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.

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.

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.