Primary Rabbit Polyclonal Antibodies Research Articles

B-011 Analytical Validation of a New Vasoactive Intestinal Peptide Radioimmunoassay

Abstract Background Vasoactive intestinal peptide (VIP), a potent vasodilator, is produced by neuronal cells located in the central nervous system, the digestive, respiratory, and urogenital tracts, and the exocrine, thyroid, and adrenal glands. Increased circulating VIP is clinically useful to detect VIP-producing tumors in patients with chronic diarrheal diseases. Objective Develop a sensitive replacement radioimmunoassay (RIA) for VIP quantitation in human plasma due to the diminished supply of the antibody used in the previous in-house method. Method Patient VIP in EDTA plasma competes with labeled (125I) VIP for a limited number of primary rabbit polyclonal antibody (BMA Biomedicals, Augst, Switzerland) binding sites. Antibody-bound VIP is then complexed with a goat anti-rabbit secondary antibody and precipitated out of the solution with the use of polyethylene glycol and centrifugation. After the supernatant is discarded, the 125I signal from the pellet is counted on the Wizard2 gamma counter (Perkin Elmer, Waltham, MA). The measured signal is inversely proportional to the amount of VIP present in the patient’s sample. Method validation of the new assay included determination of imprecision (pooled human EDTA plasma), limits of detection and quantification, analytical measurement range (AMR), accuracy by spike recovery, interferences, cross-reactivity, stability, reference interval determination, and agreement with the current Mayo laboratory-developed assay. Results Stability studies on freshly collected EDTA plasma showed VIP is stable for 3 days ambient, 7 days refrigerated, and 14 days frozen. Intra-assay imprecision studies showed 8.3, 2.3, and 1.3%CV for mean VIP concentrations of 41, 144, and 304 pg/mL. Inter-assay imprecision was 12.9, 7.6, 5.2, and 7.5%CV for mean VIP concentrations of 30, 81, 180, and 308 pg/mL. The assay limit of detection was 29 pg/mL. The limit of quantitation was 30 pg/mL (%CV = 13%) and was established via precision profile. The AMR was 30–500 pg/mL (slope of 0.98, intercept of 3.0 pg/mL, and R2 of 0.97). The mean % spike recovery using a different preparation lot of calibrator material was 89% (range 83%–93%). The assay was not affected by up to 1000 mg/dL hemoglobin, 1000 mg/dL triglycerides, or 10 mg/dL bilirubin. Peptide histidine methionine 27, neurotensin, gastric inhibitory peptide, motilin, secretin, and glucagon do not cross-react in the assay. The 97.5th reference limit was <86 pg/mL (95% CI 81–90, n = 127). Positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) with the current Mayo VIP assay were 97, 82, and 90% respectively. Conclusion We have developed a replacement VIP RIA. This assay has a lower limit of quantitation and a broader AMR than the current assay. Concordance with the current assay was acceptable.

Alterations in the spatiotemporal expression pattern of geminin during human epidermal morphogenesis

Geminin, a (25 kDa) protein, was originally identified as a key regulator of DNA replication licensing in the cell cycle and of cell fate during embryonic nervous system formation. Although geminin is involved in mechanisms underlying the regulation of transcription and patterning in embryonic development, its expression and possible significance in human epidermal morphogenesis remains unknown. Forty-one skin biopsy specimens obtained from human fetuses (10th to 23rd week of estimated gestational age) were processed for immunohistochemistry using a primary rabbit polyclonal antibody against geminin. Distinct and statistically significant qualitative and quantitative alterations in the spatiotemporal expression pattern of geminin were observed in the developing human epidermis. The highly ordered expression of geminin in different layers of fetal human epidermis reported here for the first time suggests that this protein may play a significant role in epidermal morphogenesis. However, the mechanisms underlying the alterations of the geminin expression pattern during fetal development at the molecular level remain to be elucidated. Further studies are now warranted to address whether the expression pattern of geminin in the developing human epidermis is disturbed in fetuses with genodermatoses and whether these disturbances might be important for prenatal diagnosis of genodermatoses.

Changes in the Expression of GAT1 (GABA transporter) in the Ventrolateral Part of the Solitary Tract Nucleus in Prenatal Serotonin Deficiency in Rats

Objectives. To study the expression of GAT1 (GABA transporter) at the early periods of postnatal development in the ventrolateral part of the solitary tract nucleus in prenatal serotonin deficiency in rats. Materials and methods. The endogenous serotonin level in the embryonic period was decreased by inhibition of tryptophan hydroxylase with para-chlorophenylalanine (PCPA) (Sigma, USA). GAT-1 transporter was detected using primary rabbit polyclonal antibodies (anti-GABA transporter 1, GAT1) (AbCam, UK). Results. From the early neonatal period to the beginning of juvenile age, the neuropil and the processes, terminals, and synaptic structures of the lateral and ventral parts of the solitary tract nucleus of control rats showed a gradual increase in GAT1 expression in experimental animals. The level of expression of GAT1 transporter was significantly greater in the lateral and ventral parts during postnatal weeks 1 and 2 than in controls, though by the end of week 3, i.e., by the beginning of juvenile age, it decreased to become significantly lower than in controls. Conclusions. The respiratory areas of the solitary tract nucleus in control rats in the first three weeks of postnatal development showed a gradual increase in GAT1 expression. Serotonin deficiency during the prenatal period led to impaired expression of GAT1 in the early postnatal period. These changes can lead to changes in GABA transmission, which may in turn be the cause of imbalance between inhibitory and excitatory effects in the respiratory center in the early postnatal period and, thus, may be the basis of the development of respiratory dysfunctions at early age.

2011P - Semiquantitative assessment of vimentin expression in prostate cancer (PC)

BackgroundVimentin (Vim) is one of the mesenchymal markers, that are often upregulated in cancer cells during epithelial-mesenchymal transition (EMT) – a process endowing tumor cells with invasiveness, pro-metastatic potential and, arguably, cancer stem cells (CSC) properties. It is normally seen in cytoplasm, but cell surface Vim was also detected on cells with CSC characteristics. Vim expression may be used as a surrogate marker of EMT in tumor tissue samples from patients and aid in prognosis evaluation. We aimed to characterize Vim expression in prostate cancer (PCa) using our method of semiquantitative staining assessment. MethodsVim expression was assessed in 48 cases of PCa using immunohistochemistry with primary rabbit polyclonal antibodies (ThermoFischer, 1:1000). UnoView Rabbit Detection Kit was used for visualization. Specificity of staining was confirmed by another antibody (mouse monoclonal, DAKO, 1:200) in selected cases. To assess Vim expression semiquantitatively we proposed a weighed staining index (WSI) that takes into account the proportion of stained cells as well as the ratios of staining intensities and can be applied to different cellular compartments staining separately. It combines advantages of simple ad oculus semiquantitative staining evaluation and obtaining numerical characteristics of marker expression. ResultsMedian WSI for membranous staining was 5 (0 – 52) (7, if cases without expression were excluded), for cytoplasmic – 20.5 (0 – 64) (22.5, if cases without expression were excluded). In 2 (4.2%) cases marker expression was absent in both membrane and cytoplasm. WSI in PCa was significantly higher for cytoplasmic staining. However, in 3 (6.25%) cases WSI for membranous staining was higher than that for cytoplasmic. Significant correlation was found between WSI for cytoplasmic and membranous staining (Spearman test, r=0.51, p<0,05). However, no correlation was found between WSI for both compartments and E-cadherin expression (as assessed by average staining intensity score – a method similar to WSI – using Aperio software previously). ConclusionsVim expression as a sign of EMT in the tumor is present in 95.8% cases of PCa and is more prevalent in cytoplasm. WSI is a useful tool to assess Vim (and possibly other markers) expression. Legal entity responsible for the studyM. Puchinskaya. FundingBelarusian Republican Foundation for Fundamental Research. DisclosureAll authors have declared no conflicts of interest.

Connexin 43 expression in human brain glial tumors

Тo conduct an immunohistochemical (IHC) study of the expression of connexin 43 in the samples of glial tumors of various grades: gemistocytic astrocytomas (Grade 2), oligodendrogliomas (Grade 2) and glioblastomas (Grade 4). The material investigated was fragments of human brain glial tumors (grade 2 gemistocytic astrocytomas (n=2), grade 2 oligodendrogliomas (n=2), and grade 4 glioblastomas (n=14) and those of tumor-surrounding tissue (n=4). The material was fixed in 10% buffered formalin, dehydrated, and embedded in paraffin according to the standard technique. IHC studies of the slices applied primary rabbit polyclonal antibodies against connexin 43 ('Spring Bioscience', USA) and the Dako EnVision + Peroxidase (DAB) visualization system ('Dako', Denmark). After the immunohistochemical reaction, the cell nuclei were stained with Mayer's hematoxylin. Immunohistochemistry showed the changing pattern of connexin 43 expression as compared with intact tissue in the glial tumors. Instead of the fine-granular expression in the thin cellular processes in the neuropil, the tumors mainly displayed a coarse-grained cytoplasmic and even nuclear reaction. The morphology and localization of positive structures depended on the variant of an examined tumor. In addition, the most malignant brain gliomas generally exhibited a reduction in the expression of connexin 43, i.e. its quantity is inversely proportional to the degree of malignancy of the tumor. The low connexin 43 expression levels may reflect both a reduction in astroglial functional gap junctions and semicanals and a decrease in the amount of the protein itself that has independently antioncogenic properties. The observed cytoplasmic and nuclear expression of connexin 43 is most likely to be associated with the aberrant activity of a number of kinases, such as proto-oncogene tyrosine-kinase Src or protein kinase C (PKC).

PP09.11 – 2460: Colocalization of HIF1 alpha and abnormal mitochondrial accumulations in muscle fibers in different neuromuscular diseases

Objective We perform the comparative study of immunohistochemical distribution of mitochondrial and hypoxia markers in abnormal mitochondrial clusters in striated muscle fibers of patients with myopathies. Such clusters, accordingly to recent data obtained by means of electron microscopy, play compensatory role in muscle tissue in different neuromuscular diseases. However, the regulatory impact's nature and the interaction with hypoxia are still unclear. Methods Fluorescence immunohistochemistry was performed. Primary mouse mitochondria monoclonal antibody, biotin conjugate (clone MTCO2) and Alexa Fluor 488 goat anti-mouse IgG as secondary antibody were used for mitochondria visualization. Visual image of HIF1 alpha was achieved by using primary rabbit polyclonal antibodies to HIF1 alpha and Alexa Fluor 555 goat anti-rabbit IgG secondary antibody. Paraffin embedded muscle tissue section slides obtained from 25 patients with mitochondrial myopathies, muscular dystrophies (DMD, merosinopathy, calpainopathy) and congenital myopathies (multicore, nemaline) were stained. Evaluation of the products is carried out by using The EVOS® FL Imaging System. Results Mitochondria and abnormal mitochondrial accumulations were visualized in subsarcolemmal zones of some muscle fibers using fluorescent immunohistochemistry. In the same muscle fibers we also observed increased expression HIF1 alpha; its localization matched with location of detected mitochondrial clusters, resulted in fluorescent lights overlapping and color change of subsarcolemmal zones luminescence. In the cases of muscular dystrophies it was observed that severity of pathological changes in muscles positively correlated with expression intensity of HIF1 alpha in the absence of mitochondrial marker expression. Conclusion The obtained results suggest that there is a colocalization of increased intracellular marker of hypoxia HIF1 alpha concentration and abnormal mitochondrial accumulations in different neuromuscular diseases. It can be explained by such clusters emerging in the areas of intracellular hypoxia. So hypoxia can be an important factor that stimulates mitochondrial proliferation, which may be important for tissue adaptation to different pathological conditions.

P161 – 2452: HIF1 alpha abnormal distribution in muscle fibers in the cases of nemaline myopathy

Objective The comparative study of immunohistochemical distribution of hypoxia marker HIF1 alpha and mitochondrial markers in striated muscle fibers of patients suffering from nemaline myopathy (NM) was performed for evaluation of tissue hypoxia's role in the pathogenesis of NM, characterized by rod-shaped structures, which originated from the Z-disks of the sarcomeres. Methods Fluorescence immunohistochemistry was performed. Primary mouse mitochondria monoclonal antibody, biotin conjugate (clone MTCO2) and Alexa Fluor 488 goat anti-mouse IgG as secondary antibody were used for mitochondria visualization. Visual image of HIF1 alpha was achieved by using primary rabbit polyclonal antibodies to HIF1 alpha and Alexa Fluor 555 goat anti-rabbit IgG as secondary antibody. Paraffin embedded muscle tissue section slides obtained from 3 NM patients were stained. Evaluation of the products is carried out by The EVOS® FL Imaging System. Results Mitochondria and abnormal mitochondrial accumulations were visualized in subsarcolemmal zones of some muscle fibers. In the same muscle fibers we observed increased expression of HIF1 alpha; its localization matched with location of detected mitochondrial clusters, resulted in fluorescent lights overlapping and color change of subsarcolemmal zones luminescence. Furthermore, the most intensive fluorescence and clearly delineated HIF1 alpha clusters were observed in the areas of rods accumulation, pointing pronounced tissue hypoxia in these regions. Conclusion While the diffuse subsarcolemmal zones of tissue hypoxia and increased number of mitochondrial clusters are often found in other myopathies, location of HIF1 alpha clusters in the rods areas is very unusual. Due to their brightness, HIF1 alpha clusters can be used in the quantification of muscular disorders severity at low magnification of fluorescence microscopy. No evidence whether these changes are secondary to rods accumulation or vice versa was found in the literature. In this way immunomorphological characterisation of energy muscle tissue metabolism of patients suffering from NM is of the current interest.

Abstract 393: Chymase: Augmented Renal Tubular Protein Expression in Diabetes

Chymase is a serine protease that is predominantly found stored in granules of mast cells. Chymase expression is increased in mesangial cells, podocytes, vascular smooth muscle cells, and cardiomyocytes under hyperglycemic culture conditions. We have presented evidence for enhanced intrarenal chymase-dependent Ang II formation on afferent arteriole vasoconstriction in the diabetic kidney ( Hypertension. 56: e55, 2010). We tested the hypothesis that renal chymase protein expression is enhanced in the diabetic kidney. Kidneys were harvested from littermate control ( db/m ) and type II diabetic ( db/db ) mice at 36-wk-old. Body weight was significantly lower (33 ± 3 vs 40 ± 2 g), while plasma glucose was significantly higher (552 ± 40 vs 220 ± 21 mg/dl) in diabetic (n=6) compared to control (n=7) mice. However, plasma insulin levels were not different from controls (0.77 ± 0.10 vs 0.43 ± 0.11 mg/L), indicating that the diabetic mice are transitioning from type II to type I diabetic disease. Immunohistochemistry was performed using primary rabbit polyclonal antibodies [mMCP-4 (chymase, A Husain), anti-malignant T cell amplified sequence 1 (chymase, Abcam), H + -ATPase (D Brown), and aquaporin 2 (AQP2, J Sands)]. Renal subcapsular and adipose tissue mast cell granules stained positively for toluidine blue and chymase. Chymase protein expression was localized to the apical membrane of connecting tubules and cortical collecting ducts of both control and diabetic kidneys. Chymase was co-localized to AQP2 positive and H + -ATPase negative cells indicating that chymase is specifically expressed in the principal cells of the distal nephron. Prominent chymase immunostaining was observed in the renal papilla and endothelial cells of renal arteries and arterioles in all diabetic, but none of the control kidneys. Chymase was not localized to renal vascular smooth muscle cells. The renal localization of chymase was identical using custom and commercially available polyclonal antibodies. These are the first studies to identify chymase protein expression in the principal cells of the distal nephron. These novel data suggest that the serine protease actions of chymase may be linked to altered epithelial transport mechanisms in the distal nephron in diabetic kidney disease.

Localization of thyrotropin receptor and thyroglobulin in the bovine corpus luteum

The receptor of the Thyroid Stimulating Hormone (TSHr) and thyroglobin (TGB), are two proteic factors necessary for the synthesis of hormones, in the thyrocite. In mammals, many immuno-histochemical reports indicate the presence of the TSHr in extra-thyroidal tissues, but not in the ovary. Triiodothyronine (T 3) and thyroxine (T 4) have been widely shown to affect ovarian functions and the synthesis of progesterone (P 4). The aim of this study was to determine if by immunohistochemistry techniques TSHr and TGB could be found in the bovine corpora haemorragica, lutea and albicantia. A primary rabbit polyclonal antibody against human TSHr and a primary rabbit polyclonal antibody against human TGB were employed. Furthermore, the accuracy of bovine thyroid to the antibodies used in this study was tested. A positivity reaction for the anti-TSHr serum in the large luteal cells and immunostaining of both small and large luteal cells with the anti-TGB serum occurred only in mature corpora lutea. No immunostaining was detected in stromal cells, blood and lymphatic vessels and in corpora haemorragica and albicantia. Bovine thyroid tissue showed immunostaining to both the antibodies employed. These data suggest that the luteal cells of mature corpora lutea may be involved in the synthesis of thyroid hormones, which may modulate P 4 synthesis, acting in an autocrine and paracrine way.

Unimpaired Lysosomal Acidification in Respiratory Epithelial Cells in Cystic Fibrosis

The mechanisms remain uncertain by which mutations in CFTR cause lung disease in cystic fibrosis (CF). Teichgräber et al. recently reported increased ceramide in CF lungs, which was proposed to result from defective lysosomal acidification in airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgräber, V., Ulrich, M., Endlich, N., Reithmüller, J., Wilker, B., Conceição Ce Olivereira-Munding, C., van Heeckeren, A. M., Barr, M. L., von Kürthy, G., Schmid, K. W., Weller, M., Tümmler, B., Lang, F., Grassme, H., Döring, G., and Gulbins, E. (2008) Nat. Med. 14, 382-391). Here, we measured lysosomal pH in several CFTR-expressing and -deficient cell lines, freshly isolated airway epithelial cells from non-CF and CF mice and humans, and well-differentiated primary cultures of human non-CF and CF airway epithelial cells. Lysosomal pH was measured by ratio imaging using a fluorescent pH indicator consisting of 40-kDa dextran conjugated to Oregon Green 488 and tetramethylrhodamine. In all cell types, lysosomal pH was approximately 4.5, unaffected by the thiazolidinone CFTR inhibitor CFTR(inh)-172, and increased to approximately 6.5 following bafilomycin inhibition of the vacuolar proton pump. Lysosomal pH did not differ significantly in airway epithelial cells from non-CF versus CF humans or mice. Our results provide direct evidence against the conclusions of Teichgräber et al. that lysosomal acidification is CFTR-dependent, impaired in CF, or responsible for ceramide accumulation. As such, alternative mechanisms are needed to explain increased ceramide in CF airways. Non-CFTR mechanisms, such as ClC-type chloride channels, are likely involved in maintaining electroneutrality during organellar acidification.

Intracellular localization of “soluble” adenylate cyclase in potato plants

In animal cells, a key enzyme of the adenylate cuclase system, adenylate cyclase (AC), is represented by its transmembrane (tmAC) and soluble (sAC) forms [1]. Earlier, we have established that both forms of this enzyme are present in plants as well (potato), in nuclei and chloroplasts [2]. Using immunoprecepitation and Western blotting with antibody against animal sAC, we have found five sAC isoforms with mol wts of 225, 193, 92, 68, and 60 kD [3]. However, it is not clear until now, whether sAC is present only in nuclei and chloroplasts or it is localized also in other cell compartments, which could provide for more rapid transduction of external signals. Therefore, the objective of our work was to analyze intracellular sAC localization at the level of the cell ultrastructure. In this study, we applied for the first time the method of immuno-electronic cytochemistry for the studying of sAC intracellular localization in various potato tissues and its re-dislocation in the response to biotic stress. Tube-grown potato plantlets of two cultivars contrasting in their resistance to the causal agent of the ring rot were used in experiments: cv. Lugovskoi, resistant, and cv. Luk’yanovskii, susceptible. The root segments and leaf pieces from the upper part of the shoot of control plants (with roots not treated with extracellular polysaccharides (EPS) of the pathogen) and experimental plants kept for 2 h in the 0.1% EPS solution were fixed for 2 h in the mixture of 4% formaldehyde and 1% glutaraldehyde (Sigma, United States) prepared in 0.1 M Na-phosphate buffer, pH 7.2. Thereafter, samples were postfixed in 0.8% OsO 4 in the same buffer (2 h), dehydrated by the routine procedure, and embedded in the mixture of epoxide resins, Epon and Araldite. Immunocytochemistry was performed on ultrathin sections mounted on nickel grids, using primary rabbit polyclonal antibodies (PAB) against animal sAC at 1 : 200 dilution conjugated with 10-nm colloidal gold particles and secondary goat antibodies against rabbit immunoglobulins (SAB, Sigma, United States) at 1 : 30 dilution. All procedures were performed as described in [4], with the exception of the usage of the hoarse serum at 1 : 5 dilution as a blocking solution. Two control treatments were applied: (a) ultrathin sections not treated with PAB and (b) treatment with nonimmune rabbit serum instead PAB and then with SAB saturated with such serum. Specimens stained with uranyl acetate were examined with an LEO 906E electron microscope (Carl Zeiss, Germany), and the images were shown on the computer display. To evaluate sAC partition between cell compartments, we counted the number of gold particles in them. In each treatment, we analyzed 25 images; the total number of particles on the area of each compartment was calculated per 1 µ m 2 . The results were processed statistically. We observed the presence of SAB, reflecting sAC localization, in many intracellular compartments. Thus, in the cells of the root meristem and elongation zone, gold particles were seen in the cytosol, mitochondria,

Semiautomated Multiplexed Quantum Dot-Based in Situ Hybridization and Spectral Deconvolution

Gene expression profiling has identified several potentially useful gene signatures for predicting outcome or for selecting targeted therapy. However, these signatures have been developed in fresh or frozen tissue, and there is a need to apply them to routinely processed samples. Here, we demonstrate the feasibility of a potentially high-throughput methodology combining automated in situ hybridization with quantum dot-labeled oligonucleotide probes followed by spectral imaging for the detection and subsequent deconvolution of multiple signals. This method is semiautomated and quantitative and can be applied to formalin-fixed, paraffin-embedded tissues. We have combined dual in situ hybridization with immunohistochemistry, enabling simultaneous measurement of gene expression and cell lineage determination. The technique achieves levels of sensitivity and specificity sufficient for the potential application of known expression signatures to biopsy specimens in a semiquantitative way, and the semiautomated nature of the method enables application to high-throughput studies.

185 EXPRESSION AND LOCALIZATION OF µ-OPIOID RECEPTOR IN CANINE OOCYTES

Endogenous opioid peptides (EOP), through G-protein-coupled receptors, control metabolism and many physiological and pathological conditions. Once EOP are linked to their receptors, above all µ-opioid receptor (MOR), a block of the Ca2+ channel occurs (Sciorsci et al. 2000 Immunopharm. Immunotox. 22, 575–626). The disruption of Ca2+ homeostasis interferes with many Ca2+-mediated/dependent actions. Our previous studies demonstrated the presence of MOR in human, bovine, and equine oocytes, in sperm cells of several species (equine, canine, etc.), in mare's tube, in ovine, bovine and mouse embryos. The presence of MOR on the male canine gamete lets us hypothesize its presence on the female gamete, too. In this study we demonstrated the presence of MOR on canine oocytes by immunofluorescence (IF) and western blot (WB) analysis, and we speculate on its possible functional role. Canine ovaries were obtained from healthy bitches randomly chosen among those arriving at our veterinary hospital for surgical ovariectomy without considering the period of their reproductive cycle. Oocytes were collected by ovary slicing and tested to check for the presence of MOR. For IF, oocytes were washed in 100 mm glycine in PBS and incubated for 30 min in PBS-1% BSA. Control oocytes were incubated with primary rabbit polyclonal antibody against the rat 3rd extracellular loop of MOR (Chemicon, Temecula, CA, USA). All oocytes were incubated for 2 h at room temperature with a FITC-conjugated anti-rabbit IgG-secondary antibody diluted 1:200 in Evans blue/PBS, washed, and visualized by laser scanning confocal microscope. For the WB, crude plasma membranes were obtained from pools of oocytes. They were lysed in Laemli buffer and loaded on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. After electrophoresis, proteins were electrotransferred (semi-dry apparatus, BioRad, Milano, IT) to Immobilon-P membranes (Millipore, Bedford, MA, USA). Filters were blocked for 1 h and blotted overnight at 4�C against the same primary antibody used for IF, diluted 1:7500 in blocking buffer. After washing, membranes were incubated with a 1:10 000 dilution of peroxidase-conjugated goat anti-rabbit IgG secondary antibody for 2 h at room temperature. Reactive bands were visualized by Supersignal West Pico Chemiluminescent substrate (Pierce, Milano, IT). A negative control was performed. The IF highlighted, by clear brilliant green, the MOR's localization on canine oocytes. The negative control did not present any fluorescent region or spotted coloring. The WB revealed the presence of one immunoreactive band of approximately 65 kDa, thus confirming the results obtained by IF. No reactivity was evident when the primary antibody was adsorbed with an excess of immunizing peptide. The presence of MOR on canine oocytes indicates its possible role in the modulation of oocyte metabolism. These data strongly confirm previous evidence from our research unit on the involvement of the opioidergic system during gamete development and interaction, thus allowing us to speculate on a primary role of MOR in controlling key events of the reproductive activity.

Isolation and Functional Characterization of a Novel Organic Solute Carrier Protein, hOSCP1

We succeeded in isolating a novel organic solute carrier from a human placenta cDNA library. The isolated cDNA consisted of 1137 base pairs that encoded a 379-amino acid protein, hOSCP1. Northern blot and reverse transcription PCR analyses revealed that the hOSCP1 mRNA is expressed in the placenta and testis and weakly expressed in the thymus and small intestine. When expressed in Xenopus laevis oocytes, hOSCP1 mediated the high affinity transport of p-aminohippurate (PAH) (K(m) = 35.0 +/- 7.5 microm) and tetraethylammonium (K(m) = 62.3 +/- 12.2 microm) in a sodium-independent manner. However, the hOSCP1-expressing oocyte did not mediate the transport of L-carnitine. The transport of PAH by hOSCP1 was sensitive to pH, but the tetraethylammonium was not transported at the high pH examined. hOSCP1 transported prostaglandin E(2), prostaglandin F(2alpha), estrone sulfate, glutarate, L-leucine, L-ascorbic acid, and tetracycline. Thus, hOSCP1 also showed broad substrate specificity. A wide range of structurally unrelated organic compounds inhibited the hOSCP1-mediated PAH uptake. Immunohistochemical analysis revealed that the hOSCP1 protein is localized in the basal membrane of the syncytiotrophoblast in the human placenta. Our results suggest that hOSCP1 is a novel polyspecific organic solute carrier protein responsible for drug clearance from the human placenta.

Role of Protein Phosphatase 2A in mGluR5-regulated MEK/ERK Phosphorylation in Neurons

The regulation of protein phosphorylation requires coordinated interaction between protein kinases and protein phosphatases (PPs). Recent evidence has shown that the Galphaq-protein-coupled metabotropic glutamate receptor (mGluR) 5 up-regulates phosphorylation of MAPK/ERK1/2. However, signaling mechanisms linking mGluR5 to ERK are poorly understood. In this study, roles of a major serine/threonine PP, PP2A, in this event were evaluated in cultured neurons. We found that the PP1/2A inhibitors okadaic acid and calyculin A mimicked the effect of the mGluR5 agonists (RS)-3,5-dihydroxyphenylglycine and (RS)-2-chloro-5-hydroxyphenylglycine in facilitating phosphorylation of ERK1/2 and its upstream kinase, MEK1/2, in a PP2A-dependent but not PP1-dependent manner. Co-administration of either inhibitor with an mGluR5 agonist produced additive phosphorylation of ERK1/2. Enzymatic assays showed a basal level of phosphatase activity of PP2A under normal conditions, and activation of mGluR5 selectively inhibited PP2A, but not PP1, activity. In addition, a physical association of the cytoplasmic C terminus of mGluR5 with PP2A was observed, and ligand activation of mGluR5 reduced mGluR5-PP2A binding. Additional mechanistic studies revealed that mGluR5 activation increased tyrosine (Tyr307) phosphorylation of PP2A, which was dependent on activation of a p60c-Src family tyrosine kinase, but not the epidermal growth factor receptor tyrosine kinase and resulted in dissociation of PP2A from mGluR5 and reduced PP2A activity. Together, we have identified a novel, mGluR5-triggered signaling mechanism involving use- and Src-dependent inactivation of PP2A, which contributes to mGluR5 activation of MEK1/2 and ERK1/2.

Calpain I Induces Cleavage and Release of Apoptosis-inducing Factor from Isolated Mitochondria

The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.

LEKTI demonstrable by immunohistochemistry of the skin: a potential diagnostic skin test for Netherton syndrome

Netherton syndrome (NS) is a rare autosomal recessive condition characterized by ichthyosiform erythroderma, trichorrhexis invaginata and atopic manifestations. Confirming the diagnosis may be difficult in the early stages. Mutations in the SPINK5 gene which encodes for the serine protease inhibitor LEKTI are associated with NS. These mutations create premature termination codons which result in absent or abnormal expression of LEKTI in patients with NS. To investigate the expression of LEKTI in the skin of patients with NS in comparison with normal controls and patients with other skin conditions, namely atopic dermatitis, psoriasis and nonbullous ichthyosiform erythroderma. Immunohistochemistry was performed on skin sections from four patients with NS, four normal controls, four with atopic dermatitis, two with psoriasis and two with nonbullous ichthyosiform erythroderma, using a primary rabbit polyclonal antibody against LEKTI. LEKTI was localized to the stratum granulosum in normal skin. All four skin sections from patients with NS showed absent or very reduced staining for LEKTI. Staining in the other disorders showed positive LEKTI expression in varying patterns. NS can be difficult to diagnose especially in the early stage, which can lead to inappropriate treatments particularly if it is misdiagnosed as atopic dermatitis. Immunohistochemistry of skin with an antibody against LEKTI is a potentially useful diagnostic test for NS.

Presence and localization of neurotrophins and neurotrophin receptors in rat lacrimal gland.

To determine which of the neurotrophins (NTs)-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT4)-and their receptors (NTrs), TrkA, TrkB, TrkC, and p75, are present in the adult rat lacrimal gland. RT-PCR was performed on RNA isolated from male rat lacrimal gland, using oligonucleotides specific to each NT and NTr. The presence of NT and NTr protein, was determined by Western blot analysis of lacrimal gland homogenate or membranes. The location of NTs and NTrs was determined by immunofluorescence histochemistry. Western blot analyses and immunofluorescence microscopy were performed using primary rabbit polyclonal antibodies raised against NTs and NTrs. RT-PCR showed positive bands at the appropriate sizes for NGF, BDNF, NT3, and NT4, and for the receptors TrkA, TrkB, TrkC, and p75. Western blot analysis confirmed these results, showing that the lacrimal gland expresses NGF, BDNF, NT3, and NT4 as well as the NTrs TrkA, TrkB, and TrkC and the p75 protein. NGF, BDNF, NT3, and NT4 were localized in the lacrimal gland acini with differing cellular distributions, whereas TrkA, TrkB, and TrkC, were localized in myoepithelial cell and ductal cell membranes. The protein p75 was expressed only on myoepithelial cell membranes. Members of the neurotrophin family of growth factors and their receptors are present in rat lacrimal gland, which suggests a role for NTs and their receptors in the lacrimal gland.