they originate from the EOM motor nuclei. Nevertheless, there is considerable difference of opinion concerning the nature of palisade ending function.Palisade endings in EOMs were examined in cats to test whether they display motor or sensory characteristics. We injected an anterograde tracer into the oculomotor or abducens nuclei and combined tracer visualization with immunohistochemistry and α-bungarotoxin staining. Employing immunohistochemistry, we performed molecular analyses of palisade endings and trigeminal ganglia to determine whether cat palisade endings are a cholinergic trigeminal projection.We confirmed that palisade endings are cholinergic and showed, for the first time, that they, like extraocular motoneurons, are als...

The epidemic cycle of Chlamydia pneumoniae infection was examined in two areas in eastern Finland over a period of 15 years, 1972-S7. The C. pneumoniae IgG antibody prevalence was determined with 5-3 r ear intervals in a random sample of the population aged 25-59 years. The total number of sera studied using immunofluorescence was 2387. In 1972 the antibody prevalence was 57% and it increased to GG% in 1977. Over the next 5 years the prevalence decreased to 44% in 1982, but by 1987 it had again increased to 59%. The temporal variation in prevalence was statistically significant (P < 0001) and similar for both genders. Throughout the observation period the overall prevalence Mas 7-11 % higher in men than in women (P < 0-01). The antibody prevalence increased with age, being the highest among the oldest study subjects of both genders. The periods of high and low prevalence alternated in an epidemic cycle (P < 0001) of about 10 years.

When defined in terms of markers for normal cell lineages, most invasive breast cancer cells correspond to the phenotype of the common luminal epithelial cell found in the terminal ductal lobular units. Luminal epithelial cells cultured from milk, which have limited proliferative potential, have now been immortalized by introducing the gene encoding simian virus 40 large tumor (T) antigen. Infection with a recombinant retrovirus proved to be 50-100 times more efficient than calcium phosphate transfection, and of the 17 cell lines isolated, only 5 passed through a crisis period as characterized by cessation of growth. When characterized by immunohistochemical staining with monoclonal antibodies, 14 lines showed features of luminal epithelial cells and of these, 7 resembled the common luminal epithelial cell type in the profile of keratins expressed. These cells express keratins 7, 8, 18, and 19 homogeneously and do not express keratin 14 or vimentin; a polymorphic epithelial mucin pr...

Infection of type II alveolar epithelial (ATII) cells by influenza A viruses (IAV) correlates with severe respiratory disease in humans and mice. To understand pathogenic mechanisms during IAV infection of ATII cells, murine ATII cells were cultured to maintain a differentiated phenotype, infected with IAV-PR8, which causes severe lung pathology in mice, and proteomics analyses were performed using liquid chromatography-mass spectrometry. PR8 infection increased levels of proteins involved in interferon signaling, antigen presentation, and cytoskeleton regulation. Proteins involved in mitochondrial membrane permeability, energy metabolism, and chromatin formation had reduced levels in PR8-infected cells. Phenotypic markers of ATII cells in vivo were identified, confirming the differentiation status of the cultures. Surfactant protein B had decreased levels in Pisis isiiiR8-infected cells, which was confirmed by immunoblotting and immunofluorescence assays. Analysis of ATII cell prot...

A specific and sensitive radioimmunoassay (RIA) was developed for the measurement of sodefrin, a femaleattracting decapeptide pheromone of the red-bellied newt, Cynops pyrrhogaster. Sodefrin synthesized according to the amino acid sequence of native sodefrin, isolated from the abdominal glands of the cloaca of the male newt, was used as a reference standard. An antiserum to sodefrin was produced by immunizing a rabbit with synthetic sodefrin that was extended on its Cterminus with Cys coupled to hemocyanin. For the radioligand, sodefrin N-terminally extended with Tyr was used. An aqueous extract of the abdominal glands of C. pyrrhogaster produced a displacement curve parallel to the sodefrin standard, whereas that from the swordtailed newt (Cynops ensicauda) showed no inhibition of binding in this RIA. The sensitivity of the RIA was 30.5 6 3.4 pg/100 ml assay buffer. Intraassay and interassay coefficients of variation were 1.6 and 4.5%, respectively. The RIA was used to determine sodefrin levels in the abdominal gland of the male newt. Hypophysectomy and castration greatly reduced the sodefrin content. Administration of testosterone propionate (TP) to the hypophysectomized and castrated newt increased the pheromone content in the abdominal gland. A combination of prolactin (PRL) and TP elevated the sodefrin content markedly, while PRL alone scarcely affected it. Immunoreactive sodefrin was observed in the epithelial cells of the abdominal gland of both hormonetreated and saline-injected newts. Among them, groups treated with PRL plus TP and TP alone exhibited strong immunoreactivity in their abdominal gland compared with PRL-treated and saline-injected groups.

Aging is a multifaceted process associated with various functional and structural deficits that might be evolved in degenerative diseases. It has been shown that neurodegenerative disorders are associated with alterations in Ca2+ homeostasis. Thus, in the present work, we have investigated Ca2+ signaling and apoptosis in aged striatum. Our results show that glutamate and NMDA evoke a greater Ca2+ rise in striatum slices from aged animals. However, this difference is not present when glutamate is tested in the absence of external Ca2+. Immunostaining of glutamate receptors shows that only NMDA receptors (NR1) are increased in the striatum of aged rats. Increases in mitochondrial Ca2+ content and in the reactive oxygen species levels were also observed in aged animals, which could be associated with tissue vulnerability. In addition, a decrease in the Bcl‐2 protein expression and an enhancement in apoptosis were also present in aged striatum. Together the results indicate that, in age...

We recently found that endogenous (free fatty acids) and pharmacological (thiazolidinediones) agonists of nuclear receptor Peroxisome proliferator-activated receptor (PPAR)γ stimulate renin transcription. In addition, the renin gene was identified as a direct target of PPARγ. The mouse renin gene is regulated by PPARγ through a distal enhancer direct repeat closely related to consensus PPAR response element (PPRE). In vitro studies demonstrated that PPARγ knockdown stimulated PPRE-driven transcription. These data predicted that deficiency of PPARγ would upregulate mouse renin expression. Consistent with these observations knockdown of PPARγ increased the transcription of a reporter gene driven by the mouse renin PPRE-like motif in vitro. To study the impact of PPARγ on renin production in vivo, we used a cre/lox system to generate double-transgenic mice with disrupted PPARγ locus in renin-producing juxtaglomerular (JG) cells of the kidney (RC-PPARγ fl/fl mice). We provide evidence t...

IGF-binding proteins (IGFBPs) and their proteases regulate IGFs bioavailability in multiple tissues. Pregnancy-associated plasma protein A (PAPP-A) is a protease acting by cleaving IGFBP2, 4, and 5, regulating local bioavailability of IGFs. We have previously shown that IGFs and IGFBPs are produced by human adult cardiac progenitor cells (haCPCs) and that IGF-1 exerts paracrine therapeutic effects in cardiac cell therapy with CPCs. Using immunofluorescence and enzyme immunoassays, we firstly report that PAPP-A is produced and secreted in surprisingly high amounts by haCPCs. In particular, the homodimeric, enzymatically active, PAPP-A is secreted in relevant concentrations in haCPC-conditioned media, while the enzymatically inactive PAPPA/proMBP complex is not detectable in the same media. Furthermore, we show that both homodimeric PAPP-A and proMBP can be detected as cell associated, suggesting that the previously described complex formation at the cell surface does not occur easily...

Quantitative antibody levels to three herpesviruses in acute and chronic sera from six patients with clinical signs of the acute retinal necrosis syndrome were consistent with a spe cific etiologic diagnosis only in the two cases associated with cutaneous herpes zoster. Available data on acute and convalescent anti body titers to herpes group viruses from these six patients in addition to data from 27 acute retinal necrosis cases from the literature dis closed that only 13 of the 33 patients (39%) had a diagnostic increase or decrease in herpes group viral antibody levels on serial sam pling. Three patients had nondiagnostic changes in viral antibody levels despite posi tive vitreous cultures for herpesviruses. In contrast, a review of 25 cases from the litera ture with paired antiviral serum and intraocu lar fluid antibody levels suggested a more promising approach to the etiologic diagnosis of the acute retinal necrosis syndrome. By calculating the ratio of antiviral antibodies in intraocular fluid and serum, an etiologic diag-

Aims/hypothesis Aldehyde dehydrogenase 1 (ALDH1), a human stem-cell marker, is an enzyme responsible for converting retinaldehydes to retinoic acids (RAs) to modulate cell differentiation. However, data on expression levels and functional roles of ALDH1 during human fetal pancreatic development are limited. The focus of this study was to characterise ALDH1 expression patterns and to determine its functional role in islet cell differentiation. Methods The presence of ALDH1 in the human fetal pancreas (8-22 weeks) was characterised by microarray, quantitative RT-PCR, western blotting and immunohistological approaches. Isolated human fetal islet-epithelial cell clusters were treated with ALDH1 inhibitors, retinoic acid receptor (RAR) agonists and ALDH1A1 small interfering (si)RNA. Results In the developing human pancreatic cells, high ALDH1 activity frequently co-localised with key stem-cell markers as well as endocrine transcription factors. A high level of ALDH1 was expressed in newly differentiated insulin + cells and this decreased as development progressed. Pharmacological inhibition of ALDH1 activity in human fetal islet-epithelial cell clusters resulted in reduced endocrine cell differentiation and increased cell apoptosis, and was reversed with co-treatment of RAR/RXR agonists. Furthermore, siRNA knockdown of ALDH1A1 significantly decreased RAR expression and induced cell apoptosis via suppression of the phosphoinositide 3-kinase (PI3K) pathway and activation of caspase signals. Conclusions/interpretation Our findings indicate that ALDH1 + cells represent a pool of endocrine precursors in the developing human pancreas and that ALDH1 activity is required during endocrine cell differentiation. Inhibition of ALDH1-mediated retinoid signalling impairs human fetal islet cell differentiation and survival. Keywords Aldehyde dehydrogenase 1 . Human fetal pancreas . Pancreatic endocrine cell differentiation . Retinoic acid signalling Abbreviations ALDH Aldehyde dehydrogenase ATRA All-trans retinoic acid CDA Canadian Diabetes Association cRNA Complementary RNA DEAB Diethylaminobenzaldehyde NGN3 Neurogenin 3 NKX2.2 NK2 homeobox 2 NKX6.1 NK6 homeobox 1 Electronic supplementary material The online version of this article (doi:10.1007/s00125-013-3147-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users. J

Eosinophils contain many preformed cytokines and chemokines, which are stored in specific granules along with cationic granule proteins. Mobilization and release of these granule contents can be selective and mediated by vesicular transport. We have developed a sensitive method to detect and quantitate eosinophil vesicular transport-mediated release of specific eosinophil proteins. Our EliCell assay is based on microscopic observations of individual viable eosinophils embedded in an agarose matrix that contains immobilized antibody to the protein of interest. Following stimulation of eosinophils, released protein is bound by the capture antibody at its site of release and is detected by a fluorochrome-conjugated detection antibody. We have validated this assay by evaluating interferon-g-induced release of RANTES from eosinophils. Extracellularly released RANTES was visualized as focal immunoflourescent staining and was quantitated by scoring the numbers of eosinophils releasing RANTES and by measuring the fluorescent intensity over individual eosinophils. In comparison with ELISA assays of RANTES released into supernatant fluids by interferon-g-stimulated eosinophils, EliCell assays were more sensitive enabling detection of RANTES release at earlier times and at lower levels of interferon-g stimulation. The EliCell assay provides a sensitive method to study the regulated release of eosinophil-derived cytokines, chemokines and other granule proteins.

Ghrelin is an endocrine peptide that has been identified in gastric oxyntic glands and that induces growth hormone secretion in the pituitary gland. This growth hormone secretagogue is expressed in many tissues such as stomach, pituitary gland, thyroid, testis, placenta and pancreas. Initial studies of ghrelin focused on its role as a circulating orexigenic signal. However, ghrelin has also been found to be involved in the modulation of glucose homeostasis. Although a number of studies have reported ghrelin expression in developing pancreas, the location of ghrelin-immunoreactive cells in adult pancreas (epsilon cells) remains controversial. In this study, we have analysed the distribution of pancreatic epsilon cells in adult human and rat islets by immunohistochemistry and in situ hybridisation. In humans, our immunohistochemical analysis has shown that ghrelin is expressed in glucagonsecreting cells, whereas in rats, it is present in insulinsecreting cells. Similar observations have been revealed by in situ hybridisation.

Ghrelin is an endocrine peptide that has been identified in gastric oxyntic glands and that induces growth hormone secretion in the pituitary gland. This growth hormone secretagogue is expressed in many tissues such as stomach, pituitary gland, thyroid, testis, placenta and pancreas. Initial studies of ghrelin focused on its role as a circulating orexigenic signal. However, ghrelin has also been found to be involved in the modulation of glucose homeostasis. Although a number of studies have reported ghrelin expression in developing pancreas, the location of ghrelin-immunoreactive cells in adult pancreas (epsilon cells) remains controversial. In this study, we have analysed the distribution of pancreatic epsilon cells in adult human and rat islets by immunohistochemistry and in situ hybridisation. In humans, our immunohistochemical analysis has shown that ghrelin is expressed in glucagonsecreting cells, whereas in rats, it is present in insulinsecreting cells. Similar observations have been revealed by in situ hybridisation.

The histologic and ultrastructural features of nephrogenic adenomas of the urinary bladder and urethra were studied in multiple specimens obtained from eight patients. Three of these were studied by indirect immunofluorescence for Tamm-Horsfall uromucoprotein. The lesions are uncommon benign metaplastic proliferations of urothelium, occurring most frequently in males as small cystic, papillary, or nodular masses and most often presenting with hematuria. Typically the metaplastic tubules resemble nephronic tubtdes. A florid atypical and pseudoinfiltrative proliferation of these tubules may lead to an erroneous diagnosis of adenocarcinoma. Ultrastructural features of proximal convoluted tubules were identified in some of the tubules, but resemblance to specific segments of distal tubtdes was less certain. The ultrastructural features combined with the absence of Tamm-Horsfall protein in tubular lumina or cells suggest a mesonephric rather than metanephric homology. The lesions are appropriately treated by transurethral resection or fulguration, but persistent lesions were present in three patients up to 18 years after initial treatment. Altered differentiation of the urothelium of the bladder may occur in both metaplastic and neoplastic conditions, most commonly as squamous differentiation. Glandular, mucus secreting, and intestinal dif-ferentiation are seen in the exstrophic bladder, in chronically infected or irritated bladders, and in some bladder tumors. A rare peculiar nephronic tubular type of transformation has been designated as neph-,i

In humans, most blood group substances (BGS) are expressed throughout the fetal colon but are absent from the distal portion of adult colon. Cancers of the distal colon frequently reexpress BGS thereby suggesting that these antigens behave as oncofetal antigens at this organ site. We used a sensitive immunoperoxidase method with monoclonal antibodies directed against blood groups A, B, O (H), Lewis8 and Lewis &quot; to systematically evaluate BGS expression in fetal colon, normal adult colon from immediate autopsies of kidney donors, mucosa adjacent to cancer (transi tional mucosa) and colorectal cancer tissues. In normal colon, BG-A, B, H, and Lewis &quot; were expressed in proximal but not distal colon, whereas Lewis &quot; was distributed uniformly through

Kisspeptin (KP) neurones in the rostral periventricular area of the third ventricle (RP3V) and arcuate nucleus (Arc) are important elements in the neuronal circuitry regulating gonadotropin-releasing hormone (GnRH) secretion. KP and co-synthesised neuropeptides/neurotransmitters act directly on GnRH perikarya and processes. GnRH neurones not only form the final output pathway regulating the reproductive functions of the anterior pituitary gland, but also provide neuronal input to sites within the hypothalamus. The current double-label immunohistochemical studies investigated whether GnRH-immunoreactive (IR) projections to the RP3V and/or Arc establish morphological connections with KP-IR neurones at these sites. To optimise visualisation of KP immunoreactivity in, respectively, the RP3V and Arc, ovariectomised (OVX) oestrogen-treated and OVX oil-treated female mice were studied. Confocal laser microscopic analysis of immunofluorescent specimens revealed GnRH-IR axon varicosities in ...

The ErbB-4 receptors are unique in the EGFR/ErbB family for the ability to associate with WW domain-containing proteins. To identify new ligands of the cytoplasmic tail of ErbB-4, we panned a brain cDNA phage library with ErbB-4 peptides containing sequence motifs corresponding to putative docking sites for class-I WW domains. This approach led to identification of AIP4/Itch, a member of the Nedd4-like family of E3 ubiquitin protein ligases, as a protein that specifically interacts with and ubiquitinates ErbB-4 in vivo. Interaction with the ErbB-4 receptors occurs via the WW domains of AIP4/Itch. Functional analyses demonstrate that AIP4/Itch is recruited to the ErbB-4 receptor to promote its polyubiquitination and degradation, thereby regulating stability of the receptor and access of receptor intracellular domains to the nuclear compartment. These findings expand our understanding of the mechanisms contributing to the integrity of the ErbB signaling network and mechanistically link the cellular ubiquitination pathway of AIP4/Itch to the ErbB-4 receptor.

We evaluated whether the angiotensin II (Ang II) receptors from perioperation through one-year post-transplantation predict the transplant coronary artery disease (TCAD) progression. BACKGROUND The role of Ang II receptors (type 1: AT 1 R; type 2: AT 2 R) in TCAD is uncertain. We investigated 28 heart donors and the corresponding recipients. The levels of AT 1 R and AT 2 R messenger ribonucleic acid (mRNA) were examined in lymphocytes from the donor spleen and in the donor heart at one-week and one-year posttransplantation to determine their association with the progression of TCAD, measured as changes in maximal intimal thickness (CMIT) and plaque volume (CPV) by intravascular ultrasound (IVUS) examinations. The AT 1 R mRNA in lymphocytes from the donor spleen (CMIT: r ϭ 0.73, p Ͻ 0.0001; CPV: r ϭ 0.69, p Ͻ 0.0001) and in the donor hearts at one-week (CMIT: r ϭ 0.52, p ϭ 0.005; CPV: r ϭ 0.56, p ϭ 0.002) and at one-year (CMIT: r ϭ 0.63, p Ͻ 0.0001; CPV: r ϭ 0.43, p ϭ 0.004) post-transplantation along with AT 2 R mRNA in the donor hearts at one-year post-transplantation (CMIT: r ϭ 0.3, p Ͻ 0.0001; CPV: r ϭ 0.53, p ϭ 0.009) were univariate predictors, whereas AT 1 R mRNA in lymphocytes and in the donor hearts at one-year post-transplantation proved to be multivariate predictors of the progression of TCAD. CONCLUSIONS These data suggest a role for Ang II receptors in the pathogenesis of TCAD and support a novel concept that TCAD may have its origin in the donor per se and may be modulated by the recipient's inherent biological factors. (

Neural crest-derived cells populate the thymus, and their coexistence with epithelial cells is required for proper organ development and T cell education function. We show here that epidermal growth factor (EGF), a major epithelial cell growth-enhancing agent, has a morphogenetic action to promote the expression of a neuronal phenotype (e.g., neurofilament expression) in cultured thymic epithelial cells that are characterized by a cytokeratin-positive epithelial cell background. The proliferation of such neurodifferentiated cells is also enhanced by EGF. Furthermore, the growth factor enhances cells that express the genes encoding the preprotachykinin A-generated neuropeptides and bipotential neuropoietic and lymphopoietic cytokines ciliary neurotrophic factor and interleukin-6. These cytokines also enhance the neuronal phenotype of thymic epithelial cells. Therefore, EGF appears to be a composite autocrine/paracrine neuromodulator in thymic stroma. This suggests that EGF may regula...

Rationale: Nuclear factor erythroid 2-related factor 2 (Nrf2), an important regulator of lung antioxidant defenses, declines in chronic obstructive pulmonary disease (COPD). However, Nrf2 also regulates the proteasome system that degrades damaged and misfolded proteins. Because accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and ER stress-induced apoptosis, Nrf2 may potentially prevent ER stress-mediated apoptosis in COPD. Objectives: To determine whether Nrf2-regulated proteasome function affects ER stress-mediated apoptosis in COPD. Methods: We assessed the expression of Nrf2, Nrf2-dependent proteasomal subunits, proteasomal activity, markers of ER stress, and apoptosis in emphysematous lungs of mice exposed to cigarette smoke (CS) as well as peripheral lung tissues from normal control subjects and patients with COPD. Measurements and Main Results: Compared with wild-type mice, emphysematous lungs of CS-exposed Nrf2-deficient mice exhibited markedly lower proteasomal activity and elevated markers of ER stress and apoptosis. Furthermore, compared with normal control subjects, lungs of patients with mild and advanced COPD showed a marked decrease in the expression of Nrf2-regulated proteasomal subunits and total proteasomal activity. However, they were associated with greater levels of ER stress and apoptosis markers. In vitro studies have demonstrated that enhancing proteasomal activity in Beas2B cells either by sulforaphane, an activator of Nrf2, or overexpression of Nrf2regulated proteasomal subunit PSMB6, significantly inhibited cigarette smoke condensate (CSC)-induced ER stress and cell death. Conclusions: Impaired Nrf2 signaling causes significant decline in proteasomal activity and heightens ER stress response in lungs of patients with COPD and CS-exposed mice. Accordingly, pharmacological approaches that augment Nrf2 activity may protect against COPD progression by both up-regulating antioxidant defenses and relieving ER stress.

The B cell coreceptor CD22 plays an important role in regulating signal transduction via the B cell Ag receptor. Studies have shown that surface expression of CD22 can be modulated in response to binding of ligand (i.e., mAb). Thus, it is possible that alterations in the level of CD22 expression following binding of natural ligand(s) may affect its ability to modulate the Ag receptor signaling threshold at specific points during B cell development and differentiation. Therefore, it is important to delineate the physiologic mechanism by which CD22 expression is controlled. In the current study, yeast two-hybrid analysis was used to demonstrate that CD22 interacts with AP50, the medium chain subunit of the AP-2 complex, via tyrosine-based internalization motifs in its cytoplasmic domain. This interaction was further characterized using yeast two-hybrid analysis revealing that Tyr 843 and surrounding amino acids in the cytoplasmic tail of CD22 comprise the primary binding site for AP50. Subsequent studies using transfectant Jurkat cell lines expressing wild-type or mutant forms of CD22 demonstrated that either Tyr 843 or Tyr 863 is sufficient for mAb-mediated internalization of CD22 and that these motifs are involved in its interaction with the AP-2 complex, as determined by coprecipitation of ␣-adaptin. Finally, experiments were performed demonstrating that treatment of B cells with either intact anti-Ig Ab or F(ab) 2 blocks ligand-mediated internalization of CD22. In conclusion, these studies demonstrate that internalization of CD22 is dependent on its association with the AP-2 complex via tyrosine-based internalization motifs.

Regulatory T cells (Treg cells) are an important area of investigation in human health and disease. In this study, the trajectory of percentage of Treg cells (defined as CD4+CD25+Foxp3+CD127lymphocytes) was measured in the blood of 208 women during pregnancy and up to 3 additional times in the postpartum period (1, 6 and 12 months postpartum). Whether the trajectory was affected by gravidity, parity, neonatal sex, pet exposure, maternal atopic and asthma status, smoking, maternal race or other pregnancy factors was examined. Multilevel models were fit using full maximum likelihood methods and included both random and fixed effects. Overall, percentages of Treg cells increased from the prenatal to the postpartum period. Among women who were not atopic, nulliparous women had lower percentages of Treg cells over time compared with parous women. Atopic women with pets in the home during pregnancy had lower percentages of Treg cells than atopic women who did not have pets. The trajectory was not affected by the other factors investigated. We conclude that within-woman change in percentages of Treg cells may vary by time in relation to delivery, as well as by maternal atopic status and exposure to pets and number of prior births. The data did not indicate an overall decline in Treg cells in the postpartum period. Future work to better identify the role of Treg cells in successful pregnancy would ideally include a set of well characterized women sampled serially starting prior to pregnancy and throughout the postpartum period.

In previous reports we proposed a new genus for Rhabdoviridae and described neurotropic preference and gross neuropathology in newborn albino Swiss mice after Curionopolis and Itacaiunas infections. In the present report a timecourse study of experimental encephalitis induced by Itacaiunas and Curionopolis virus was conducted both in vivo and in vitro to investigate cellular targets and the sequence of neuroinvasion. We also investigate, after intranasal inoculation, clinical signs, histopathology and apoptosis in correlation with viral immunolabeling at different time points. Curionopolis and Itacaiunas viral antigens were first detected in the parenchyma of olfactory pathways at 2 and 3 days post-inoculation (dpi) and the first clinical signs were observed at 4 and 8 dpi, respectively. After Curionopolis infection, the mortality rate was 100% between 5 and 6 dpi, and 35% between 8 and 15 dpi after Itacaiunas infection. We identified CNS mice cell types both in vivo and in vitro and the temporal sequence of neuroanatomical olfactory areas infected by Itacaiunas and Curionopolis virus. Distinct virulences were reflected in the neuropathological changes including TUNEL immunolabeling and cytopathic effects, more intense and precocious after intracerebral or in vitro inoculations of Curionopolis than after Itacaiunas virus. In vitro studies revealed neuronal but not astrocyte or microglial cytopathic effects at 2 dpi, with monolayer destruction occurring at 5 and 7 dpi with Curionopolis and Itacaiunas virus, respectively. Ultrastructural changes included virus budding associated with interstitial and perivascular edema, endothelial hypertrophy, a reduced and/or collapsed small vessel luminal area, thickening of the capillary basement membrane, and presence of phagocytosed apoptotic bodies. Glial cells with viral budding similar to oligodendrocytes were infected with Itacaiunas virus but not with Curionopolis virus. Thus, Curionopolis and Itacaiunas viruses share many pathological and clinical features present in other rhabdoviruses but distinct virulence and glial targets in newborn albino Swiss mice brain.

Retinal pigment epithelium (RPE)-derived membranous debris named blebs, may accumulate and contribute to sub-RPE deposit formation, which is the earliest sign of age-related macular degeneration (AMD). Oxidative injury to the RPE might play a significant role in AMD. However, the underlying mechanisms are unknown. We previously reported that hydroquinone (HQ), a major pro-oxidant in cigarette smoke, foodstuff, and atmospheric pollutants, induces actin rearrangement and membrane blebbing in RPE cells as well as sub-RPE deposits in mice. Here, we show for the first time that phosphorylated Heat shock protein 27 (Hsp27), a key regulator of actin filaments dynamics, is up-regulated in RPE from patients with AMD. Also, HQ-induced nonlethal oxidative injury led to Hsp27mRNA up-regulation, dimer formation , and Hsp27 phosphorylation in ARPE-19 cells. Furthermore, we found that a cross talk between p38 and extracellular signal-regulated kinase (ERK) mediates HQ-induced Hsp27 phosphorylation and actin aggregate formation, revealing ERK as a novel upstream mediator of Hsp27 phosphorylation. Finally, we demonstrated that Hsp25, p38, and ERK phosphorylation are increased in aging C57BL/6 mice chronically

Thanatophoric dysplasia (TD), the commonest lethal skeletal dysplasia in humans, is accounted for by recurrent mutations in the fibroblast growth factor receptor 3 gene (FGFR 3), causing its constitutive activation in vitro. Taking advantage of medical abortion of 18 TD fetuses, cartilage sections were studied for FGFR 3 gene expression by in situ hybridization and immunohistochemistry. Specific antibodies revealed high amounts of FGFR 3 in cartilage of TD fetuses with no increased level of the corresponding mRNA. The specific signal was mainly detected in the nucleus of proliferative and hypertrophic chondrocytes. Based on this observation and the abnormal expression of collagen type X in hypertrophic TD chondrocytes, we suggest that constitutive activation of the receptor through formation of a stable dimer increases its stability and promotes its translocation into the nucleus, where it might interfere with terminal chondrocyte differentiation.

The up-regulation of various matrix metalloproteinases (MMP), certain cell receptors such as integrins and CD44, and the SIBLING family of integrin-binding glycophosphoproteins have been reported separately and in various combinations for many types of tumors. The mechanisms by which these different proteins may be interacting and enhancing the ability of a cancer cell to survive and metastasize have become an interesting issue in cancer biology. Dentin matrix protein 1 (DMP1) has been known for a number of years to bind to CD44 and ArgGlyAsp sequence–dependent integrins. This SIBLING was recently shown to be able to specifically bind and activate proMMP-9 and to make MMP-9 much less sensitive to inhibition by tissue inhibitors of metalloproteinases and synthetic inhibitors. In this study, we used a modified Boyden chamber assay to show that DMP1 enhanced the invasiveness of the MMP-9 expressing colon cancer cell line, SW480, through Matrigel in a dose-dependant manner. DMP1 (100 nm...

Asbestos fibers, silica dust and glass fibers induced aggregation of the pericellular matrix fibronectin when added to the growth medium of cultured human fibroblasts as judged by immunofluorescence microscopy. Direct binding experiments and analysis of the bound proteins by (SDS‐polyacrylamide gel electrophoresis) revealed an adsorption of purified plasma fibronectin to glass fibers, amosite, crocidolite and silica but not to anthophyllite and chrysotile A and B. The results suggest that aggregation of the pericellular fibronectin by these fibrogenic materials may be involved in the initial stages of the development of fibrosis in vivo.

We have compared murine cytomegalovirus (MCMV) antibody determination by enzyme-linked immuno- sorbent assay (ELISA) and indirect immunofluorescence assay. A comparison of antibody detection with 146 serum samples at a 1:20 dilution showed 100% agreement (60 negatives and 86 positives) between the assays. There was close agreement of endpoint determinations of sera by both methods. After experimental MCMV infection, antibody to MCMV was detected by both assays as early as day 7, and high titers persisted as late as 6 months. In contrast to immunocompetent littermates, athymic nude mice did not develop antibody after infection. Mice lacking antibody detectable by ELISA were susceptible to lethal MCMV challenge. In a survey of animals from five commercial sources, MCMV antibody was not detected unless mice were experimentally infected. MCMV antibody determination by ELISA is a convenient method, comparable to the indirect immunofluorescence assay in sensitivity and specificity.

Heme oxygenase (HO)-1 is upregulated by many stressful stimuli, including arsenic. A GT-repeat ((GT)n) polymorphism in the HO-1 gene promoter inversely modulates the levels of HO-1 induction. Previous HO-1 (GT)n polymorphism studies in relation to cancer risk have shown disparate results. We prospectively investigated the associations between HO-1 (GT)n polymorphism and cancer risk related to arsenic from drinking water. Totally, 1,013 participants from community-based cohorts of arseniasis-endemic areas in Taiwan were followed for 13 years. Allelic polymorphisms were classified into long (L, ≥27 (GT)n) and short (S, &lt;27 (GT)n). Newly developed cases were identified through linkage with National Cancer Registry of Taiwan. Multivariate Cox proportional hazard methods were used to evaluate effects of the HO-1 polymorphism alone or combined with arsenic exposure. Results showed that participants with the S/S genotype had an increased risk of Bowen&#39;s disease (HR=10.49; 95% CI: 2....

Background. Bilateral ureteral obstruction (BUO) is characterized by the development of hemodynamic and tubular lesions. However, little is known about the expression of organic anion renal transporters. The objective of this work was to study the renal excretion of p-aminohippurate (PAH) and the cortical renal expression of the organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) in BUO rats. Methods. Male Wistar rats underwent bilateral obstruction of the proximal ureters for 24 hours (BUO) or sham operation. After 24 hours of ureteral releasing, the following studies were performed: PAH renal excretion employing conventional clearance techniques and OAT1 and OAT3 abundance (homogenates, intracellular and basolateral plasma membrane fractions from renal cortex) using immunoblotting and immunocytochemical techniques (light microscopic and confocal immunofluorescence microscopic analysis). Results. BUO rats showed a lower renal excretion of PAH. In obstructed kidneys, immunoblotting revealed a significant decrease in the abundance of both OAT1 and OAT3 in basolateral plasma membranes from renal cortex. An increase of OAT1 expression was observed in homogenates and in intracellular membrane fractions in kidneys from BUO rats compared with sham-operated ones, indicating an internalization of this carrier. Immunocytochemical techniques confirmed these results. On the contrary, OAT3 expression was reduced both in homogenates and in intracellular membrane fractions in obstructed kidneys. Conclusion. BUO was associated with down-regulation of OAT1 and OAT3 in basolateral plasma membranes from proximal tubule cells, thus these carriers may play important roles in the impaired organic anion excretion displayed in the obstructed kidney. Urinary tract obstruction is a serious and common clinical condition, which is associated with increased intralu-

During continuous proliferation, cyclin D1 protein is induced to high levels in a Ras-dependent manner as cells progress from S phase to G2 phase. To understand the mechanism of the Ras-dependent cyclin D1 induction, cyclin D1 mRNA levels were determined by quantitative image analysis following fluorescent in situ hybridization. Although a slight increase in mRNA expression levels was detected during the S/G2 transition, this increase could not explain the more robust induction of cyclin D1 protein levels. This suggested the involvement of posttranscriptional regulation as a mechanism of cyclin D1 protein induction. To directly test this hypothesis, the cyclin D1 transcription rate was determined by run-on assays. The transcription rate of cyclin D1 stayed steady during the synchronous transition from S the G2 phase. We further demonstrated that cyclin D1 protein levels could increase during G2 phase in the absence of new mRNA synthesis. a-Amanitin, a transcription inhibitor, did not suppress cyclin D1 protein elevation as the cells progressed from S to G2 phase, even though the inhibitor was able to completely block cyclin D1 protein induction during reentry into the cell cycle from quiescence. The half life of cyclin D1 protein was shortest during S phase indicating that a change in protein stability might play a role in post-translational induction of cyclin D1 in G2 phase. These data indicate a fundamental difference in the regulation of cyclin D1 production during continuous cell cycle progression and re-initiation of the cell cycle.

HLA class I phenotyping was performed using T-lymphocyte populations isolated by immunomagnetic beads (IMBs) coated with monoclonal antibodies with specificity for CD2, CD4 or CD8. The results were compared to those obtained using density gradient-separated lymphocytes (PBL). The typing trays were read by the automated simultaneous double-fluorescence (SDF) technique previously established in our laboratory using an Astroscan 2100 system. The aims of the present study were to establish whether the advantages of IMB lymphocyte separation and automated plate reading by SDF were complementary and whether the results obtained by IMB-SDF and PBL-SDF were concordant. Similarity coefficients for paired results obtained by IMB-SDF and PBL-SDF varied between 0.825 using anti-CD8-coated IMBs and 0.914 using anti-CD4coated IMBs with a consistent excess of stronger results observed with the PBL-SDF technique. The variations observed did not result in incorrect phenotype assignment but would significantly influence a cross-matching test. These results illustrate the feasibility of using IMB-separated lymphocytes for HLA phenotyping by SDF.

Cryostat sections of fresh frozen tissues were used in a prospective study of blood group H and A antigen fluorescence in 73 transitional cell carcinomas of the bladder. The aim was to evaluate antigen expression without subjecting the tumour tissues to organic solvents that extract blood group active glycolipids. Deletion of the genetically predicted antigen was twice as common in tumours of pTl or greater stage than those of pTa stage and also twice as common in poorly differentiated than in moderately well differentiated tumours. The considerable heterogeneity and overlap, however, in patterns of reactivity in tumours of various histopathological stages and grades and the effect of secretor status on antigenicity meant that there was no obvious antigenic feature that correlated precisely with invasive stage or differentiation grade. It remains to be determined whether the antigen positive and antigen negative tumours represent different disease entities with differing clinical courses. Our results indicate, however, that studies of the blood group antigens in urinary tract tumours are more likely to be of value in research into biochemical disorders in the neoplastic process than in routine clinical assessment as a guide to treatment. Tumours of the transitional epithelium of the bladder often recur, and up to 30% of superficial tumours eventually become invasive. ' 2 It is highly desirable at the time of diagnosis to distinguish the superficial tumours that will have a relatively benign course and require simple endoscopic follow up from those that will eventually invade and metastasise and may require more aggressive treatment. There are at present no reliable criteria for such a distinction. Thus molecular markers have been sought in the hope that they would accurately predict bladder tumour behav- iour and permit new strategies of management. Many studies have concentrated on the blood group A, B, and H antigens, which are normally expressed on epithelial tissues, but which may be absent or reduced in several types of epithetial carcinomas.3 Several retro- spective studies using paraffin embedded bladder tumours have suggested that loss or reduction of the predicted antigen(s), as judged by the red cell adher- ance test, is associated with, and even precedes, invasive or metastatic recurrence.46 Other investigators using different methods have found no

Mutation of the retinoblastoma (RB) tumor suppressor gene is strongly linked to osteosarcoma formation. This observation and the documented interaction between the retinoblastoma protein (pRb) and Runx2 suggests that pRb is important in bone development. To assess this hypothesis, we used a conditional knockout strategy to generate pRb-deficient embryos that survive to birth. Analysis of these embryos shows that Rb inactivation causes the abnormal development and impaired ossification of several bones, correlating with an impairment in osteoblast differentiation. We further show that Rb inactivation acts to promote osteoblast differentiation in vitro and, through conditional analysis, establish that this occurs in a cell-intrinsic manner. Although these in vivo and in vitro differentiation phenotypes seem paradoxical, we find that Rb-deficient osteoblasts have an impaired ability to exit the cell cycle both in vivo and in vitro that can explain the observed differentiation defects. ...

The accumulation of neurofibrillary tangles in Alzheimer's disease (AD) propagates with characteristic spatiotemporal patterns which follow brain network connections, implying trans-synaptic transmission of tauopathy. Since misfolded tau has been shown to transmit across synapses in AD animal models, we hypothesized that synapses in AD patients may contain misfolded tau. By immunofluorescence imaging of bipartite synapses from AD subjects, we detected tau protein in 38.4% of presynaptic and 50.9% of postsynaptic terminals. The pre/post distribution for hyperphosphorylated tau was 26.9%/30.7%, and for misfolded tau 18.3%/19.3%. In the temporal cortex, microscopic aggregates of tau, containing ultra-stable oligomers, were estimated to accumulate within trillions of synapses, outnumbering macroscopic tau aggregates such as tangles by 10000 fold. Non-demented elderly also showed considerable synaptic tau hyperphosphorylation and some misfolding, implicating the synapse as one of the first subcellular compartments affected by tauopathy. Misfolding of tau protein appeared to occur in situ inside synaptic terminals, without mislocalizing or mistrafficking. Misfolded tau at synapses may represent early signs of neuronal degeneration, mediators of synaptotoxicity, and anatomical substrates for transmitting tauopathy, but its actual role in these processes remain to be elucidated.