Phosphorylation profile of human AQP2 in urinary exosomes by LC-MS/MS phosphoproteomic analysis.

BACKGROUND: Aquaporin-2 (AQP2) is a key water channel protein which determines the water permeability of the collecting duct. Multiple phosphorylation sites are present at the C-terminal of AQP2 including S256 (serine at 256 residue), S261, S264 and S/T269, which are regulated by vasopressin (VP) to modulate AQP2 trafficking. As the dynamics of these phosphorylations have been studied mostly in rodents, little is known about the phosphorylation of human AQP2 which has unique T269 in the place of S269 of rodent AQP2. Because AQP2 is excreted in urinary exosomes, the phosphoprotein profile of human AQP2 can be easily examined through urinary exosomes without any intervention. METHODS: Human urinary exosomes digested with trypsin or glutamyl endopeptidase (Glu-C) were examined by the liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) phosphoproteomic analysis. RESULTS: The most dominant phosphorylated AQP2 peptide identified was S256 phosphorylated form (pS256), followed by pS261 with less pS264 and far less pT269, which was confirmed by the western blot analyses using phosphorylated AQP2-specific antibodies. In a patient lacking circulating VP, administration of a VP analogue showed a transient increase (peak at 30-60 min) in excretion of exosomes with pS261 AQP2. CONCLUSION: These data suggest that all phosphorylation sites of human AQP2 including T269 are phosphorylated and phosphorylations at S256 and S261 may play a dominant role in the urinary exosomal excretion of AQP2.

AQP2 in human urine is predominantly localized to exosomes with preserved water channel activities.

BACKGROUND: AQP2 water channel is critical for urinary concentration in the kidney. Interestingly, AQP2 is abundantly excreted in the urine as extracellular vesicles (EVs), which is known to be a useful biomarker for water-balance disorders although the character of AQP2-enriched EVs is poorly understood including water channel function. METHODS: Human urine EVs were obtained by a differential centrifugation method. AQP2-bearing EVs were isolated by immunoprecipitation with an AQP2-specific antibody, and the proteins in the EVs were analyzed by LC-MS/MS proteomic analysis. Osmotic water permeability (Pf) of the AQP2-rich EVs was measured by a stopped-flow method monitoring scattered light intensity in response to outwardly directed osmotic gradient. RESULTS: Sequential centrifugation of human urine showed that AQP2 was present predominantly (80%) in low-density EVs (160,000 g), whereas negligible amount in high-density EVs (17,000 g). Proteomic analysis of the AQP2-bearing EVs identified 137 proteins, mostly in the endosome pathway, including the components of ESCRT (endosomal sorting complex required transporter)-I, II, III. Pf value of the 160,000 g EVs was 4.75 ± 0.38 × 10-4 cm s-1 (mean ± SE) with the activation energy of 3.51 kcal mol-1 which was inhibited with 0.3 mM HgCl2 by 63%, suggesting a channel-mediated water transport. Moreover, Pf value showed a significant correlation with the abundance of AQP2 protein in EVs. CONCLUSION: Taken together, AQP2 is localized predominantly to urinary exosomes with preserved water channel activities.

Genital Autocleaning in the Male Cricket Gryllus bimaculatus (2): Rhythmic Movements of the Genitalia and Their Motor Control.

Three types of genital movement, their neural controls, and functional roles were investigated to gain a better understanding of the mechanism underlying autocleaning in the male cricket. The membrane complex consisting of the median pouch and genital chamber floor shows peculiar undulation that is composed of two types of movements: a right-left large shift and small crease-like movements. The large shift was caused by contraction of a pair of muscles (MPA) located anterior to the median pouch, while the crease-like movements were caused by numerous muscle fibers extending over the membrane complex. The MPA and muscle fibers were each innervated by efferent neurons in the terminal abdominal ganglion. Experiments with artificial dirt mimicking a foreign object revealed that the crease-like movements were responsible for dirt transport, while the large shift participated in sweeping the dirt into the lateral pouch as a trash container. On the other hand, the dorsal pouch serving as a template for the spermatophore showed a jerky bending movement. Simultaneous monitoring of the membrane complex and dorsal pouch activities suggested that their movements cooperate to enable the efficient evacuation of waste in the dorsal pouch. Based on the results, we conclude that genital autocleaning supports the production of the spermatophore.

Genital Autocleaning in the Male Cricket Gryllus bimaculatus (1): Structure and Function of the Genital Membrane.

We found that the genitalia of the male cricket Gryllus bimaculatus are equipped with an autocleaning system. The cricket keeps its genitalia clean by removing foreign matter and endogenous waste. Morphological study showed that the membrane complex consists of a median pouch and a genital chamber floor covered by small scales, each of which has a base of approximately 10 µm in width and a fringe with 5-10 spines 3-20 µm in length. The scales are arranged symmetrically about the midline, curving gradually in the lateral direction and continuing to the lateral pouch serving as a trash container. Observation of cleaning revealed that a small piece of artificial dirt placed on the membrane complex was conveyed over a distance of 1.3 mm to the lateral pouch in 12 minutes. Inspection of the dorsal pouch just after spermatophore extrusion in the mating stage revealed that there were patchy remnants of spermatophore material on the inner surface of the pouch, but that these were evacuated in a few minutes. Surgical elimination of the median pouch caused the formation of abnormal spermatophores with the sperm tube and attachment plate being deformed. These results suggest that genital autocleaning is indispensable for the production of a normal spermatophore in the male cricket.

Calling and courtship behaviors initiated by male-male contact via agonistic encounters in the cricket Gryllus bimaculatus.

We investigated the stridulatory courtship initiated by male-male contact after agonistic encounters and the effect of dominance status on subsequent reproductive behavior in the cricket Gryllus bimaculatus. When two male crickets were kept together in a small area, their dominance status was quickly established through fighting or non-fighting interactions. Approximately 10 min after pairing, most dominant males produced calling and/or courtship songs in the presence of subordinate males. This behavior appeared to be triggered by some contact chemicals on the body surface of the males. Stimulation using the forewing of a neutral male induced courtship in dominant males at a higher level compared with neutral males which were not previously paired with males. These observations suggest that the sexual motivation in dominant males increased because of previous agonistic interactions. In contrast, subordinate males remained silent. Stimulation using the male forewing induced stridulation to a lesser degree in subordinate males than in neutral males, suggesting decreased sexual motivation in subordinate males. Furthermore, only 40% of subordinate males exhibited courtship behavior under triadic conditions (dominant male, subordinate male and female) in contrast with 100% in male-female pairs. This result reveals that subordinate males, being less sexually motivated, are continuously suppressed in their courtship by intermittent attacks by the nearby dominant males. In the other triadic condition where the males were allowed to copulate, 65% of the dominant males copulated, while none of the subordinates did. These results suggest that dominant males have a greater chance to copulate and produce offspring.

Hepatitis B surface antigen incorporated in dissolvable microneedle array patch is antigenic and thermostable.

Alternatives to syringe-based administration are considered for vaccines. Intradermal vaccination with dissolvable microneedle arrays (MNA) appears promising in this respect, as an easy-to-use and painless method. In this work, we have developed an MNA patch (MNAP) made of hydroxyethyl starch (HES) and chondroitin sulphate (CS). In swines, hepatitis B surface antigen (HBsAg) formulated with the saponin QS-21 as adjuvant, both incorporated in HES-based MNAP, demonstrated the same level of immunogenicity as a commercially available aluminum-adjuvanted HBsAg vaccine, after two immunizations 28 days apart. MNAP application was associated with transient skin reactions (erythema, lump, scab), particularly evident when the antigen was delivered with the adjuvant. The thermostability of the adjuvanted antigen when incorporated in the HES-based matrix was also assessed by storing MNAP at 37, 45 or 50 °C for up to 6 months. We could demonstrate that antigenicity was retained at 37 and 45 °C and only a 10% loss was observed after 6 months at 50 °C. Our results are supportive of MNAP as an attractive alternative to classical syringe-based vaccination.

Copulation in the cricket is performed by chain reaction.

The male and female genitalia are finely designed to match each other for copulation in the cricket Gryllus bimaculatus. Copulatory acts of the male, stereotyped and time-fixed, are elicited by stimulation of mechanoreceptors on particular regions of the abdomen, cerci and genitalia. Sequential execution of each motor act proceeds as a chain reaction in which one act stimulates some receptors which in turn elicits another act and so on, while the female remains immobile on the male's back. Each key stimulus for a motor act appears as a result of the male's own act, except for copulatory papilla protrusion by the female. The final sequence of spermatophore extrusion and transfer are irreversible fixed motor actions which are triggered when the female copulatory papilla stimulates the epiphallic hairs. They proceed without continual central drive from the brain, and apparently without sensory feedback. In addition, they are well coordinated with movement and posture in the entire body. Some neural mechanisms of controlling mating behavior and switching the reproductive cycle are discussed.

Morphological and functional recovery of the planarian photosensing system during head regeneration.

When exposed to light, planarians display a distinctive light avoidance behavior known as negative phototaxis. Such behavior is temporarily suppressed when animals are decapitated, and it is restored once the animals regenerate their heads. Head regeneration and the simple but reproducible phototactic response of planarians provides an opportunity to study the association between neuronal differentiation and the establishment of behavior in a simple, experimentally tractable metazoan. We have devised a phototaxis assay system to analyze light response recovery during head regeneration and determined that light evasion is markedly re-established 5 days after amputation. Immunohistological and in situ hybridization studies indicate that the photoreceptors and optic nerve connections to the brain begin by the fourth day of cephalic regeneration. To experimentally manipulate the light response recovery, we performed gene knockdown analysis using RNA interference (RNAi) on two genes (1020HH and eye53) previously reported to be expressed at 5 days after amputation and in the dorso-medial region of the brain (where the optic nerves project). Although RNAi failed to produce morphological defects in either the brain or the visual neurons, the recovery of the phototactic response normally observed in 5-day regenerates was significantly suppressed. The data suggest that 1020HH and eye53 may be involved in the functional recovery and maintenance of the visual system, and that the phototaxis assay presented here can be used to reliably quantify the negative phototactic behavior of planarians.

Pharmacokinetic and pharmacodynamic profiles of canagliflozin in Japanese patients with type 2 diabetes mellitus and moderate renal impairment.

BACKGROUND AND OBJECTIVES: This study examined the effects of moderate renal impairment on the pharmacokinetics and pharmacodynamics of canagliflozin in Japanese patients with type 2 diabetes mellitus. METHODS: Japanese patients with stable type 2 diabetes (12 with moderate renal impairment and 12 with normal renal function or mild renal impairment) were eligible. This was an open-label, randomized, two-way crossover, two-sequence, single-dose study performed at a single center in Japan. The subjects were hospitalized for the pharmacodynamic/pharmacokinetic evaluations. Twenty-four patients received a single dose each of canagliflozin 100 and 200 mg before breakfast in a crossover manner with a 14-day washout between doses. The main outcome measures were pharmacokinetics of canagliflozin and its main metabolites (M5 and M7) in plasma and urine, and change from baseline in 24-h urinary glucose excretion (ΔUGE24 h). RESULTS: There was no significant effect of moderate renal impairment on the maximum canagliflozin concentration. The ratios of least square means (90 % confidence intervals [CIs]) of moderate renal impairment relative to normal renal function or mild renal impairment were 0.982 (0.821-1.173) and 0.989 (0.827-1.182) for the 100 and 200 mg doses, respectively. The canagliflozin area under the plasma concentration-time curve was greater in those with moderate renal impairment than in those without, after both canagliflozin doses (ratio of least square means [90 % CI] 1.258 [1.061-1.490] and 1.216 [1.026-1.441]). ΔUGE24 h increased after administration of both doses, but in patients with moderate renal impairment, the increase was approximately 70 % of that in patients with normal renal function or mild renal impairment. The incidence of adverse events was low and no patient developed hypoglycemia. CONCLUSION: The pharmacokinetics of canagliflozin are affected by renal function, with slight decreases in renal clearance observed. No effect of renal impairment on the maximum concentration was observed. Renal impairment reduced the ability of canagliflozin to promote urinary glucose excretion.

Aromatic foldamers with iminodicarbonyl linkers: their structures and optical properties.

[structure: see text] Carboxamides possessing naphthalene rings connected by multiple iminodicarbonyl linkers were synthesized. These molecules forced the naphthalene rings to be placed in the positions facing each other, and they form helical foldamers both in solution and in the crystalline state. Their folding structures were investigated by single-crystal X-ray analysis and (1)H NMR spectroscopy. Their absorption and fluorescence spectra showed a red shift as the number of naphthalene moieties increased. This remarkable change is based on the intramolecular interaction between naphthalene moieties. Helicity of the foldamer can be controlled by the introduction of chiral auxiliaries at imide nitrogen atoms, which results in an observation of induced circular dichroism.

Auto-spermatophore extrusion in male crickets.

The reproductive cycle of the male cricket consists of the mating stage and the sexually refractory stage. The latter is further divided into the first refractory stage (RS1) from spermatophore extrusion in copulation to spermatophore preparation after copulation, and the second refractory stage (RS2) from spermatophore preparation to recommencement of a calling song. RS2 is time-fixed and unaffected by the female or by stress, hence RS2 is assumed to be controlled by the reproductive timer. Previously, we suggested that the timer is located in the terminal abdominal ganglion (TAG), because functional inactivation of the TAG by local cooling lengthened RS2 in proportion to cooling time. To obtain further evidence of timer localization and to examine the operation of the timer in dissected animals, we investigated the characteristics of auto-spermatophore extrusion, a phenomenon in which males eject the mature spermatophore themselves without any prior courtship. The occurrence of auto-spermatophore extrusion was 100% in dissected males with the TAG separated, compared to 1.7% in intact males. The time interval (SPaSE) between spermatophore preparation and auto-spermatophore extrusion was comparable to RS2 measured by the calling song. Spike recording from a genital motor neurone in the separated TAG indicated that burst discharge associated with auto-spermatophore extrusion occurred with a SPaSE comparable to RS2. Other efferent neurones, some of which were identified as dorsal unpaired median (DUM) neurones, showed a time-dependent spike frequency increase during SPaSE. These results strengthen our previous conclusion that the reproductive timer is located within the TAG, and demonstrate that the timer functions normally even when the TAG is separated from the central nervous system.