Intracellular cAMP controls a physical association of V-1 with CapZ in cultured mammalian endocrine cells

V-1, an ankyrin repeat protein with the activity to control tyrosine hydroxylase (TH) gene expression and transmitter release in PC12D cells, associates with CapZ, an actin capping protein, and thereby regulates actin polymerization in vitro. In this study, immunoprecipitation and Western blot analysis showed that V-1 was physically associated with CapZ-beta in PC12D transfectants overexpressing V-1. These proteins were co-localized in the soma of Purkinje cells of rat cerebellum as assayed by immunohistochemistry. Furthermore, in the V-1 transfectants, the amount of CapZ which physically associated with V-1 was steeply reduced at 2h after treatment with forskolin, but was thereafter increased to reach its initial level at 12h after forskolin-treatment. These results suggest that the association of V-1 with CapZ is controlled by a cAMP-dependent signalling pathway probably to play a functional role in the regulatory mechanism of actin dynamics in the endocrine system and the central nervous system.     

Contraction and stiffness changes in collagenous arm ligaments of the stalked crinoid Metacrinus rotundus (Echinodermata)

Shortening and stiffness were measured simultaneously in the aboral ligament of arms of sea lilies. Arm pieces were used from which oral tissues (including muscles) were removed, leaving only collagenous ligaments connecting arm ossicles. Chemical stimulation by means of artificial seawater with an elevated concentration of potassium caused both a bending movement and stiffness changes (either softening or stiffening). The movement lasted for 1.5-10 min, and bent posture was maintained. The observation that contraction was not necessarily associated with softening provided evidence against the hypothesis that the shortening of the aboral ligaments was driven by the elastic components that had been charged by the oral muscles and released their strain energy at the softening of the aboral ligaments. The speed of ligamental shortening was slower by at least one order of magnitude than that of muscles. Acetylcholine (10(-5)-10(-3) M) caused both contraction and softening. We conclude that the aboral ligament shows two mechanical activities based on different mechanisms: one is active contraction and the other is connective tissue catch in which passive mechanical properties show mutability. We suggest that there is neural coordination between the two mechanisms.     

Evidence for the presence of a threshold weight for entering diapause in the yellow-spotted longicorn beetle, Psacothea hilaris

Under long-day conditions larvae of Psacothea hilaris (Coleoptera: Cerambycidae) pupate after the 4th or 5th instar, while under short-day conditions they undergo 2-4 nonstationary supernumerary molts and eventually enter diapause. To explore the possibility of a threshold weight for entering diapause, P. hilaris larvae were deprived of food on days 0 (day of ecdysis), 4 or 8 of the 4th, 5th and 6th instars under short-day conditions. Within the first 40 days of starvation, 60% of the larvae starved starting on day 0 of the 4th instar died, but all the larvae starved at later stages survived. The incidence of diapause in these survivors was determined by the occurrence of pupation after a temporary chilling at 15 degrees C for 15 days. Diapause incidence increased as the onset of starvation was delayed; from 11% in the larvae starved on day 0 of the 5th instar to 100% in the larvae starved on day 4 and day 8 of the 6th instar. Analysis of the relationship between the initial weight of a respective larva at the onset of starvation and its pupation success revealed that none of the larvae weighing 690 mg did. This finding suggests the presence of a threshold weight (about 600 mg), below which larvae are incapable of entering diapause. We discuss these findings with reference to the life history of P. hilaris.     

Expression of ameloblastin during enamel formation in a crocodile

Ameloblastin is an enamel-specific protein that plays critical roles in enamel formation, as well as adhesion between ameloblasts and the enamel matrix, as shown by analyses of ameloblastin-null mice. In the present study, we produced two distinct antibodies that recognize the N-terminus and C-terminus regions of caiman ameloblastin, in order to elucidate the fate of ameloblastin peptides during tooth development. An immunohistochemical study using the antibodies showed that caiman ameloblastin was a tooth-specific matrix protein that may initially be cleaved into two groups, N- and C-terminal peptides, as shown in mammals. The distribution of the N-terminal peptides was much different from that of the C-terminal peptides during enamel formation; however, it was similar to that of mammalian ameloblastin. Although ameloblastin is thought to have a relationship with the enamel prismatic structure in mammals, in the caiman, which has non-prismatic enamel, functional ameloblastin has no relationship with any enamel structure. Consequently, it is suggested that ameloblastin has kept its original functions during the evolutionary transition from reptiles to mammals and that it has been conserved in both lineages during more than 200 million years of evolution. Our results support the notion that ameloblastin acts as a factor for ameloblast adhesion to enamel matrix, because distribution of the C-terminal peptides was consistently restricted on the surface layers of enamel matrix specimens ranging from immature to nearly completely mature. The principal molecules that provide the adhesive function are presumably C-terminal peptides.     

Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2-and TLR4-independent signaling

Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P. gingivalis cells and activation of nuclear factor-kappaB in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P. gingivalis, they responded to gingipain-deficient P. gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P. gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P. gingivalis but have inhibitory effects on TLR2-and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P. gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P. gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of P. gingivalis that were able to induce TLR2-and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P. gingivalis to escape from the innate immune system.     

Parkin potentiates ATP-induced currents due to activation of P2X receptors in PC12 cells

Loss-of-function mutations of the parkin gene causes an autosomal recessive juvenile-onset form of Parkinson's disease (AR-JP). Parkin was shown to function as a RING-type E3 ubiquitin protein ligase. However, the function of parkin in neuronal cells remains elusive. Here, we show that expression of parkin-potentiated adenosine triphosphate (ATP)-induced currents that result from activation of the P2X receptors which are widely distributed in the brain and involved in neurotransmission. ATP-induced inward currents were measured in mock-, wild-type or mutant (T415N)-parkin-transfected PC12 cells under the conventional whole-cell patch clamp configuration. The amplitude of ATP-induced currents was significantly greater in wild-type parkin-transfected cells. However, the immunocytochemical study showed no apparent increase in the number of P2X receptors or in ubiquitin levels. The increased currents were attenuated by inhibition of cAMP-dependent protein kinase (PKA) but not protein kinase C (PKC) or Ca2+ and calmodulin-dependent protein kinase (CaMKII). ATP-induced currents were also regulated by phosphatases and cyclin-dependent protein kinase 5 (CDK5) via dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32), though the phosphorylation at Thr-34 and Thr-75 were unchanged or rather attenuated. We also tried to investigate the effect of alpha-synuclein, a substrate of parkin and also forming Lysine 63-linked multiubiquitin chains. Expression of alpha-synuclein did not affect the amplitude of ATP-induced currents. Our finding provides the evidence for a relationship between parkin and a neurotransmitter receptor, suggesting that parkin may play an important role in synaptic activity.     

On the respiratory mechanism during underwater oviposition in a damselfly Calopteryx cornelia Selys

Calopteryx cornelia females oviposit almost exclusively underwater in forest streams. Field observation showed that the duration of uninterrupted submerged oviposition ranged between 20 and 120 min and the number of eggs laid was linearly related to the time spent underwater. By holding a damselfly under water in a small jar, we measured the maximum 'submergence potential', which was defined as the time elapsed between placing the insect underwater and asphyxiation. A series of experiments showed that there was no gender difference in the submergence potential. This was about 120 min if a damselfly was allowed to change its position while under water. The submergence potential was shorter if the damselflies were kept motionless, if air bubbles trapped on the wing surfaces were removed by coating with Vaseline or if the water was hypoxic. By contrast, submergence potential was longer if a part of the wings were kept above the water surface, or if the water was agitated using a magnetic stirrer. These results suggest that ovipositing C. cornelia females depend for oxygen on the physical-gill action of the thin air layer trapped on the body and wing surfaces. Respiration capacity under water is not likely to be a limiting factor for ovipositing females during the production of a single clutch.     

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.