María Isabel Arenas

Nestin, a neuroectodermal stem cell marker molecule, is expressed in Leydig cells of the human testis and in some specific cell types from human testicular tumours.

The intermediate filament protein nestin is predominantly expressed in some stem/progenitor cells and appears to be a useful molecular tool to characterise tumours originating from precursor cells of neuroectodermal and mesenchymal lineages. Leydig cells originate in the adult testis by differentiation from stem cells and express a variety of neural and neuroendocrine markers. The possible expression of the neural stem cell marker nestin in Leydig cells and testicular tumour cells was determined by analysing the patterns of nestin expression in normal and pathological human testes by Western blot and immunohistochemical methods. In normal testis, nestin was found in some vascular endothelial cells, a subset of peritubular spindle-shaped cells and some Leydig cells; spermatogenic and Sertoli cells were unstained. In normal Leydig cells, nestin was distributed in the perinuclear cytoplasm and accumulated in the crystalloids of Reinke with ageing. In non-tumour pathologies (cryptorchidism, impaired spermatogenesis), the seminiferous tubules were immunonegative, whereas hyperplastic Leydig cells showed cytoplasmic immunolabelling. In testicular malignancies, nestin was localised in the Sertoli cells of the seminiferous tubules affected with intratubular germ cell neoplasia, in the hyperplastic Leydig cells associated with these tumours and in some components (mesenchymal and neuroepithelial cells) of teratomas; spermatocytic and non-spermatocytic seminomas were unstained. Some vascular endothelial cells were immunolabelled in all tumour samples. Thus, nestin is expressed in a population of normal and hyperplastic Leydig cells and in Sertoli cells in the presence of intratubular germ-cell neoplasia. Nestin may be a good marker for identifying components of testicular teratomas.

A lectin histochemistry comparative study in human normal prostate, benign prostatic hyperplasia, and prostatic carcinoma.

The partial oligosaccharide sequences of glycoconjugates and the nature of their glycosidic linkages were investigated in normal human prostate, benign prostatic hyperplasia (BPH) and prostatic carcinoma by means of lectin histochemistry, using light microscopy and Western blot analysis. The labeling pattern of BPH differed from that of normal prostate in having more intense staining with DSA, HPA, UEA-I and AAA, and in showing lesser staining with WGA and SBA. Prostatic carcinoma differed from normal prostates in displaying the more intense labeling with PNA, DSA, SBA, DBA, UEA-I and AAA, and in having lesser labeling with WGA. The main differences in labeling pattern between prostatic carcinoma and BPH were that the latter specimens showed more marked staining with PNA, DSA, DBA, SBA, UEA-I and AAA, and lesser staining with WGA and HPA. The staining patterns of SNA, MAA, ConA, LCA and GNA were similar in all three groups of specimens. For most of the lectins studied, including those showing a similar immunohistochemical staining in the three groups of specimens studied, the Western blot analysis showed differences in the banding pattern among normal, hyperplastic, and carcinomatous prostates. Present results suggest that the glycosylation of proteins was modified in both BPH and prostatic carcinoma. In BPH a strong expression of N-acetylgalactosamine residues occurred, while in prostatic carcinoma an increase of sialic aci, galactose and fucose residues was observed. No changes in mannose residues were detected.

DAX-1 expression in human breast cancer: comparison with estrogen receptors ER-α, ER-β and androgen receptor status

BackgroundSo far there have been no reports on the expression pattern of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) in human breast cells and its relationship to the estrogen receptors, ER-α and ER-β, and the androgen receptor (AR).MethodsIn this study we evaluated, by immunohistochemistry and Western blot analysis, the presence and distribution of DAX-1 in benign breast disease (BBD), in situ carcinoma (CIS), and ductal and lobular breast carcinomas.ResultsIn BBD and breast carcinomas, DAX-1 was present in both the nuclei and the cytoplasm of epithelial cells, although in infiltrative carcinomas the percentage of nuclear immunoreaction was higher than in CIS. An important relation was observed between DAX-1 and AR expression and between this orphan receptor and nodal status.ConclusionDAX-1 might modify the AR and ER-β intracellular location, and because a direct positive relation between the expression of these three receptors was found it could be assumed that the presence of DAX-1 in neoplastic cells might indicate a possible failure of endocrine therapies.

Androgen receptor (AR), estrogen receptor‐alpha (ER‐α) and estrogen receptor‐beta (ER‐β) expression in the testis of the newt, Triturus marmoratus marmoratus during the annual cycle

Expression of androgen receptor (AR), estrogen receptor alpha (ER‐α) and estrogen receptor beta (ER‐β) in the testis of the marbled newt (Triturus marmoratus marmoratus) was investigated, with special attention to changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. Primordial germ cells, primary and secondary spermatogonia and spermatocytes showed a positive reaction to the 3 receptor antibodies during the annual reproductive cycle. Follicular cells were positive to AR, ER‐α and ER‐β during the spermiogenesis and quiescence periods in the glandular tissue. Interstitial cells showed reactivity to AR, ER‐α and ER‐β in the spermiogenesis and the quiescence periods, and presented no labelling to these receptors in the proliferative period. These findings suggest that, as in mammals, there is an androgen‐estrogen regulation of the function and development of the newt testis.

PCPH/ENTPD5 Expression Enhances the Invasiveness of Human Prostate Cancer Cells by a Protein Kinase Cδ–Dependent Mechanism

Previous reports showed that PCPH is mutated or deregulated in some human tumors, suggesting its participation in malignant progression. Immunohistochemical analyses showed that PCPH is not expressed in normal prostate, but its expression increases along cancer progression stages, being detectable in benign prostatic hyperplasia, highly expressed in prostatic intraepithelial neoplasia, and remaining at high levels in prostate carcinoma. Experiments designed to investigate the contribution of PCPH to the malignant phenotype of prostate cancer cells showed that PCPH overexpression in PC-3 cells, which express nearly undetectable PCPH levels, increased collagen I expression and enhanced invasiveness, whereas shRNA-mediated PCPH knockdown in LNCaP cells, which express high PCPH levels, down-regulated collagen I expression and decreased invasiveness. PCPH regulated invasiveness and collagen I expression by a mechanism involving protein kinase C delta (PKC delta): (a) PCPH knockdown in LNCaP cells decreased PKC delta levels relative to control cells; (b) PKC delta knockdown in LNCaP cells recapitulated all changes caused by PCPH knockdown; and (c) forced expression of PKC delta in cells with knocked down PCPH reverted all changes provoked by PCPH down-regulation and rescued the original phenotype of LNCaP cells. These results strongly suggested that the expression level and/or mutational status of PCPH contributes to determine the invasiveness of prostate cancer cells through a mechanism involving PKC delta. Data from immunohistochemical analyses in serial sections of normal, premalignant, and malignant prostate specimens underscored the clinical significance of our findings by showing remarkably similar patterns of expression for PCPH and PKC delta, thus strongly suggesting their likely coregulation in human tumors.

IL-2, Its Receptors, and bcl-2 and bax Genes in Normal, Hyperplastic and Carcinomatous Human Prostates: Immunohistochemical Comparative Analysis

Abstract The presence of interleukin-2 (IL-2) and its receptors (Rα, Rβ, Rγ), and their relationship with the products of bcl-2 and bax genes was studied in normal prostates, benign prostatic hyperplasia (BPH), and prostatic cancer (PC) by ELISA, Western blot, and immuno-histochemistry. A comparative semiquantitative immunohistochemical study was also performed. For all the antibodies assayed, immunoreactions were found in the epithelium and some stromal cells in the three types of specimens studied. These immunoreactions were much more higher in PC samples than in normal prostates. In BPH, immunoreactions were similar to that of normal prostates (bax), similar to that of PC (IL-2 and its three receptors), or intermediate between that of normal prostates and that of PC (bcl-2). Immunoexpressions of IL-2 and its receptors were found in the epithelial basal cells and some stromal cell of normal prostates and might be related to the control of the proliferation-apoptosis equilibrium. The increased expressions of IL-2 and its receptors in the epithelium of prostates in BPH, associated with increased bcl-2 expression which would account for the decrease in the apoptosis index that has been reported in this disorder. The increased expression of both bcl-2 and bax in PC might be involved in the higher apoptosis index reported in PC specimens. Since IL-2 administration seems to have an anti-tumour effect, the increased expression of this interleukin in BPH and PC could be interpreted as an attempt to hinder cell proliferation which would only be efficient at high doses.

Algoriphagus hitonicola sp. nov., isolated from an athalassohaline lagoon.

A Gram-negative, heterotrophic, aerobic, reddish-orange-pigmented, non-spore-forming, non-motile bacterial strain, designated 7-UAH(T), was isolated from salty water from the athalassohaline lagoon at El Hito, located in central Spain. The strain grew optimally at 37 degrees C and pH 7.0 and in the presence of 2.5 % NaCl. A polyphasic taxonomic study was carried out in order to characterize the strain in detail. Phylogenetic analyses based on 16S rRNA gene sequence comparisons indicated that strain 7-UAH(T) clustered within the branch constituted by species of the genus Hongiella, which were recently transferred to the genus Algoriphagus. Analysis of the polar lipid profile and DNA G+C content also supported placement of strain 7-UAH(T) within the genus Algoriphagus. On the basis of its phenotypic, chemotaxonomic and phylogenetic distinctiveness, strain 7-UAH(T) represents a novel species of the genus Algoriphagus, for which the name Algoriphagus hitonicola sp. nov. is proposed. The type strain is 7-UAH(T) (=CECT 7267(T) =CCM 7449(T)).

Halomonas ilicicola sp. nov., a moderately halophilic bacterium isolated from a saltern

A Gram-negative, heterotrophic, aerobic, pale orange-pigmented, non-endospore-forming and motile bacterial strain, designated strain SP8(T), was isolated from a salty water sample from the solar salterns of Santa Pola, located on the Mediterranean coast of Spain. The strain grew optimally at 37 degrees C, pH 6.5 and in the presence of 10 % NaCl. A polyphasic taxonomic study was conducted in order to characterize the strain in detail. Phylogenetic analyses based on 16S rRNA gene sequence comparisons indicated that strain SP8(T) clustered within the branch constituted by species of the genus Halomonas. The closest phylogenetic neighbours of strain SP8(T) were Halomonas muralis LMG 20969(T) (96.0 % sequence similarity), Halomonas pantelleriensis AAP(T) (95.9 %) and Halomonas campaniensis 5AG(T) (95.8 %). Phenotypic features, the fatty acid profile and the DNA G+C content of the novel strain further supported its placement in the genus Halomonas. On the basis of phenotypic, chemotaxonomic and phylogenetic distinctiveness, it is suggested that strain SP8(T) represents a novel species for which the name Halomonas ilicicola sp. nov. is proposed. The type strain is SP8(T) (=CECT 7331(T)=CCM 7522(T)=DSM 19980(T)).

Human pregnane X receptor is expressed in breast carcinomas, potential heterodimers formation between hPXR and RXR-alpha

BackgroundThe human pregnane X receptor (hPXR) is an orphan nuclear receptor that induces transcription of response elements present in steroid-inducible cytochrome P-450 gene promoters. This activation requires the participation of retinoid X receptors (RXRs), needed partners of hPXR to form heterodimers. We have investigated the expression of hPXR and RXRs in normal, premalignant, and malignant breast tissues, in order to determine whether their expression profile in localized infiltrative breast cancer is associated with an increased risk of recurrent disease.MethodsBreast samples from 99 patients including benign breast diseases, in situ and infiltrative carcinomas were processed for immunohistochemistry and Western-blot analysis.ResultsCancer cells from patients that developed recurrent disease showed a high cytoplasmic location of both hPXR isoforms. Only the infiltrative carcinomas that relapsed before 48 months showed nuclear location of hPXR isoform 2. This location was associated with the nuclear immunoexpression of RXR-alpha.ConclusionBreast cancer cells can express both variants 1 and 2 of hPXR. Infiltrative carcinomas that recurred showed a nuclear location of both hPXR and RXR-alpha; therefore, the overexpression and the subcellular location changes of hPXR could be considered as a potential new prognostic indicator.

Oral administration of bisphenol A induces high blood pressure through angiotensin II/CaMKII-dependent uncoupling of eNOS

Bisphenol A (BPA) is found in human urine and fat tissue. Higher urinary BPA concentrations are associated with arterial hypertension. To shed light on the underlying mechanism, we orally administered BPA (4 nM to 400 μM in drinking water) to 8‐wk‐old CD11 mice over 30 d. Mice developed dosage‐dependent high blood pressure (systolic 130±12 vs. 170±12 mmHg; EC50 0.4 μM), impairment of acetylcholine (AcH)‐induced carotid relaxation (0.66±0.08 vs. 0.44±0.1 mm), a 1.7‐fold increase in arterial angiotensin II (AngII), an 8.7‐fold increase in eNOS mRNA and protein, and significant eNOS‐dependent superoxide and peroxynitrite accumulation. AngII inhibition with 0.5 mg/ml losartan reduced oxidative stress and normalized blood pressure and endothelium‐dependent relaxation, which suggests that AngII uncouples eNOS and contributes to the BPA‐induced endothelial dysfunction by promoting oxidative and nitrosative stress. Microarray analysis of mouse aortic endothelial cells revealed a 2.5‐fold increase in expression of calcium/calmodulin‐dependent protein kinase II‐α (CaMKII‐α) in response to 10 nM BPA, with increased expression of phosphorylated‐CaMKII‐α in carotid rings of BPA‐exposed mice, whereas CaMKII‐α inhibition with 100 nM autocamptide‐2‐related inhibitor peptide (AIP) reduced BPA‐mediated increase of superoxide. Administration of CaMKII‐α inhibitor KN 93 reduced BPA‐induced blood pressure and carotid blood velocity in mice, and reverted BPA‐mediated carotid constriction in response to treatment with AcH. Given that CaMKII‐α inhibition prevents BPA‐mediated high blood pressure, our data suggest that BPA regulates blood pressure by inducing AngII/CaMKII‐α uncoupling of eNOS.—Saura, M., Marquez, S., Reventun, P., Olea‐Herrero, N., Arenas, M.I., Moreno‐Gómez‐Toledano, R., Gómez‐Parrizas, M., Muñóz‐Moreno, C., González‐Santander, M., Zaragoza, C., Bosch, R.J. Oral administration of bisphenol A induces high blood pressure through angiotensin II/CaMKII‐dependent uncoupling of eNOS. FASEB J. 28, 4719–4728 (2014). www.fasebj.org