Functional roles of the neuropeptide PACAP in brain and pancreas

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide implicated in a broad variety of physiological processes. To assess PACAP's function in vivo, we recently generated PACAP knockout mice (PACAP(-/-)) and transgenic mice overexpressing PACAP specifically in the pancreas (PACAP-Tg). In PACAP(-/-) mice, we have demonstrated a marked phenotypic changes including a high early mortality rate, increased novelty-seeking behavior and abnormal explosive jumping in a novel environment, as well as reduced female fertility. In this paper, we reevaluated these phenotypes in terms of the genetic background of the mice. Genetic background appears to modulate critically the magnitude but not the general nature of the PACAP-null phenotype. In PACAP-Tg mice, we have recently demonstrated that enhanced glucose-induced insulin secretion with normal glucose tolerance, amelioration of streptozotocin-induced diabetes with increased beta-cell proliferation, and a trend towards an increase in total islet mass with age. Here we show that PACAP(-/-) mice exhibit significantly impaired glucose-induced insulin secretion but still have normal glucose tolerance. These observations suggest that PACAP may play important roles in and beyond the regulation of insulin release. Taken together, the mutant phenotypes revealed both expected and unexpected roles of PACAP in the brain and pancreatic functions.     

Comparative genetic studies on the APRR5 and APRR7 genes belonging to the APRR1/TOC1 quintet implicated in circadian rhythm, control of flowering time, and early photomorphogenesis

In Arabidopsis thaliana, a number of circadian-associated factors have been identified. Among those, TOC1 (TIMING OF CAB EXPRESSION 1) is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as Arabidopsis PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). Nonetheless, it is not very clear whether or not the APRR family members other than APRR1/TOC1 are also implicated in the mechanisms underlying the circadian rhythm. To address this issue further, here we characterized a set of T-DNA insertion mutants, each of which is assumed to have a severe lesion in each one of the quintet genes (i.e. APRR5 and APRR7). For each of these mutants (aprr5-11 and aprr7-11) we demonstrate that a given mutation singly, if not directly, affects the circadian-associated biological events simultaneously: (i) flowering time in the long-day photoperiod conditions, (ii) red light sensitivity of seedlings during the early photomorphogenesis, and (iii) the period of free-running rhythms of certain clock-controlled genes including CCA1 and APRR1/TOC1 in constant white light. These results suggest that, although the quintet members other than APRR1/TOC1 may not be directly integrated into the framework of the central oscillator, they are crucial for a better understanding of the molecular mechanisms underlying the Arabidopsis circadian clock.     

Characterization of plant circadian rhythms by employing Arabidopsis cultured cells with bioluminescence reporters

Recent intensive studies have begun to shed light on the molecular mechanisms underlying the plant circadian clock in Arabidopsis thaliana. During the course of these previous studies, the most powerful technique, elegantly adopted, was a real-time bioluminescence monitoring system of circadian rhythms in intact plants carrying a luciferase (LUC) fusion transgene. We previously demonstrated that Arabidopsis cultured cells also retain an ability to generate circadian rhythms, at least partly. To further improve the cultured cell system for studies on circadian rhythms, here we adopted a bioluminescence monitoring system by establishing the cell lines carrying appropriate reporter genes, namely, CCA1::LUC and APRR1::LUC, with which CCA1 (CIRCADIAN CLOCK-ASSOCIATED1) and APRR1 (or TOC1) (ARABIDOPSIS PSEUDO-RESPONSE REGULATORS1 or TIMING OF CAB EXPRESSION1) are believed to be the components of the central oscillator. We report the results that consistently supported the view that the established cell lines, equipped with such bioluminescence reporters, might provide us with an advantageous means to characterize the plant circadian clock.     

Defects in reproductive functions in PACAP-deficient female mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved neuropeptide and widely expressed in both brain and peripheral tissues, including several reproductive organs (e.g., testis and ovary). PACAP stimulates syntheses of several sexual hormones and steroids, suggesting it has possible roles in reproductive function. In this study, the role of PACAP in female reproductive functions such as fertility, mating behavior and maternal behaviors were investigated by using mice lacking PACAP (PACAP(-/-)). PACAP(-/-) females showed reduced fertility (the number of parturitions relative to the number of pairings). Mating experiments using vasectomized males revealed that mating frequency and its intervals in some PACAP(-/-) females were quite different (zero to eight times/4 weeks), whereas the frequency was relatively constant (two to three times/4 weeks) in wild-type females. In PACAP(-/-) females, maternal crouching behavior tended to decrease compared to wild-type females, although the influence of litter size on maternal behavior needs to be considered. These data suggest a role for endogenous PACAP in female reproductive processes.     

Involvement of intracellular Ca2+ elevation but not cyclic AMP in PACAP-induced p38 MAP kinase activation in PC12 cells

We have recently shown that in PC12 cells, pituitary adenylate cyclase-activating polypeptide (PACAP) and NGF synergistically stimulate PACAP mRNA expression primarily via a mechanism involving a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Here we have analyzed p38 MAPK activation by PACAP and the mechanism underlying this action of PACAP in PC12 cells. PACAP increased phosphorylation of p38 MAPK with a bell-shaped dose-response relationship and a maximal effect was obtained at 10(-8) M. PACAP (10(-8) M)-induced p38 MAPK phosphorylation was already evident at 2.5 min, maximal at 5 min, and rapidly declined thereafter. PACAP-induced p38 MAPK phosphorylation was potently inhibited by depletion of Ca(2+) stores with thapsigargin and partially inhibited by the phospholipase C inhibitor U-73122, L-type voltage-dependent calcium channel inhibitors nifedipine and nimodipine, and the Ca(2+) chelator EGTA, whereas the protein kinase C inhibitor calphostin C, the protein kinase A inhibitor H-89, the cAMP antagonist Rp-cAMP, and the nonselective cation channel blocker SKF96365 had no effect. These results indicate that PACAP activates p38 MAPK in PC12 cells through activation of a phospholipase C, mobilization of intracellular Ca(2+) stores, and Ca(2+) influx through voltage-dependent Ca(2+) channels, but not cyclic AMP-dependent mechanisms.     

Fission yeast Mor2/Cps12, a protein similar to Drosophila Furry,is essential for cell morphogenesis and its mutation induces Wee1-dependent G(2) delay

Fission yeast cells identify growing regions at the opposite ends of the cell, producing the rod-like shape. The positioning of the growth zone(s) and the polarized growth require CLIP170-like protein Tip1 and the Ndr kinase Orb6, respectively. Here, we show that the mor2/cps12 mutation disrupts the localization of F-actin at the cell ends, producing spherical cells and concomitantly inducing a G(2) delay at 36 degrees C. Mor2 is important for the localization of F-actin at the cell end(s) but not at the medial region, and is essential for the restriction of the growth zone(s) where Tip1 targets. Mor2 is homologous to the Drosophila Furry protein, which is required to maintain the integrity of cellular extensions, and is localized at both cell ends and the medial region of the cell in an actin-dependent fashion. Cellular localization of Mor2 and Orb6 was interdependent. The tyrosine kinase Wee1 is necessary for the G(2) delay and maintenance of viability of the mor2 mutant. These results indicate that Mor2 plays an essential role in cell morphogenesis in concert with Orb6, and the mutation activates the mechanism coordinating morphogenesis with cell cycle progression.     

Analysis of Genetic Polymorphism of Deoxyribonuclease I in Ovambo and Turk Populations Using a Genotyping Method

Deoxyribonuclease I (DNase I) polymorphism has been used as a valuable marker in genetic and clinical investigations. Six codominant alleles are known for DNase I, DNASE1*1, *2, *3, *4, and the recently discovered alleles *5 and *6. To detect these two new alleles, we added a new DNase I genotyping method based on both an allele-specific amplification and mismatched polymerase chain reaction (PCR). These methods were used to examine the distribution of DNase I genotypes in unrelated individuals from bloodstains of Ovambo and Turkish populations. The DNASE1*1 allele was found to be most dominant in the Ovambos. In contrast, Turks showed the highest allele frequency for DNASE1*2. This study is the first to demonstrate that there is a certain genetic heterogeneity in the worldwide distribution of DNase I polymorphism using the genotyping method of human DNase I polymorphism with PCR.     

Olfactory epithelium consisting of supporting cells and horizontal basal cells in the posterior nasal cavity of mice

The olfactory epithelium of mice generally consists of olfactory cells, progenitors of olfactory cells (globose basal cells), supporting cells, and horizontal basal cells. However, in the dorsal fossa (the roof) of the posterior nasal cavity of mice, we found seven epithelial patches consisting of only non-neuronal cell types, i.e., supporting cells and horizontal basal cells, among the normal olfactory epithelium. The supporting cells occupied three or four layers in the apical to middle regions; in the basal region, horizontal basal cells were localized in a single row adjacent to the basement membrane. Bowman's gland ducts were also present in the epithelium. Neuronal cells (olfactory cells and globose basal cells) were totally absent. The ultrastructure of the supporting cells, horizontal basal cells, and Bowman's glands was essentially similar to that in the normal olfactory epithelium. In the early postnatal period (P1-P7), cell types in the epithelium were the same as those in the normal olfactory epithelium. From P10 to P21, olfactory cells and globose basal cells had disappeared from the olfactory epithelium. At this period, the number of TUNEL-positive cells was significantly higher than that in the surrounding olfactory epithelium; ultrastructurally, many apoptotic figures were observed. This suggests that the epithelium consisting of supporting cells and horizontal basal cells is generated by the apoptotic death of olfactory cells and globose basal cells during postnatal development.