High-speed camera recordings uncover previously unidentified elements of zebrafish mating behaviors integral to successful fertilization.

The mating behavior of teleost fish consists of a sequence of stereotyped actions. By observing mating of zebrafish under high-speed video, we analyzed and characterized a behavioral cascade leading to successful fertilization. When paired, a male zebrafish engages the female by oscillating his body in high frequency (quivering). In response, the female pauses swimming and bends her body (freezing). Subsequently, the male contorts his trunk to enfold the female's trunk. This behavior is known as wrap around. Here, we found that wrap around behavior consists of two previously unidentified components. After both sexes contort their trunks, the male adjusts until his trunk compresses the female's dorsal fin (hooking). After hooking, the male trunk slides away from the female's dorsal fin, simultaneously sliding his pectoral fin across the female's gravid belly, stimulating egg release (squeezing/spawning). Orchestrated coordination of spawning presumably increases fertilization success. Surgical removal of the female dorsal fin inhibited hooking and the transition to squeezing. In a neuromuscular mutant where males lack quivering, female freezing and subsequent courtship behaviors were absent. We thus identified traits of zebrafish mating behavior and clarified their roles in successful mating.

Measurements of ionic concentrations along with endocochlear potential in wild-type and claudin 14 knockout mice.

OBJECTIVE: To examine whether the changes in endolymphatic ion concentrations were involved in hair cells degeneration in claudin-14 knockout (KO) mice (Cldn14-/-), we measured the endocochlear potential (EP) along with concentrations of K+, Na+, H+, or Ca2+ ([K]e, [Na]e, pHe, [Ca]e) in Cldn14-/-, in which hair cells were selectively damaged, and compared with measurements in wild type mice (Wt). METHODS: We used the Cldn14-/- from 3 weeks of age, in which the auditory brain responses (ABR) was severely diminished. Using double-barreled ion-selective microelectrodes, we measured [K]e, [Na]e, pHe, and [Ca]e in both Wt and Cldn14-/- at 8-10 weeks of age. RESULTS: (1) In Wt, the EP was +92mV. [K]e, [Na]e, pHe, and [Ca]e were 169mM, ∼1.0mM, 7.50, and 395nM, respectively. In the Cldn14-/-, the EP was +96mV. [K]e, [Na]e, pHe, and [Ca]e were 167mM, ∼1.0mM, 7.73, and 179nM, respectively. No significant differences in the above values were observed between Wt and Cldn14-/-. (2) A significant linear correlation between EP and [Ca]e (R=0.93) was observed for both Wt and Cldn14-/-, but no correlation was observed between EP and K+, Na+, or H+. CONCLUSION: These findings suggest that (1) the changes in endolymphatic ion concentrations might not be involved in hair cells degeneration in Cldn14-/-, (2) [Ca]e might be regulated by EP in both Wt and Cldn14-/-.

"Slow" skeletal muscles across vertebrate species.

Skeletal muscle fibers are generally classified into two groups: slow (type I) and fast (type II). Fibers in each group are uniquely designed for specific locomotory needs based on their intrinsic cellular properties and the types of motor neurons that innervate them. In this review, we will focus on the current concept of slow muscle fibers which, unlike the originally proposed version based purely on amphibian muscles, varies widely depending on the animal model system studied. We will discuss recent findings from zebrafish neuromuscular junction synapses that may provide the framework for establishing a more unified view of slow muscles across mammalian and non-mammalian species.

Zebrafish mutants of the neuromuscular junction: swimming in the gene pool.

This review provides an overview of zebrafish mutants with dysfunctional acetylcholine receptors or related proteins at the neuromuscular junction (NMJ). The NMJ, which has served as the classical model of the chemical synapse, uses acetylcholine as the neurotransmitter, and mutations of proteins involved in the signaling cascade lead to a variety of behavioral phenotypes. Mutants isolated after random chemical mutagenesis screening are summarized, and advances in the field resulting from these mutants are discussed.

Procaterol-stimulated increases in ciliary bend amplitude and ciliary beat frequency in mouse bronchioles.

The beating cilia play a key role in lung mucociliary transport. The ciliary beating frequency (CBF) and ciliary bend amplitude (CBA) of isolated mouse bronchiolar ciliary cells were measured using a light microscope equipped with a high-speed camera (500 Hz). Procaterol (aß(2)-agonist) increased CBA and CBF in a dose dependent manner via cAMP. The time course of CBA increase is distinct from that of CBF increase: procaterol at 10 nM first increased CBA and then CBF. Moreover, 10 pM procaterol increased CBA, not CBF, whereas 10 nM procaterol increased both CBA and CBF. Concentration-response studies of procaterol demonstrated that the CBA curve was shifted to a lower concentration than the CBF curve, which suggests that CBA regulation is different from CBF regulation. Measurements of microbead movements on the bronchiole of lung slices revealed that 10 pM procaterol increased the rate of ciliary transport by 37% and 10 nM procaterol increased it by 70%. In conclusion, we have shown that increased CBA is of particular importance for increasing the bronchiolar ciliary transport rate, although CBF also plays a role in increasing it.

Morphology and infectivity of virus that persistently caused infection in an AGS cell line.

A recent report has indicated that proteins and genes of simian virus 5 (SV5) are detected in a human gastric adenocarcinoma (AGS) cell line, which is widely provided for oncology, immunology, and microbiology research. However, the production of infective virions has not been determined in this cell line. In this study, the morphology and infectivity of the virus particles of the AGS cell line were studied by light and electron microscopy and virus transmission assay. The virus particles were approximately 176.0 ± 41.1 nm in diameter. The particles possessed projections 8-12 nm long on the surface and contained a nucleocapsid determined to be 13-18 nm in width and less than 1,000 nm in length. The virus was transmissible to the Vero cell line, induced multinuclear giant cell formation, and reproduced the same shape of antigenic virions. In this study, the persistently infected virus in the AGS cell line was determined to be infective and form reproducible virions, and a new morphological feature of SV5 was determined.

Membrane potential modulation of ionomycin-stimulated Ca(2+) entry via Ca (2+)/H (+) exchange and SOC in rat submandibular acinar cells.

Ionomycin (IM) at 5 microM mediates the Ca(2+)/H(+) exchange, while IM at 1 microM activates the store-operated Ca(2+) entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar cells by increasing extracellular K(+) concentration ([K(+)](o)). IM (5 microM, the Ca(2+)/H(+) exchange) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)) to an extremely high value at 151 mM [K(+)](o). However, with increasing [K(+)](o), the rates of Ca(2+) entry decreased in a linear relationship. The reversal potential (E (rev)) for the Ca(2+)/H(+) exchange was +93 mV, suggesting that IM (5 microM) exchanges 1 Ca(2+) for 1 H(+). Thus, depolarization decreases the Ca(2+) influx via the Ca(2+)/H(+) exchange because of its electrogenicity (1 Ca(2+) for 1 H(+)). On the other hand, IM (1 microM, the SOCs) abolished an increase in [Ca(2+)](i) at 151 mM [K(+)](o). With increasing [K(+)](o), the rate of Ca(2+) entry immediately decreased linearly. The E (rev) for the SOC was +3.7 mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca(2+) over Na(+) (P (Ca)/P (Na) = 8.2). Moreover, an increase in extracellular Ca(2+) concentration (20 mM) enhanced the Ca(2+) entry via the SOCs at 151 mM [K(+)](o), suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca(2+) entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca(2+)](i) via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca(2+)](i) via two pathways depending on its concentration, the exchange of 1 Ca(2+) for 1 H(+) at 5 muM and the SOCs at 1 microM.

HCO(3) (-)-dependent transient acidification induced by ionomycin in rat submandibular acinar cells.

Ionomycin (IM, 5 microM), which exchanges 1 Ca2+ for 1 H+, changed intracellular pH (pHi) with Ca2+ entry into rat submandibular acinar cells. IM-induced changes in pHi consisted of two components: the first is an HCO3--dependent transient pHi decrease, and the second is an HCO3--independent gradual pHi increase. IM (1 microM), which activates store-operated Ca2+ channels, induced an HCO3--dependent and transient pHi decrease without any HCO3--independent pHi increase. Thus, a gradual pHi increase was induced by the Ca2+/H+ exchange. The HCO3--dependent and transient pHi decrease induced by IM was abolished by acetazolamide, but not by methyl isobutyl amiloride (MIA) or diisothiocyanatostilbene disulfonate (DIDS), suggesting that the Na+/H+ exchange, the Cl-/HCO3- exchange, or the Na+-HCO3- cotransport induces no transient pHi decrease. Thapsigargin induced no transient pHi decrease. Thus, IM, not Ca2+ entry, reduced pHi transiently. IM reacts with Ca2+ to produce H+ in the presence of CO2/HCO3-: [H-IM]-+Ca2++CO2<-->{H-Ca-IM]+.HCO3-+H+. In this reaction, a monoprotonated IM reacts with Ca2+ and CO2 to produce an electroneutral IM complex and H+, and then H+ is removed from the cells via CO2 production. Thus, IM transiently decreased pHi. In conclusion, in rat submandibular acinar cells IM (5 microM) transiently reduces pHi because of its chemical characteristics, with HCO3- dependence, and increases pHi by exchanging Ca2+ for H+, which is independent of HCO3-.

Ciliary beat frequency controlled by oestradiol and progesterone during ovarian cycle in guinea-pig Fallopian tube.

The ciliary beat frequency (CBF) of guinea-pig fimbria during the ovarian cycle was measured by video microscopy using a high-speed camera (500 Hz). In the follicular phase, with increasing concentrations of beta-oestradiol ([betaE(2)]) and a low concentration of progesterone ([PRG]), CBF increased from 13.5 to 16 Hz. In the ovulatory phase, with further increase of [betaE(2)], CBF decreased gradually from 16 to 13.5 Hz. In the early luteal phase, with low [PRG] and [betaE(2)], CBF increased to 17 Hz; however, in the middle luteal phase, with increasing [PRG], CBF decreased (12 Hz), and in the late luteal phase, with decreasing [PRG], CBF increased to 15 Hz. Then, in the resting phase, with low [betaE(2)] and [PRG], CBF decreased immediately to 14 Hz. The CBF of the fimbria was measured in guinea-pigs treated with beta-oestradiol benzoate (betaE(2)B) or medroxyprogesterone (mPRG). A low dose of betaE(2)B increased CBF to 14.5 Hz, whereas a high dose decreased it to 11 Hz. A betaE(2) receptor blocker, ICI-182,780, abolished the betaE(2)B-induced CBF changes and maintained CBF at 12.0 Hz. Medroxyprogesterone decreased CBF to 12.5 Hz, and mifepristone (a PRG receptor blocker) abolished the mPRG-induced CBF decrease and maintained CBF at 15 Hz. The addition of both blockers increased CBF to 18 Hz, suggesting that activation of betaE(2) or PRG receptors decreases the CBF of the fimbria. In conclusion, a moderate [betaE(2)] increase maintains a high CBF (15.5 Hz) in the follicular phase, and then further [betaE(2)] increase decreases CBF to 13.5 Hz in the ovulatory phase. In the early and late luteal phase, low [betaE(2)] and [PRG] increase CBF to 17 and 15 Hz, respectively, and in the middle luteal phase a high [PRG] decreases CBF (to 12 Hz). Thus, the CBF of the fimbria was controlled by signals via betaE(2) and PRG receptors in guinea-pigs.

Reactivation from latency displays HIV particle budding at plasma membrane, accompanying CD44 upregulation and recruitment.

BACKGROUND: It has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV. RESULTS: We observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-alpha stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1) and U1 cells (latently infected U937 cells with HIV-1) displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-alpha, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane. CONCLUSION: Our study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute infection. Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.

Nanotransportation system for cholera toxin in Vibrio cholerae 01.

Vibrio cholerae (V. cholerae) cholera toxin (CT), which causes a severe watery diarrheal illness, is secreted via the type II secretion machinery; it remains unclear, however, how this toxin is transported toward the machinery. In this study, we determined that the pH-dependent intrabacterial transport system correlates with the priming of CT secretion by V. cholerae. The secretion and production of V. cholerae treated at different pHs were examined by enzyme immunoassay. The localization of the CT was analyzed by immunoelectron microscopy. The CT secretion level rapidly increases in the alkaline-pH-treated V. cholerae but does so more slowly in neutral- and acidic-pH-treated V. cholerae. The CT was found to be densely localized near the membrane in the alkaline-pH-treated bacterial cytoplasm, suggesting that the CT shifts from the center to the peripheral portion of the cytoplasm following an extracellular rise in pH. The shift was observed in V. cholerae treated at alkaline pH for more than 10 min. The pH treatment did not enhance CT production at the same stage at which secretion and intrabacterial transport of the CT were enhanced. We propose that V. cholerae possesses a pH-dependent intrabacterial nanotransportation system that probably accelerates priming for CT secretion.

Modulation of human immunodeficiency virus type 1 infectivity through incorporation of tetraspanin proteins.

Accumulating evidence indicates that human immunodeficiency virus type 1 (HIV-1) acquires various cellular membrane proteins in the lipid bilayer of the viral envelope membrane. Although some virion-incorporated cellular membrane proteins are known to potently affect HIV-1 infectivity, the virological functions of most virion-incorporated membrane proteins remain unclear. Among these host proteins, we found that CD63 was eliminated from the plasma membranes of HIV-1-producing T cells after activation, followed by a decrease in the amount of virion-incorporated CD63, and in contrast, an increase in the infectivity of the released virions. On the other hand, we found that CD63 at the cell surface was preferentially embedded on the membrane of released virions in an HIV-1 envelope protein (Env)-independent manner and that virion-incorporated CD63 had the potential to inhibit HIV-1 Env-mediated infection in a strain-specific manner at the postattachment entry step(s). In addition, these behaviors were commonly observed in other tetraspanin proteins, such as CD9, CD81, CD82, and CD231. However, L6 protein, whose topology is similar to that of tetraspanins but which does not belong to the tetraspanin superfamily, did not have the potential to prevent HIV-1 infection, despite its successful incorporation into the released particles. Taken together, these results suggest that tetraspanin proteins have the unique potential to modulate HIV-1 infectivity through incorporation into released HIV-1 particles, and our findings may provide a clue to undiscovered aspects of HIV-1 entry.

Intrabacterial proton-dependent CagA transport system in Helicobacter pylori.

Helicobacter pylori CagA modifies the signalling of host cells and causes gastric diseases. Although CagA is injected into gastric epithelial cells through the type IV secretion machinery, it remains unclear how CagA is transported towards the machinery in the bacterial cytoplasm. In this study, it was determined that the proton-dependent intracytoplasmic transport system correlates with the priming of CagA secretion from H. pylori. The cytotoxicity of neutral-pH- and acidic-pH-treated H. pylori was examined in the AGS cell line. The amount of phosphorylated CagA in AGS cells incubated with acidic-pH- and neutral-pH-treated H. pylori was determined by enzyme immunoassay and Western blot. The production of CagA and adherence of the treated bacteria were examined by enzyme immunoassay and light microscopy, respectively. To clarify how CagA is transported towards the inner membrane of the treated bacteria, the localization of CagA was analysed by immunoelectron microscopy. The proportion of hummingbird cells in the AGS cell line rapidly increased following the inoculation of acidic-pH-treated H. pylori but increased more slowly with neutral-pH-treated H. pylori, and the phenomenon correlated with the amount of phosphorylated CagA in AGS cells. CagA was densely localized near the inner membrane in the acidic-pH-treated bacterial cytoplasm, but this localization was not observed in the neutral-pH-treated bacterial cytoplasm, suggesting that CagA shifts from the centre to the peripheral portion of the cytoplasm as a result of an extracellular decrease in pH. This phenomenon depended on the presence of UreI, a proton-dependent urea channel, but not on the presence of urea. The pH treatments did not enhance CagA production or the adherence of the bacterium to AGS cells. The authors propose that H. pylori possesses a proton-dependent intracytoplasmic transport system that probably accelerates priming for CagA injection.

Evaluation of disinfective potential of reactivated free chlorine in pooled tap water by electrolysis.

Tap water is one of the causative factors of hospital infections. We examined the disinfective potential of electrolysis and mechanism of disinfection, and clarified the disinfective effect of electrolysis on tap water contaminated with bacteria, and discussed its clinical applications. Tap waters artificially contaminated with Pseudomonas aeruginosa, Escherichia coli, Legionella pneumophila, and Staphylococcus aureus could be sterilized by electrolysis at 20-30 mA for 5 min. A high-density suspension (10(6) CFU/ml) of a spore forming bacterium, Bacillus subtilis was not completely sterilized by electrolysis at 50 mA up to 30 min, but a low-density suspension (10(5) CFU/ml) was totally sterilized by electrolysis at 50 mA for 5 min. Electrolyzed P. aeruginosa changed morphologically, that is, there was bleb formation on the cell wall and irregular aggregation of cytoplasmic small granules. Moreover, cytoplasmic enzyme, nitrate reductase, was inactivated by the electrolysis. On the other hand, genomic DNA of the electrolyzed bacteria was not degenerated, therefore, their DNA polymerase activity was not completely inactivated. Consequently, the major agent in electrolysis for bactericidal action was considered to be free chlorine, and the possible bactericidal mechanism was by destruction of bacterial membranes, followed by the aggregation of peripheral cytoplasmic proteins. Electrolysis of tap water for both disinfecting contaminating bacteria and increasing the disinfectant capacity was considered effective with some limitations, particularly against high-density contamination by spore-forming bacteria. In clinical settings, electrolysis of tap water is considered effective to disinfect water for hand washing in operation theatres, and bathing water for immunocompromised hosts.