Overexpression of cyclin‐dependent kinase 4 protein in extramammary Paget's disease

Cyclin‐dependent kinase 4 and 6 (CDK4/6) plays an important role in cell cycle progression, and the CDK4/6–cyclin D1 complex controls the cell cycle transition from G₁ phase to S phase. CDK4 is enhanced in several types of cancers and CDK4/6 inhibitors attenuate the proliferation of several types of cancer in vitro/in vivo. The purpose of our study was to investigate the expression pattern of CDK4 and evaluate its clinical importance in extramammary Paget's disease (EMPD). Almost all EMPD tissues were positive for CDK4, and metastatic lesions had a similar immunostaining intensity to primary lesions. In addition, CDK4 protein levels were positively correlated with those of cyclin D1 protein. Taken together, CDK4 may assume a crucial role in EMPD progression.     

Role of Neurokinin B and Dynorphin A in pituitary gonadotroph and somatolactotroph cell lines

The role of Neurokinin B (NKB) and Dynorphin A (Dyn) in the regulation of the hypothalamic pituitary axis is an important area of recent investigation. These peptides are critical for the rhythmic release of GnRH, which subsequently stimulates the secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The present study utilized the gonadotroph cell line LβT2 and the somatolactotroph GH3 cell line to examine the possible role of these peptides in pituitary hormone secretion. The NKB receptor (NK3R) and the Dyn receptor (the κ-opiate receptor (KOR)) were both detected in LβT2 cells and GH3 cells. NKB, by itself, failed to increase gonadotropin LHβ and FSHβ promoter activities and did not modulate the effects of GnRH on gonadotropin promoter activity. In GH3 cells, NKB significantly increased TRH-induced PRL promoter activity although NKB alone did not have an effect on basal PRL promoter activity. Dyn had no effect on gonadotropin promoters alone or in combination with GnRH stimulation. PRL promoters stimulated by TRH were not significantly changed by Dyn. TRH-induced PRL promoter activity was further increased in the presence of higher concentrations of NKB, whereas Dyn did not have a significant effect on the PRL promoter even at a high concentration. In addition, TRH-induced ERK (Extracelluar signal-regulated kinase) activation was enhanced in the presence of NKB. Our current study demonstrated that NKB had a stimulatory effect on PRL expression in a PRL-producing cell, but had no effect on gonadotropin secretion from a gonadotroph cell line.     

Detection of mutans streptococci in plaque samples from Mongolian preschool and school children

Mutans streptococci, in particular Streptococcus mutans and Streptococcus sobrinus, are generally considered to be the principal microbial pathogen of dental caries. The objective of the study was to isolate S. mutans and S. sobrinus, identify them by PCR, and to compare their presence with the caries status and caries risk in Mongolian preschool and school children. Forty one preschool children aged 3–5 years and 40 school children aged 12–15 years were enrolled in this study. As assessed using Cariostat test, 75.6% of preschool children had high caries risk and 37.5% of school children had high caries risk. In preschool children, the prevalence of S. mutans and S. sobrinus were 100% and 36.6%, respectively; 63.4% were positive for S. mutans alone and 36.6% were positive for both S. mutans and S. sobrinus. In school children, the prevalence of S. mutans and S. sobrinus were 100% and 25.0%, respectively; 75.0% carried S. mutans alone and 25.0% had both S. mutans and S. sobrinus. The percentage of children positive for both S. mutans and S. sobrinus in the high caries risk group were significantly higher than those in the low risk group of either preschool (42.0% vs. 10.0%, P< 0.001) or school children (46.6% vs. 12.0%, P<0.001). Moreover, the caries status of children positive for both S. mutans and S. sobrinus were significantly higher than those positive for S. mutans alone (P< 0.01 for preschool children, and P< 0.05 for school children).     

Prolonged stimulation with thyrotropin-releasing hormone and pituitary adenylate cyclase-activating polypeptide desensitize their receptor functions in prolactin-producing GH3 cells

We used somatolactotroph GH3 cells to examine changes in response to stimulation with thyrotropin-releasing hormone (TRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) after sustained treatment with these peptides. TRH and PACAP increased prolactin promoter activity in mock- and PACAP type 1 receptor (PAC1R)-transfected cells. When the cells were pretreated with TRH for 48 h, the response of the prolactin promoter to both TRH and PACAP was diminished. Similarly, in PAC1R-transfected GH3 cells pretreated with PACAP, the effects of TRH and PACAP on the prolactin promoter were eliminated. The stimulation of prolactin mRNA expression by TRH and PACAP was eliminated by prolonged pretreatment with these peptides in PAC1R-transfected cells. Both the serum response element (SRE) promoters and cAMP response element (CRE) promoters were activated by TRH and PACAP in either mock- or PAC1R-transfected cells. Pretreatment for 48 h with TRH also eliminated the effects of TRH and PACAP on the SRE and CRE promoters, and pretreatment of PAC1R-transfected cells with PACAP for 48 h reduced the responses of the SRE and CRE promoters to TRH and PACAP. These observations demonstrated that sustained stimulation with TRH and PACAP desensitizes their own and each other’s receptors.     

Stimulatory effect of pituitary adenylate-cyclase activating polypeptide (PACAP) and its PACAP type I receptor (PAC1R) on prolactin synthesis in rat pituitary somatolactotroph GH3 cells

In this present study, we investigated the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor, PACAP type I receptor (PAC1R) on prolactin synthesis in pituitary somatolactotroph GH3 cells. PACAP increased prolactin promoter activity up to 1.3 ± 0.1-fold. This increase, while significant, was less than the increase resulting from thyrotropin-releasing hormone (TRH) stimulation. By transfection of a PAC1R expression vector to the cells, the response to PACAP on prolactin promoter activity was dramatically potentiated to a degree proportional to the amount of PAC1R transfected. In the PAC1R expressing GH3 cells, TRH and PACAP alone increased prolactin promoter up to 3.3 ± 0.3-fold and 4.9 ± 0.2-fold, respectively, and combined treatment with TRH and PACAP further increased prolactin promoters up to 6.8 ± 0.6-fold. PACAP binds both Gs- and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/extracellular signal-regulated kinase (ERK) signaling pathways. PACAP increased ERK phosphorylation in PAC1R expressing cells to the same degree as TRH. Combined treatment with TRH and PACAP had a synergistic effect on ERK activation. GH3 cells produce both prolactin and growth hormone. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin and attenuated growth hormone mRNA expression. PACAP increased both prolactin and growth hormone mRNA levels, particularly in PAC1R expressing cells. In addition, increasing amount of PAC1R in GH3 cells potentiated the action of TRH on prolactin promoter activity, as well as on ERK phosphorylation. PAC1R was induced by PACAP itself, but not by TRH. Our current study demonstrates that PACAP and its PAC1R, functions as a stimulator of prolactin alone or with TRH in prolactin producing cells.     

Mucosal immune features to phosphorylcholine by nasal Flt3 ligand cDNA-based vaccination

Phosphorylcholine (PC) is an immunodominant epitope in some pathogens including Streptococcus pneumoniae and it is well-known that PC-specific antibodies (Abs) play a key role in the induction of protective immunity against pneumococcal infection. In this study, we examined whether nasal administration of DNA plasmid encoding Flt3 ligand gene (pFL) as a mucosal adjuvant plus PC-conjugated keyhole limpet hemocyanin (PC-KLH), would elicit PC-specific immune responses, and characterized mucosal immune responses to PC induced by this nasal vaccination. Nasal immunization with pFL plus PC-KLH enhanced induction of PC-specific IgA and IgM Abs in airway secretions when compared with mice given PC-KLH with or without empty plasmid gene (pORF) as controls; in addition to the mucosal immune responses, PC-specific immune responses in serum were also induced. Furthermore, the mucosal and serum IgA and IgM Abs in mice given pFL plus PC-KLH nasally, exhibited high-specificity for the PC molecule. Of interest, the PC-specific Abs bound dose-dependently to anti-T15 idiotype (AB1-2). Thus, the inhibition of S. pneumoniae colonization on the nasal cavity and lungs after nasal challenge with the live organism was significantly elicited in mice immunized with pFL plus PC-KLH compared to that of mice immunized with antigen with pORF. Taken together, these findings show that nasal administration of pFL with PC-KLH elicited T15-like anti-PC IgA and IgM Abs in the respiratory tracts, and further attenuated S. pneumoniae colonization on the respiratory tracts. Nasal administration of Flt3 ligand cDNA with PC may contribute to the development of nasal vaccination for prevention of S. pneumoniae infection.     

Pulsatile kisspeptin effectively stimulates gonadotropin-releasing hormone (GnRH)-producing neurons

Hypothalamic kisspeptin is integral to the hypothalamic–pituitary–gonadal axis by stimulating gonadotropin-releasing hormone (GnRH) release. GnRH is released from the hypothalamus in a pulsatile manner and determines the output of the gonadotropins. However, the effect of kisspeptin on GnRH-secreting cells remains unknown. In an experiment using static cultures of GT1-7 cells, kisspeptin did not significantly increase GnRH mRNA expression. However, when kisspeptin was administered to the cells in a pulsatile manner, GnRH mRNA expression was significantly increased. Primary cultures of fetal rat brain containing GnRH-expressing neurons responded to kisspeptin and increased GnRH mRNA expression by 1.65?±?0.27-fold in the static condition. When cells were stimulated with kisspeptin in a pulsatile manner, GnRH mRNA expression was increased by up to 2.40?±?0.21-fold. In perifused GT1-7 cells, pulsatile, but not continuous kisspeptin stimulation, effectively stimulated GnRH mRNA expression. To assess the level of stimulation of GnRH neurons by kisspeptin, the expression of c-fos was examined. In GT1-7 cells, kisspeptin stimulation in the static condition failed to increase c-fos mRNA expression. However, pulsatile kisspeptin stimulation increased c-fos mRNA by 2.31?±?0.47-fold. Similar to the phenomenon observed in GT1-7 cells, pulsatile, but not static, kisspeptin stimulation significantly increased c-fos mRNA expression in the primary cultures of fetal rat brain. These observations suggest that pulsatile kisspeptin more effectively stimulates GnRH-producing cells to increase the production of GnRH.