Spiroquinazolinones as novel, potent, and selective PDE7 inhibitors. Part 1

The synthesis and SAR studies of spiroquinazolinones as novel PDE7 inhibitors are discussed. The best compounds from the series displayed nanomolar inhibitory affinity and were selective versus other PDE isoenzymes.     

Galphaq-coupled receptor internalization specifically induced by Galphaq signaling. Regulation by EBP50

In the present report, we investigated the effect of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) expression on the agonist-induced internalization of the thromboxane A(2) beta receptor (TPbeta receptor). Interestingly, we found that EBP50 almost completely blocked TPbeta receptor internalization, which could not be reversed by overexpression of G protein-coupled receptor (GPCR) kinases and arrestins. Because we recently demonstrated that EBP50 can bind to and inhibit Galpha(q), we next studied whether Galpha(q) signaling could induce TPbeta receptor internalization, addressing the long standing question about the relationship between GPCR signaling and their internalization. Expression of a constitutively active Galpha(q) mutant (Galpha(q)-R183C) resulted in a robust internalization of the TPbeta receptor, which was unaffected by expression of dominant negative mutants of arrestin-2 and -3, but inhibited by expression of EBP50 or dynamin-K44A, a dominant negative mutant of dynamin. Phospholipase Cbeta and protein kinase C did not appear to significantly contribute to internalization of the TPbeta receptor, suggesting that Galpha(q) induces receptor internalization through a phospholipase Cbeta- and protein kinase C-independent pathway. Surprisingly, there appears to be specificity in Galpha protein-mediated GPCR internalization. Galpha(q)-R183C also induced the internalization of CXCR4 (Galpha(q)-coupled), whereas it failed to do so for the beta(2)-adrenergic receptor (Galpha(s)-coupled). Moreover, Galpha(s)-R201C, a constitutively active form of Galpha(s), had no effect on internalization of the TPbeta, CXCR4, and beta(2)-adrenergic receptors. Thus, we showed that Galpha protein signaling can lead to internalization of GPCRs, with specificity in both the Galpha proteins and GPCRs that are involved. Furthermore, a new function has been described for EBP50 in its capacity to inhibit receptor endocytosis.     

Lysosomal and proteasomal degradation play distinct roles in the life cycle of Cx43 in gap junctional intercellular communication-deficient and -competent breast tumor cells

The present study was designed to determine the specific roles played by lysosomes and proteasomes in the degradation of Cx43 in both gap junctional intercellular communication-deficient MDA-MB-231 and -competent BICR-M1Rk cells. In MDA-MB-231 cells, immunolocalization and brefeldin A protein transport blocking studies revealed that there was a propensity for newly synthesized Cx43 to be transported to lysosomes. On the other hand, light and electron microscopic analysis of BICR-M1Rk cells showed that Cx43 gap junctions were prevalent with a subpopulation of intracellular Cx43 localized to lysosomes. In both cell types, Western blots revealed a notable increase in total cellular Cx43 in response to lysosome inhibitors. Interestingly, lactacystin inhibition of proteosomal degradation in MDA-MB-231 cells resulted in a marked increase in phosphorylated Cx43 at the expense of non-phosphorylated Cx43, and this change corresponded with an increase in "oversized" gap junction plaques. In BICR-M1Rk cells, lactacystin treatment partially prevented the BFA-induced loss of gap junctions. Together, our data suggests that lysosomes play a key role in not only degrading internalized gap junction in BICR-M1Rk cells but also in degrading Cx43 delivered from early secretory compartments to lysosomes in MDA-MB-231 cells. Overall proteasomal degradation regulates the stability of phosphorylated Cx43 and appears to promote the internalization of Cx43 from the cell surface.     

Benign paratesticular fibrous pseudotumor with malignant clinical features

Paratesticular tumors are rare intrascrotal masses that grow mainly in structures containing connective tissue around the testis, epididymis, or spermatic cord. We report here on a case of a 55-year-old man with a mass in the hemiscrotum that was found to be a paratesticular fibrous pseudotumor in the orchiectomy specimen pathologic examination. From this case report and literature review, a diagnostic and therapeutic strategy is demonstrated for the management of this uncommon lesion.     

Concomitant sertoli and leydig cell tumor of the testis: a case report

A rare intratubular gonadal stromal tumor was present in the testis of a 45-year-old man who was admitted to our hospital with the chief complaint of gradual enlargement of the left testis. Tumoral markers were negative and no extension was observed. The tumor comprised an intratubular mixture of two types of tumor cells with intercellular junctions: the predominant tumor cells were consistent with a Sertoli cell origin and cells comprising the minor population consistent with a Leydig cell origin. The patient is disease free after 6-month follow-up. The case is considered to be a testicular mixed tubular Sertoli-Leydig cell tumor. It highlights a rare type of primary tumor of the testis that features a good prognosis.     

Role of the Rab11-associated intracellular pool of receptors formed by constitutive endocytosis of the beta isoform of the thromboxane A2 receptor (TP beta)

Intracellular trafficking pathways of G protein-coupled receptors (GPCRs), following their agonist-induced endocytosis and their consequences on receptor function, are the subject of intense research efforts. However, less is known regarding their constitutive endocytosis. We previously demonstrated that the beta isoform of the thromboxane A(2) receptor (TPbeta) undergoes constitutive and agonist-induced endocytosis. Constitutive endocytosis of GPCRs can lead to the formation of an intracellular pool of receptors from which they can recycle back to the cell surface. In the present report, we show with the help of two TPbeta mutants (TPbeta-Y339A and TPbeta-I343A) specifically deficient in constitutive endocytosis that this intracellular pool of receptors serves to maintain agonist sensitivity over prolonged receptor stimulation in HEK293 cells. Second messenger generation by the TPbeta-Y339A and TPbeta-I343A mutants was drastically reduced compared to the wild-type receptor as suggested by dose-response and time-course experiments of inositol phosphates production following agonist treatment, despite normal coupling between the receptors and the Galpha(q) protein. Moreover, second messenger production after receptor activation was dramatically reduced when cells were pretreated with monensin, a recycling inhibitor. Receptor cell surface expression and endocytosis experiments further revealed that the small GTPase Rab11 protein is a determinant factor in controlling TPbeta recycling back to the cell surface. Co-localization experiments performed by immunofluorescence microscopy indicated that both constitutive and agonist-triggered endocytosis resulted in targeting of TPbeta to the Rab11-positive recycling endosome. Thus, we provide evidence that constitutive endocytosis of TPbeta forms a pool of receptors in the perinuclear recycling endosome from which they recycle to the cell surface, a process involved in preserving receptor sensitivity to agonist stimulation.     

Design and validation of an orally administrated active L. fermentum-L. acidophilus probiotic formulation using colorectal cancer Apc Min/+ mouse model

Probiotics have been shown to have beneficial properties in attenuating the risk of colorectal cancer (CRC) development. However, functional evidence to support such effects for some probiotic bacteria are relatively unknown. Here, we document a significant antioxidant, anti-proliferative and pro-apoptotic activities of Lactobacillus acidophilus ATCC 314 and Lactobacillus fermentum NCIMB 5221 on CRC cells, particularly when used in combination (La-Lf). Furthermore, a superior synergistic activity on the inhibition of tumor growth and modulation of cell proliferation and epithelial markers in the Apc ^Min/+ CRC mouse model was explored, based on the expression levels of Ki-67, E-cadherin, β-catenin, and cleaved caspase-3 (CC3) proteins. The anti-cancer activity of La-Lf co-culture was significantly enhanced in vitro with significant reduced proliferation (38.8 ± 6.9 %, P = 0.009) and increased apoptosis (413 RUL, P < 0.001) towards cancer cells, as well as significant protection of normal colon cell growth from toxic treatment (18.6 ± 9.8 %, P = 0.001). La-Lf formulation (10¹⁰cfu/animal/day) altered aspects of intestinal tumorigenesis by significantly reducing intestinal tumor multiplicity (1.7-fold, P = 0.016) and downregulating cellular proliferation markers, including β-catenin (P = 0.041) and Ki-67 (P = 0.008). In conclusion, La-Lf showed greater protection against intestinal tumorigenesis supporting a potential use as a biotherapeutic for the prevention of CRC.