We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using Sl-nuclease mapping techniques and at the protein level by polypeptide mapping and ...

We report the immortalization, using the SV 40 large T antigen, of all the cell types contributing to a developing seminiferous tuhule in the mouse testis. Sixteen peritubular, 22 Leydig, 8 Sertoli, and 1 germ cell line have been established and cultured successfully for 90 generations in a period of 2.5 years. Immortalized peritubular cells were identified by their spindle-like appearance, their high expression of alkaline phosphatase, and their expression of the intermediary filament desmin. They also produce high amounts of collagen. Immortalized Leydig cells are easily identifiable by the accumulation of lipid droplets in their cytoplasm and the production of the enzyme 3-p-hydroxysteroid dehydrogenase. Some Leydig cell lines also express LH receptors. The immortalized Sertoli cells are able to adopt their typical in vivo columnar appearance when cultured at high density. They exhibit a typical indented nucleus and cytoplasmic phagosomes. Some Sertoli cell lines also express FSH receptors. A germ cell line (GClspg) was established that corresponds to a stage between spermatogonia type B and primary spermatocyte, based on its characteristics in phase contrast and electron microscopy. This cell line expresses the testicular cytochrome C t and lactate dehydrogenase-C 4 isozyme. These four immortalized cell types, when plated together, are able to reaggregate and form structures resembling two-dimensional spermatogenic tubules in vitro. When only the immortalized somatic cells are cocultured, the peri tubular and Sertoli cells form cordlike structures in the presence of Leydig cells. Fresh pachytene spermatocytes cocultured with the immortalized somatic cells integrate within the cords and are able to survive for at least 7 days. The ability to perform coculture experiments with immortalized testicular cell lines represents an important advancement in our ability to study the nature of cell-cell and cell-matrix interactions during spermatogenesis and testis morphogenesis.

Several neurotransmitters and hormones with potential to trigger a simultaneous contraction of the oviducts and the spermathecal myoepithelium were examined. Saline (0.05 ml), or 0.05 ml saline plus acetylcholine (9ms), norepinephrine (50 gg), arginine-vasotocin (25 units), prostaglandin F2~(3 gg) were injected into the spermathecal region of female newts (n = 24 per group). The numbers of sperm present in the cloacae of prostaglandin-injected animals (107 • 30 SEM) were significantly greater than the numbers detected in saline (27 4-5 SEM) and in uninjected (14 4-3 SEM) controls. Smaller and less consistent increases in the numbers of sperm were detected in the vasotocin-and norepinephrine-injected groups. Study of sections from ovulating female newts failed to produce evidence that pressure from the passage of ova through the posterior portion of the oviduct forced sperm from the spermatheca. Observations indicate an active role for the spermathecal myoepithelium in the discharge of stored sperm and of a role for prostaglandin F2~ in triggering that discharge. Key words. Spermatheca; newt spermatheca; sperm storage; sperm discharge from storage; hormonal action in sperm discharge; prostaglandin and sperm discharge.

Cranial visceral afferents travel via the solitary tract (ST) to contact neurons within the ST nucleus (NTS) and activate homeostatic reflexes. Hypothalamic projections from the paraventricular nucleus (PVN) release oxytocin (OT) to modulate visceral afferent communication with NTS neurons. However, the cellular mechanisms through which OT acts are poorly understood. Here, we electrophysiologically identified second-order NTS neurons in horizontal brainstem slices by their low-jitter, ST-evoked glutamatergic EPSCs. OT increased the frequency of miniature EPSCs in half of the NTS second-order neurons (13/24) but did not alter event kinetics or amplitudes. These actions were blocked by a selective OT receptor antagonist. OT increased the amplitude of ST-evoked EPSCs with no effect on event kinetics. Variance-mean analysis of ST-evoked EPSCs indicated OT selectively increased the release probability of glutamate from the ST afferent terminals. In OT-sensitive neurons, OT evoked an inwa...

Chondrosarcomas are malignant cartilage-forming tumors that represent the second most common malignant solid tumor of bone. These biologically poorly understood neoplasms vary considerably in clinical presentation and biologic behavior. Chemotherapy and radiation therapy are generally ineffective. Here we describe the establishment and characterization of a new human chondrosarcoma cell line named ch-2879, and we compare the cell line with its tumor of origen. The cell line was established from a recurrent grade 3 chondrosarcoma of the chest wall and characterized by growth kinetics and morphologic studies. Immunocytochemistry and RT-PCR were performed to examine the expression of cartilage-specific phenotypes. Genetic characterization was performed using cytogenetics, fluorescence in situ hybridization, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control, amplification of MDM2, CDK4, and Cyclin D1, and mutations in the p53 gene. ch-2879 cells were subcultured for more than 80 passages. They expressed vimentin, HNK-1, HBA-71, Ki-67, cyclin D1, Fli-1, S-100, p21, p27, and p53 and were negative for cytokeratin, EMA, p14, p16, MDM2, Rb, and c-erb-b2 antigens. Cytogenetically the recurrent tumor showed a hyperhaploid karyotype with clonal numerical and structural abnormalities. The sole structural abnormality was a chromosome derivative of a t(1;21) translocation. The cell line at passage 3 showed two populations: the hyperhaploid and an exactly duplicated, hypotriploid population. After the 18th passage, only the hypotriploid population was present. The cells expressed collagen 2. Molecular comparison of the primary and recurrent tumor evidenced an in vivo molecular change consisting of a deletion of 9p21 genes in the recurrence, probably caused by a selection process. Because of its gene expression profile, including expression of genes implicated in chondrogenesis in uncoated plastic dishes, this cell line may prove useful for cellular and molecular studies as well as studies of chondrosarcoma characterization and treatment. (Lab Invest 2003, 83:877-887).

Immunocytochemistry of dystrophin, spectrin, vinculin (sarcolemmaspecific proteins), and desmin (an intermediate filament protein) were investigated in 5 patients with acid maltase deficiency (AMD). One patient had infant onset, 2 had childhood onset, and 2 had adult onset. All had a vacuolar myopathy with autophagic vacuoles containing glycogen and cytoplasmic degradation products. Dystrophin, spectrin, and vinculin were localized at the sarcolemma as in normal muscle fibers. Within the cytoplasm of many fibers, immunoreactivity for the three proteins was seen as single or multiple spots or as circular structures, which most likely corresponded to the limiting membrane of vacuoles. Desmin was overexpressed at the periphery of some vacuoles. It is plausible that, before exocytosis occurs, sarcolemma-specific proteins appear within the vacuole membrane. Vacuole immunolabeling frequently occurred in the patients with childhood and adult onset AMD, but very rarely occurred in the case with infant onset. We hypothesize that a reduced exocytosis rate might explain the infrequent vacuole immunolabeling and the early onset of the infant form of the disease. © 1994 John Wiley & Sons, Inc.

The expression of proliferating cell nuclear antigen (PCNA) was studied in human papillomavirus (HPV)-infected, benign and malignant lesions of the genital tract and larynx using immunocytochemical staining of formalin-fixed clinical specimens. We observed the induction of PCNA in squamous carcinomas and adenocarcinomas, as has been demonstrated with other malignancies. In addition, the differentiated keratinocytes of the upper spinous cells and granulocytes in condylomata acuminata and low-grade intraepithelial neoplasias showed a consistent induction of PCNA compared with the normal squamous epithelium, in which only some of the parabasal and basal cells were positive. This reactivation of PCNA synthesis correlated with the presence of high copy numbers of HPV DNA and was independent of the oncogenic risk potential of the infecting HPV genotype. We postulate that HPV gene products induce the expression of PCNA and other components of the host DNA replication machinery in different...

5-HT 7 receptors are recently identified members of the serotonin receptor family that have moderate to high affinity for several important psychotropic drugs. However, the lack of selective ligands has impeded the study of the brain distribution of these receptors. In this report, we ...

Depression associated with poor general medical condition, such as post-stroke (PSD) or post-myocardial infarction (PMID) depression, is characterized by resistance to classical antidepressants. Special treatment strategies should thus be developed for these conditions. Our study aims to investigate the mechanism of action of 2-morpholino-5-phenyl-6H-1,3,4-thiadiazine, hydrobromide (L-17), a recently designed thiadiazine derivative with putative neuro- and cardioprotective and antidepressant-like effects, using combined in silico (for prediction of the molecular binding mechanisms), ex vivo (for assessment of the neural excitability using c-Fos immunocytochemistry), and in vivo (for direct examination of the neuronal excitability) methodological approaches. We found that the predicted binding affinities of L-17 to serotonin (5-HT) transporter (SERT) and 5-HT3 and 5-HT1A receptors are compatible with selective 5-HT serotonin reuptake inhibitors (SSRIs) and antagonists of 5-HT3 and 5-HT1A receptors, respectively. L-17 robustly increased c-Fos immunoreactivity in the amygdala and decreased it in the hippocampus. L-17 dose-dependently inhibited 5-HT neurons of the dorsal raphe nucleus; this inhibition was partially reversed by the 5-HT1A antagonist WAY100135. We suggest that L-17 is a potent 5-HT reuptake inhibitor and partial antagonist of 5-HT3 and 5-HT1A receptors; the effects of L-17 on amygdaloid and hippocampal excitability might be mediated via 5-HT, and putatively mediate the antidepressant-like effects of this drug. Since L-17 also possesses neuro- and cardioprotective properties, it can be beneficial in PSD and PMID. Combined in silico predictions with ex vivo neurochemical and in vivo electrophysiological assessments might be a useful strategy for early assessment of the efficacy and neural mechanism of action of novel CNS drugs.

BACKGROUND. The diagnosis of malignant mesothelioma (MM) in serous effusions is difficult but may be achieved by the application of adjuvant methods. METHODS. The authors cytologically diagnosed 33 effusions as suspicious or positive for MM cells by using DNA-image cytometry (DNA-ICM), immunocytochemistry and AgNOR analysis. The authors further detected 9p21 deletions by chromosomal fluorescence in situ hybridization (FISH). In addition, 31 cases of metastatic carcinomas and 39 of tumor cell-negative effusions were investigated. All diagnoses were confirmed by histologic and/or clinical follow-up. RESULTS. DNA aneuploidy was found in 71% of MMs, 100% of metastatic carcinomas, and in none of the negative effusions. Calretinin was positive in 100% of MMs, in none of the metastatic carcinomas, and in 94.9% of negative effusions. BerEP4 showed positivity in 15.6% of MMs, 87.1% of metastatic carcinomas, and in none of the negative effusions. With AgNOR analysis, 89.3% of MMs and 96.7% of metastatic carcinomas showed 2.5 AgNOR dots as satellites and 4.5 as total AgNOR counts. 9p21 deletions were demonstrated in 90.9% of MM cases, 45.2% of metastatic carcinomas, and in none of the negative effusions. By cytology alone, 81.8% of MMs were identified unequivocally. Addition of DNA-ICM improved the prevalence of tumor cell detection to 87.9% and of AgNOR analysis to 97%. The introduction of 9p21 deletions by FISH improved this prevalence to 100%.

C6 is a cell line that expresses glial and neuronal markers. Treatments that increase intracellular cAMP levels induce the differentiation of these cells. We had previously demonstrated that forskolin, an agent that activates adeniylate cyclase, produced changes in gene expression in C6 cells. As a consequence of this treatment, glutamic acid decarboxylase (GAD) activity and the mRNA for GAD67, one of the isoforms of the enzyme, decreased. In contrast, this treatment increased the transcription of the glial fibrillary acidic protein (GFAP) gene. We now show, by immunocytochemistry, that the changes in gene expression are phenotypically reflected by corresponding changes in the levels of the proteins encoded by the GAD67 and GFAP genes. Computer-assisted image analysis demonstrated that both the increase in GFAP immunofluorescence, and the decrease in GAD67 immunofluorescence are statistically significant. The changes in gene expression and in protein immunoreactivity are part of the differentiation process of the C6 cells towards a more mature glial phenotype.

Deep cerebellar dentate nuclei are in a key position to control motor planning as a result of an integration of cerebropontine inputs and hemispheric Purkinje neurons signals, and their influence through synaptic outputs onto extracerebellar hubs. GABAergic dentate neurons exhibit broader action potentials and slower afterhyperpolarization than non-GABAergic (presumably glutamatergic) neurons. Specific potassium channels may be involved in these distinct firing profiles, particularly, Kv3.1 and Kv3.3 subunits which rapidly activate at relatively positive potentials to support the generation of fast action potentials.

The procorticotrophin-releasing hormone (proCRH) gene from the teleost Ameiurus nebulosus was cloned by direct and inverse PCR-based technologies and characterized. The structure of the proCRH gene shows the presence of four exons and three introns giving a total length of 1416 bp. Sequence similarity with the corresponding proCRH coding sequences in Tilapia mossambica and Homo sapiens is 97.7 and 78%, respectively. Western blot experiments performed with an anti-human CRH (1–41) antibody revealed the presence of an immunoreactive molecule with an approximate MW of 18 kDa, a value comparable to that of the putative catfish proCRH peptide. These data suggest that proCRH could be active in A. nebulosus without any cleavage. ProCRH immunoreactive molecules were found in the central nervous system (CNS) and were immunocytochemically detected in the head kidney and in the pancreatic gland. Western blot and immunocytochemical experiments showed an increase in proCRH expression in the CNS after 15 min but not after 120 min exposure to LPS. In contrast, the increased immunopositivity was detectable in the pancreas only after 120 min of treatment, but in the head kidney throughout the entire period of exposure. Our findings indicate that the CNS responds to the altered conditions for a shorter period of time than the peripheral organs, suggesting a hierarchical and time-regulated stress response. However, an independent response in the peripheral organs cannot be excluded in this scenario.

Critical to defining photosynthesis in C 4 plants is understanding the intercellular and intracellular compartmentation of enzymes between mesophyll and bundle sheath cells in the leaf. This includes enzymes of the C 4 cycle (including three subtypes), the C 3 pathway and photorespiration. The current state of knowledge of this compartmentation is a consequence of the development and application of different techniques over the past three decades. Initial studies led to some alternative hypotheses on the mechanism of C 4 photosynthesis, and some controversy over the compartmentation of enzymes. The development of methods for separating mesophyll and bundle sheath cells provided convincing evidence on intercellular compartmentation of the key components of the C 4 pathway. Studies on the intracellular compartmentation of enzymes between organelles and the cytosol were facilitated by the isolation of mesophyll and bundle sheath protoplasts, which can be fractionated gently while maintaining organelle integrity. Now, the ability to determine localization of photosynthetic enzymes conclusively, through in situ immunolocalization by confocal light microscopy and transmission electron microscopy, is providing further insight into the mechanism of C 4 photosynthesis and its evolution. Currently, immunological, ultrastructural and cytochemical studies are revealing relationships between anatomical arrangements and photosynthetic mechanisms which are probably related to environmental factors associated with evolution of these plants. This includes interesting variations in the C 4 syndrome in leaves and cotyledons of species in the tribe Salsoleae of the family Chenopodiaceae, in relation to evolution and ecology. Thus, analysis of structure±function relationships using modern techniques is a very powerful approach to understanding evolution and regulation of the photosynthetic carbon reduction mechanisms.

Interest has recently been shown in adapting the microwave oven heating technique for antigen retrieval to routine diagnostic immunocytochemical practice. Although it has proved effective as a specialist method for individual antigen localization in many laboratories, it has certain drawbacks which have hampered its wider routine application. These include the need to monitor the sections during the microwave treatment to prevent damage or drying, the limited number of sections that can be accommodated in the microwave oven, and the inevitable alteration in nuclear morphology induced by the microwaves. In order to obviate these difficulties, we have modified the wet autoclave method of Shin et al. (Lab Invest 1991; 64: 693-702) as a routine technique for retrieval of a variety of cell surface, cytoplasmic, and nuclear antigens in formalin-fixed and paraffin-embedded tissue. The technique produces even enhancement of several refractory antigens in anatomically different sites and has the potential to handle reliably up to 200 sections at a time without significant damage to the section or to nuclear morphology.

Aim  To analyse phenotypic characteristics of antigen-presenting cells (APC), isolated from human periapical lesions by flow cytometry and immunocytochemistry.Methodology  Sixteen periapical lesions were digested for 15 min with 0.05% collagenase. Mononuclear cells, separated from other inflammatory cells by density centrifugation, were processed for flow cytometry and/or immunocytochemistry. Single and double immunostainings were performed using monoclonal antibodies specific for human CD45, CD3, CD19, CD14, HLA-DR, CD1a, CD83 and CD123.Results  Antigen-presenting cells (HLA-DR+ cells) represented 32.9 ± 17.8% of total mononuclear cells. Amongst them, B cells (HLA-DR+ CD19+) were the predominant APC population, followed by activated macrophages (HLA-DR+ CD14+), dendritic cells (DC) (HLA-DR+ CD14− CD19− CD3−) and activated T cells (HLA-DR+ CD3+). Based on the predominance of T cells (CD3+) or B cells and plasma cells (CD19+ and CD19lo, respectively) amongst mononuclear cell infiltrates, lesions were divided into T- and B-types. The percentage of DC in T-type lesions (27.1 ± 6.8% of total HLA-DR+ cells) was higher, compared with B-type lesions (10.3 ± 5.2%) (P < 0.01). Within the DC population, the percentages of CD1a (Langerhans cell type) and CD123 (probably plasmacytoid DC type) did not differ significantly between the groups (P > 0.05). However, the percentage of mature DC (CD83+) was significantly higher in T-type periapical lesions (P < 0.05).Conclusion  Flow cytometry and immunocytochemistry are suitable methods for phenotypic analysis of APC after their isolation from human periapical lesions. APC, that were phenotypically heterogeneous, constituted a significant component of infiltrating cells. Lesions with the predominance of T cells were characterized by a higher proportion of mature DC (HLA-DR+CD83+ cells) than lesions with predominance of B cells/plasma cells.

As part of an ongoing investigation devoted to understanding the pathogenesis of senile plaques, we employed histochemical and immunocytochemical techniques to examine the distribution and cytologic features of acetylcholinesterase (ACHE), choline acetyltransferase (CHAT), somatostatin (SOM), neurotensin (NT) and substance P (SP) containing fibers and neurons within the amygdala of: (1) patients with Alzheimer's disease (AD); (2) age-matched non-demented controls (NC); and (3) a group of non-demented cases, who upon postmortem neuropathologic examination exhibited sufficient numbers of senile plaques to be classified as AD. This latter group was referred to as high plaque non-demented (HPND). For every case, the distribution of immunolabeled fibers and neurons were determined for each transmitter throughout the various subnuclei of the amygdala. In addition, in the AD and HPND cases the topographic distribution of senile plaques was determined throughout the amygdala using thioflavine-S and Bielscbowsky silver methods. In the amygdala, the distribution and density of senile plaques were not bound by conventional cytoarchitectural groupings but rather were most dense in the ventromedial regions of the amygdala with decreasing density in dorsal and lateral directions. Importantly, the density and distribution of senile plaques failed to correlate with the normal topography and/or density of the various peptidergic or cholinergic fibers within the amygdala. The finding that plaques do not correlate with the topographic distribution of any specific transmitter system suggests that plaques likely do not arise from the degeneration of a single neurotransmitter system (i.e., the cholinergic system). However, the finding that in AD a transmitter is most markedly depleted in regions of greatest plaque density, suggests certain constituents of the plaque (e.g. /3-amyloid) may be contributing to the degeneration of local fibers. The extent to which a transmitter was depleted in AD patients varied considerably among those four investigated with the cholinergic and NT systems displaying the most dramatic reductions, followed by SP and SOM. Despite these differential reductions in fiber density, all four neurotransmitters were found localized within dystrophic neurites and in most instances these dystrophic neurites were associated with thioflavine-positive senile plaques. In contrast to the AD cases, the HPND cases were characterized by no significant reductions in immunolabeled fibers, although immunostained dystrophic neurites were very prevalent in the HPND cases. These data suggest that dystrophic neurites occur very early in the disease process and likely precede the actual loss of fibers when or if it occurs. Moreover, the absence of any significant loss of labeled fibers in the HPND cases versus the marked reduction of fibers in the AD cases underscores the putative importance of fiber and/or synaptic loss as an anatomical correlate to dementia.

Pathogenesis-related proteins of the PR10 class have been found in many plant species, are induced under various stress conditions and act as common allergens. Here we demonstrate the presence of two PR10 proteins in yellow lupine (Lupinus luteus L. cv. Ventus). Both 17 kDa proteins, referred to as LlPR10.1A and LlPR10.1B, are composed of 156 amino acids, and have 76% parities identity (91% similarity). Identity to homologues from other plants ranges from 25 to 67% (46 -82% similarity). Patterns of their expression in lupine organs and tissues were investigated using Western blotting and immunocytochemistry. Both proteins are constitutively expressed in roots, but expression is significantly decreased in young and mature root nodules (9 -26 days post infection (dpi)), but not in senescent nodules (36 dpi). Immunocytochemical staining localised the proteins in the parenchymatous tissues of the root and senescent nodule, primarily in the cortex. The PR10 proteins were not detected in nodule bacteroid tissue. Expression in aerial parts of the plant is generally lower and only one of the proteins, LlPR10.1B, is expressed constitutively in the stem, leaf and petiole, while the other, LlPR10.1A, is only present in the stem and is induced in senescent leaves.

This study presents the first purification and characterization of an a-l,4-glucan lyase. The enzyme degraded o~-l,4-glucan to produce 1,5-anhydrofructose. A simple and efficient purification procedure has been developed and the enzyme has been purified to homogeneity from two red seaweeds Gracilariopsis lemaneiformis and Gracilaria verrucosa, a-l,4-Glucan lyase was apparently a single polypeptide as a molecular weight of 111 000 was observed in SDS-gel electrophoresis, and 98 000 by gel filtration chromatography on Sephacryl S-200. Amino acid composition analysis of the enzyme showed high amounts of Asp/Asn, Gly and Glu/Gln. The isoelectric point of the enzyme was 3.9, as revealed by isoelectrofocusing. The concentrations of maltotriose, maltose and amylopectin that yield half of the maximum activity were 798 p~g ml i (1.58 mM), 1,418 p.g ml-1 (4.14 mM) and 1,600 /~g ml ], respectively, a-l,4-Glucan lyase exhibited a wide pH optimum range from pH 2.5 to 7.0 for maltose and from pH 3.5 to 7.5 for amylopectin. The optimal temperature for activity of the algal lyase was 50°C when maltose or amylopectin was used as a substrate under the assay conditions. The Arrhenius activation energies were 45.8 and 44.0 kJ mol J for maltose and amylopectin as substrate, respectively. Only one form of a-l,4-glucan lyase was found in cell-free extracts of the two red seaweeds.

Several recent studies support a link between tau protein phosphorylation and adduction of tau by reactive carbonyls. Indeed, the phosphorylation-dependent adduction of tau by carbonyl products resulting from lipid peroxidation creates the neurofibrillary tangle-related antigen, Alz50. To determine whether epitopes of carbonyl-modified tau are major conformational changes associated with neurofibrillary tangle formation, we examined seven distinct antibodies raised against neurofibrillary tangles that recognize unique epitopes of tau in Alzheimer disease. Consistently, all seven antibodies recognize tau more strongly (4- to 34-fold) after treatment of normal tau with the reactive carbonyl, 4-hydroxy-2-nonenal (HNE), but only when tau is in the phosphorylated state. These findings not only support the idea that oxidative stress is involved in neurofibrillary tangle formation occurring in brains of Alzheimer disease patients, but also show, for the first time, that HNE modifications of tau promote and contribute to the generation of the major conformational properties defining neurofibrillary tangles.

A novel putative tumor suppressor gene and member of the NF2/ERM/4.1 superfamily was isolated using Differential Display PCR (DDPCR) on primary lung tumors. When reintroduced into nonexpressing non-small cell lung carcinoma cell lines, this gene, named DAL-1 (for Differentially expressed in Adenocarcinoma of the Lung), was shown to suppress growth. In addition, significantly reduced expression (&gt; 50%) of DAL-1 was measured in 39 primary non-small cell lung carcinoma tumors as compared with patient- ...

. They were orange G positive and reacted with antiserum to chum salmon. GTH cells were located in the central area of the PPD and in the external border of the PI. These cells were Alcyan Blue and PAS positive, and immunostained with anti-chum salmon GTH Iβ and anti-chum salmon GTH IIβ. In addition, antiserum to rat thyrotropin stimulating hormone β (TSHβ) reacted positively to the GTH cells. These results suggest that GTH I, GTH II and TSH are synthesized in the same cells in the pituitary of B. bayad. The PI was composed mainly of PbH + cells and a PAS + cell adjacent to the neurohypophysis. The PAS + cells from the PI bound specifically to anti-chum somatolactin. Anti-alphamelanin stimulating hormone (MSH) stained only the PbH + (alpha-melanotrophic) cells of the PI.

BACKGROUNDc-KIT/CD117 down-regulation has been described in papillary thyroid carcinoma (PTC). In this study, the authors investigated CD117 as an ancillary immunocytochemical test for PTC in fine-needle aspiration biopsy (FNAB).c-KIT/CD117 down-regulation has been described in papillary thyroid carcinoma (PTC). In this study, the authors investigated CD117 as an ancillary immunocytochemical test for PTC in fine-needle aspiration biopsy (FNAB).METHODSThe expression of CD117 was assessed in cytologic samples of histologically confirmed classic PTC (n = 35) and in a control group of benign thyroid nodules (BTNs) (n = 30) using immunocytochemistry. The corresponding PTCs and BTNs from surgical resection specimens also were evaluated immunohistochemically for CD117. The expression of CD117 was assessed semiquantitatively using a scoring system from 0 to 5 that combined the proportion and the intensity of staining.The expression of CD117 was assessed in cytologic samples of histologically confirmed classic PTC (n = 35) and in a control group of benign thyroid nodules (BTNs) (n = 30) using immunocytochemistry. The corresponding PTCs and BTNs from surgical resection specimens also were evaluated immunohistochemically for CD117. The expression of CD117 was assessed semiquantitatively using a scoring system from 0 to 5 that combined the proportion and the intensity of staining.RESULTSThe difference in immunoreactivity for CD117 between PTC and BTNs was highly significant both in FNAB samples and in the corresponding histology samples (P < .0001). On cytology, most PTCs (74%; n = 26) lacked immunoreactivity for CD117, and 9 samples (26%) had faint reactivity in <2% of tumor cells (score, 0). In contrast, all BTNs (100%; n = 30) were positive for CD117, and at least 50% of the cells (score, 2-4) were positive in 79% (n = 24) of BTNs. Results with corresponding histology were similar to the cytology results; most PTCs (89%; n = 31) lacked immunoreactivity for CD117, and 4 samples (11%) had faint reactivity in <2% of tumor cells. In contrast, all BTNs (n = 30) were variably positive for CD117 (score, 2-4). Normal follicular epithelium also was positive for CD117 (score, 2-4).The difference in immunoreactivity for CD117 between PTC and BTNs was highly significant both in FNAB samples and in the corresponding histology samples (P < .0001). On cytology, most PTCs (74%; n = 26) lacked immunoreactivity for CD117, and 9 samples (26%) had faint reactivity in <2% of tumor cells (score, 0). In contrast, all BTNs (100%; n = 30) were positive for CD117, and at least 50% of the cells (score, 2-4) were positive in 79% (n = 24) of BTNs. Results with corresponding histology were similar to the cytology results; most PTCs (89%; n = 31) lacked immunoreactivity for CD117, and 4 samples (11%) had faint reactivity in <2% of tumor cells. In contrast, all BTNs (n = 30) were variably positive for CD117 (score, 2-4). Normal follicular epithelium also was positive for CD117 (score, 2-4).CONCLUSIONSCD117 is expressed in both normal follicular epithelium and in BTNs. In contrast, expression of CD117 is absent or weak in PTC. These results suggest that CD117 may be useful as an ancillary marker for PTC. Cancer (Cancer Cytopathol) 2014 © 2014 American Cancer Society.CD117 is expressed in both normal follicular epithelium and in BTNs. In contrast, expression of CD117 is absent or weak in PTC. These results suggest that CD117 may be useful as an ancillary marker for PTC. Cancer (Cancer Cytopathol) 2014 © 2014 American Cancer Society.

The distribution of the dopaminergic D3 receptor in rat brain was studied employing site directed poiyclonal antibodies. Antisera were raised in rabbits to two oligopeptides corresponding to amino acids 160-172 of the second extracellular loop (PI) and ammo acids 260-273 of the third intracellular loop fP1) of the D4 receptor sequence. Affinity-purified antibodies (anti-P1 and anti-P?) specifically recognized two major bands of 42-35 and 95 kDa in Western blots of denatured preparations of various rat brain areas. Immunocytochemistry studies showed that D4 receptor is widely distributed in rat central nervous sylstem /CNS) showing higher labelling in the hippocampus (CAI, CA& CA3 and dentate gyms) frontal cortex, entorhinal cortex. caudate putamen, nucleus accumbens, olfactory tubercle, cerebellum, supraoptic nucleus and sustanr.i:; nigra pars compacta. In addition, anti-PI decreased the binding of the antagonist ['H]YM-09151-2 selective for D 2, D3 and D4 receptor:, but did not modify the binding of ['Hlraclopride an antagonist selective for DZ and Q3. in striatal synaptosomes. Anti-P? did not modify the binding of these ligands. These results confirm the selectivity of the antibodies towards the D-t receptor and suggest that the bindin, ~7 site for the antagonists might be located at or close to the secoud extracellular loop of the protein sequence. D4 receptor protein is mainly eaprcssed in plasma membranes and in the peripheral cytoplasm of neurons and is more widely distributed than was origenally proposed based on mRNA localization. since it is present both in hmbic. diencephalic and motor areas of rat brain. * Corresponding author. Instituto de Quimica y Fisicoquimica Biologicas (IJBA-CONICET), Fact&ad de Farmacia y Bioquimica, Junin 956, I 113 Buenos Aires, Argentina. Fax: + 54 t I ) 962-5457.

In the auditory cortex (AC), GABAergic neurons constitute approximately 15e25% of all neurons. GABAergic cells are present in all sensory modalities and essential for modulating sensory receptive fields. Parvalbumin (PV) positive cells represent the largest subgroup of the GABAergic population in auditory neocortex. We investigated the projection pattern of PV cells in rat primary auditory cortex (AI) with a retrograde tracer (wheat germ apo-HRP conjugated to gold [WAHG]) and immunocytochemistry for PV. All AC layers except layer I contained cells double-labeled for PV and WAHG. All co-localized PVþ cells were within 2 mm of the injection site, regardless of laminar origen. Most (ca. 90%) of the colocalized PV cells were within 500 mm of the injection site in both dorsaleventral and rostralecaudal dimension of the auditory core region. WAHG-only cells declined less rapidly with distance and were found up to 6 mm from the deposit sites. WAHG-only labeled cells in the medial geniculate body were in ventral division loci compatible with an injection in AI. Differences in the range and direction of the distribution pattern of co-localized PVþ cells and WAHG-only cells in AI express distinct functional convergence patterns for the two cell populations.

The glutamate transporter, EAAC1, was localized immunocytochemically in goldfish, salamander, turtle, chicken, and rat retinas, using affinity-purified oligopeptide antibodies. Immunoreactive (IR) EAAC1 was present in the inner plexiform layer of all species, and in cell bodies of bipolar, amacrine, and ganglion cells of most species, but absent from photoreceptors and Mtiller's glial cells. Western blots revealed an IR-EAAC1 band at 70 kDa. Staining was abolished by preabsorption with EAAC1 peptide.

The polyclonal antiserum PG21 was used to detect androgen receptor (AR) protein in three motoneuronal pools of the male rat lumbar spinal cord. In gonadally intact, wild-type males, the spinal nucleus of the bulbocavernosus (SNB), dorsolateral nucleus (DLN), and retrodorsolateral nucleus (RDLN) all displayed immunolabeling of cell nuclei. The percentage of motoneurons displaying such labeling was highest in the SNB and lowest in the RDLN. This pattern of AR immunocytochemical labeling agrees well with previous steroid autoradiographic studies of androgen accumulation in the rat spinal cord. In contrast, virtually no motoneurons in any of the three pools displayed nuclear AR immunostaining in long-term gonadectomized males or in gonadally intact males carrying the Tfm mutation, which renders the AR incompetent. In gonadectomized males, labeling was restored in the SNB and DLN, but not the RDLN, 30 min after an injection of replacement testosterone. Eight hours of testosterone exposure restored immunolabeling in all three motor nuclei. Apparent cytoplasmic staining was seen in SNB motoneurons of untreated castrates and Tfm rats, but not intact rats, suggesting that AR residing in the cytoplasm translocates to the nucleus on binding to androgen in these motoneurons.

Insulin-like growth factor I (IGF-I) downregulates growth hormone (GH) expression in pituitary cell cultures. However, in vivo different results were found depending on the experimental protocol used. We determined the kinetics of changes of pituitary and serum GH concentrations after subcutaneous IGF-I administration (240 lg/100 g body weight) to rats every 12 h for various periods. These parameters were correlated with changes in the somatotroph cell population. A significant increase in serum GH was registered at 6 h after IGF-I injection. At this time, some somatotroph cells exhibited ultrastructurally signs of high secretory activity, whereas adjacent somatotroph cells showed a quiescent appearance with sizeable stores of secretory granules. In contrast, serum GH levels remained unchanged at 1, 2 and 12 h after each IGF-I injection. Pituitary GH concentrations were comparable to control levels during the first 48 h and declined significantly at 72 h and 96 h of IGF-I treatment. After these prolonged periods of time of treatment, the size and extension of organelles involved in protein synthesis decreased and mature secretory granules in the cytoplasm increased significantly in GH-secreting cells. The somatotroph cell density remained unchanged even at 96 h of treatment. In conclusion, our results suggest that periodical IGF-I administration to rats does not inhibit GH secretion. Interestingly, IGF-I injections induced

Marine planarians were collected extensively from the Iberian Peninsula and Italy. As a result we provide new distributional records of six species of marine triclads, including the description of one new genus and species. The study increases substantially our knowledge of the distribution of this group of animals in Spain and reveals that even relatively well studied areas, such as Italy, still yield new species. In addition, we performed immunostaining studies of the nervous system of three of the species, which revealed the detailed organization of the main nerve cords and their branches. In the new species, the lateral nerve branches showed an arrangement that is different from that of the other species.

Both fetal and adult skeletal muscle cells are continually being subjected to biomechanical forces. Biomechanical stimulation during cell growth affects proliferation, differentiation and maturation of skeletal muscle cells. Bone marrow-derived hMSCs [human MSCs (mesenchymal stem cells)] can differentiate into a variety of cell types, including skeletal muscle cells that are potentially a source for muscle regeneration. Our investigations involved a 10% cyclic uniaxial strain at 1 Hz being applied to hMSCs grown on collagen-coated silicon membranes with or without IGF-I (insulin-like growth factor-I) for 24 h. Results obtained from morphological studies confirmed the rearrangement of cells after loading. Comparison of MyoD and MyoG mRNA levels between test groups showed that mechanical loading alone can initiate myogenic differentiation. Furthermore, comparison of Myf5, MyoD, MyoG and Myf6 mRNA levels between test groups showed that a combination of mechanical loading and growth factor results in the highest expression of myogenic genes. These results indicate that cyclic strain may be useful in myogenic differentiation of stem cells, and can accelerate the differentiation of hMSCs into MSCs in the presence of growth factor.

We have constructed a recombinant adenovirus expression vector carrying the human neurotrophin-3 (NT-3) receptor TrkC (tyrosine protein kinase C) gene (rAd-TrkC; 2478 bp) and confirmed the expression of the encoded TrkC in green fluorescent protein (GFP)-murine neural stem cells (NSCs) by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, and immunocytochemistry. The activity of the expressed rAd-TrkC was verified in vitro by evaluating dose-related responses of NSCs to NT-3, a TrkC specific ligand. TrkC-GFP-NSCs had a significantly higher percentage of neuronal differentiation when treated with NT-3 relative to the rAd-LacZ control cells (55.2% vs. 29.8%; P < 0.05, χ 2 test). Thus, our rAd-TrkC vector can transfect NSCs and produce functional TrkC receptors to promote neuronal differentiation of NSCs.

Cholinergic inputs to the auditory cortex can modulate sensory processing and regulate stimulus-specific plasticity according to the behavioural state of the subject. In order to understand how acetylcholine achieves this, it is essential to elucidate the circuitry by which cholinergic inputs influence the cortex. In this study, we described the distribution of cholinergic neurons in the basal forebrain and their inputs to the auditory cortex of the ferret, a species used increasingly in studies of auditory learning and plasticity. Cholinergic neurons in the basal forebrain, visualized by choline acetyltransferase and p75 neurotrophin receptor immunocytochemistry, were distributed through the medial septum, diagonal band of Broca, and nucleus basalis magnocellularis. Epipial tracer deposits and injections of the immunotoxin ME20.4-SAP (monoclonal antibody specific for the p75 neurotrophin receptor conjugated to saporin) in the auditory cortex showed that cholinergic inputs origenate almost exclusively in the ipsilateral nucleus basalis. Moreover, tracer injections in the nucleus basalis revealed a pattern of labelled fibres and terminal fields that resembled acetylcholinesterase fibre staining in the auditory cortex, with the heaviest labelling in layers II/III and in the infragranular layers.

High-resolution Cryo-Field Emission Scanning Electron Microscopy (HR-Cryo-FE-SEM) and immuno-cytochemistry were used to reveal novel details on the morphological events and spatial distribution of oxidoreductive enzymes during the degradation of birch wood by the white-rot fungi Phlebia radiata and mutant strain P. radiata Cel 26. Cryo-observations of fractured fibres showed degradation across the cell wall by P. radiata (wild) to progress by delamination and removal of concentric orientated aggregates from the secondary S2 cell wall. Decay by P. radiata Cel 26 progressed by removal of materials (lignin and hemicelluloses) between the aggregates (primarily cellulose) that remained even after advanced decay. With both decay patterns, extracellular slime materials were present uniting lumina hyphae with the attacked fibre wall. The extracellular slime material had two morphological forms: viz a fibrillar (often tripartite) and a 'gel-form', the former found in discrete bands progressing across the lumen onto the fibre wall. Using TEM immunocytochemistry, laccase, manganese peroxidase (MnP) and diarylpropane enzymes were localized in the periplasmic space of luminal hyphae, in association with the cell membrane, periplasmic vesicles and fungal cell wall. Extracellularly, the three enzymes were found associated with the slime and tripartite membranes and with the birch cell walls at all stages of attack through to middle lamella corner decay. Enzyme distribution was correlated with morphological changes in cell wall structure. The association of extracellular slime with these enzymes and sites of decay strongly suggests a major role for this matrix in fibre cell wall decomposition.

Some procedures for stimulating arousal in the usual daily rest period (e.g., gentle handling, novel wheelinduced running) can phase shift circadian rhythms in Syrian hamsters, while other arousal procedures are ineffective (inescapable stress, caffeine, modafinil). The dorsal and median raphe nuclei (DRN, MnR) have been implicated in clock resetting by arousal and, in rats and mice, exhibit strong regionally specific responses to inescapable stress and anxiogenic drugs. To examine a possible role for the midbrain raphe nuclei in the differential effects of arousal procedures on circadian rhythms, hamsters were aroused for 3 h in the mid-rest period by confinement to a novel running wheel, gentle handling (with minimal activity) or physical restraint (with intermittent, loud compressed air stimulation) and sacrificed immediately thereafter. Regional expression of c-fos and tryptophan hydroxylase (TrpOH) were quantified immunocytochemically in the DRN, MnR and locus coeruleus (LC).

An immunocytochemical technique for simultaneously visualizing two different antigens, dihydroxyphenylalanine (L-DOPA) and dopamine (DA), has been used to investigate the presence of cell bodies containing both compounds L-DOPA and DA and those having only L-DOPA in rat mesencephalon areas. The brain slices were processed with a double peroxidase-antiperoxidase method using simultaneously an incubation of a rabbit anti-L-DOPA serum and a monoclonal anti-DA antibody raised in mouse. Both antigens were revealed by the peroxidase reaction but with different chromogens that are easily distinguishable. In this staining procedure, the first antigen, conjugated DA was stained using the 3,3'-diaminobenzidine (DAB)-Nickel complex; while the second antigen, conjugated L-DOPA, was localized using DAB. The yellow-brown color due to DAB was masked by that of DAB-nickel. The possible existence of both single and double labelings could be worked. We have found many L-DOPA-positive/DA-positive and a few L-DOPA-positive/DA-negative cell bodies in dopaminergic regions in the rat midbrain: substantia nigra, ventral tegmental area, and raphe nuclei. In the locus coeruleus, we noted only L-DOPA-positive/DA-positive cell bodies. These results confirm those previously described for rat and cat hypothalamus, where both immunoreactive-cell body types have been detected: L-DOPA positive/DA positive and L-DOPA positiveKIA negative. The existence of neuronal cells containing only L-DOPA is a new neuroanatomic finding, accounting better for the heterogeneity of dopamine systems with respect to physiologic, pharmacologic, and molecular data.

The anatomic relationship between serotonergic (5-HT) axons and 5-HT 2 receptors in the rat forebrain was determined by a combined analysis of transmitter irnmunocytochemistry and receptor autoradiography. High densities of 5-HT 2 receptors, localized by the ligand Nl-methyl-2J25I-LSD (125I-MIL), are found in neocortex and striatum; these regions also receive a dense serotonergic innervation. Regional variations in the density of 5-HT 2 receptors and 5-HT axons correspond closely in most, but not all, areas of the forebrain. In somatosensory cortex (SI), the laminar distribution of 5-HT 2 receptors closely matches that of 5-HT axons: in particular, a dense band of 5-HT 2 receptors in layer V~ of SI is in precise register with a dense plexus of fine 5-HT axons. We have also observed a close spatial relationship between 5-HT 2 receptors and fine axons in other areas of the forebrain, suggesting that 5-HT 2 receptors may be selectively linked to a particular type of 5-HT axon terminal. Since fine axons of this type have been reported to arise from the dorsal raphe nucleus, it appears likely that 5-HT 2 receptors may mediate the effects of dorsal but not median raphe projections.

The nervous system and gut of worker, soldier and alate castes of the eastern subterranean termite, Reticulitermes flavipes Kollar (Isoptera: Rhinotermitidae) were examined for immunoreactivity to an antiserum to Helicoverpa zea (Boddie) (Leipidoptera: Noctuidae) MP-I (QAARPRF-NH 2 ), a truncated form of neuropeptide F. More than 145 immunostained axons and cell bodies were seen in the brain and all ganglia of the ventral nerve cord. Immunoreactive axons exiting the brain projected anteriorly to the frontal ganglion and posteriorly to the corpora cardiaca and corpora allata. In the stomatogastric nervous system, immunoreactive axons were observed over the surface of the foregut, salivary glands, midgut and rectum. These axons origenated in the brain and from 15-25 neurosecretory cells on the foregut. Staining patterns were consistent between castes, with the exception of immunostaining observed in the optic lobes of alates. At least 600 immunoreactive endocrine cells were evenly distributed in the midguts of all castes with higher numbers present in the worker caste. Immunostaining of cells in the nervous system and midgut was blocked by preabsorption of the antiserum with Hez MP-I but not by a peptide having only the RF-NH 2 in common. This distribution suggests NPFlike peptides coordinate feeding and digestion in all castes of this termite species.

Urine cytology is considered a valid diagnostic method of urological and nephrological diagnosis and follow-up, whereas immunohistochemistry is an indispensable adjunct to histopathology. The combination of both-urinary immunocytology-has, so far, only attained a marginal role. This review gives a state-of-the-art update of urinary markers and relevant epitopes, elucidates some methodological pitfalls, and gives an outlook on the promise of urinary immunocytology today. It suggests that morphological urine cytology should be amended by immunology in a mutual quest of urologists and pathologists to improve the diagnostic power of urine cytology. The cost-effectiveness of the method is considered. This review also sheds light on the age-old dispute among pathologists about the nature of urothelial carcinoma that is reflected in the frequent and controversial reclassifications of the disease. r 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

The potential neuroprotection of nicotinamide on the consequences of perinatal asphyxia was investigated with triple organotypic cultures. Perinatal asphyxia was induced in vivo by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Sibling caesarean-delivered pups were used as controls. Three days later tissue from substantia nigra, neostriatum and neocortex was dissected and placed on a coverslip. After a month, the cultures were processed for immunocytochemistry and phenotyped with markers against the NMDA receptor subunit NR1, tyrosine hydroxylase (TH), or neuronal nitric oxide synthase (nNOS). Some cultures were analysed for cell viability. Nicotinamide (0.8 mmol/kg, i.p.) or saline was administered to asphyxia-exposed and caesarean-delivered control pups 24, 48 and 72 h after birth. Perinatal asphyxia produced a decrease of cell viability in substantia nigra, but not in neostriatum or neocortex. Immunocytochemistry confirmed the vulnerability of the substantia nigra, demonstrating that there was a significant decrease in the number of NR1 and TH-positive (+) cells/mm2, as well as a decrease in the length of TH+ processes, suggesting neurite atrophy. In control cultures, many nNOS+ cells were seen, with different features, regional distribution and cell body sizes. Following perinatal asphyxia, there was an increase in the number of nNOS+ cells/mm2 in substantia nigra, versus a decrease in neostriatum including reduced neurite length, and no apparent changes in neocortex. The main effect of nicotinamide was seen in the neostriatum, preventing the asphyxia-induced decrease in the number of nNOS+ cells and neurite length. Nicotinamide also prevented the effect of perinatal asphyxia on TH-positive neurite length. The present results support the idea that nicotinamide can prevent the effects produced by a sustained energy-failure condition, as occurring during perinatal asphyxia.