Mechanism of porphyrin-induced sonodynamic effect: possible role of hyperthermia

The biological effects of ultrasound have been investigated vigorously for various applications including the thermal coagulation of tissues, the opening of tight junctions, and localized gene or drug introduction. The synergistic cell killing effect of ultrasound and porphyrin derivatives, the so-called sonodynamic effect, holds promise for cancer treatment. Although several models to explain the sonodynamic effect have been proposed, its exact mechanism, especially in vivo, remains unknown. We examined the effect of a porphyrin derivative, protoporphyrin IX, on ultrasound-induced killing of HeLa cells. In some experiments, the intracellular protoporphyrin IX concentration was increased by 5-aminolevulinic acid treatment of the cells. Although extracellular protoporphyrin IX showed an enhanced cell killing effect by microbubble-enhanced ultrasound, intracellular protoporphyrin IX did not. On the other hand, intracellular protoporphyrin IX enhanced the cell killing effect of hyperthermia, which can be produced by ultrasound exposure, in a moderately acidic environment (pH 6.6). Because porphyrin derivatives are generally imported into the intracellular component in vivo, our results suggest that hyperthermia caused by ultrasound may play an important role in the sonodynamic effect induced by porphyrin derivatives.     

Tissue-specific DNA-PK-dependent H2AX phosphorylation and gamma-H2AX elimination after X-irradiation in vivo

Histone H2AX rapidly undergoes phosphorylation at Ser139 (γ-H2AX) in response to DNA double-strand breaks. Although ATM kinase and DNA-PK phosphorylate Ser139 of H2AX in culture cells, the regulatory mechanism of γ-H2AX level remains unclear in vivo. Here, we detected the phosphorylation of H2AX and the elimination of γ-H2AX in the mouse skin after X-irradiation. Furthermore, following X-irradiation, the level of γ-H2AX also increased in mice lacking either ATM or DNA-PK. Although the elimination after X-irradiation was detected in the skin of these mutant mice, the elimination in DNA-PK-deficient mice was slower than that in C3H and ATM knockout mice, suggesting that a fraction of γ-H2AX in the skin is eliminated in a DNA-PK-dependent manner. Although the DNA-PK-dependent elimination of γ-H2AX was also detected in the liver, kidney, and spleen, the DNA-PK-dependent phosphorylation of H2AX was detected in the spleen only. These results suggest that the regulatory mechanism of γ-H2AX level is tissue-specific.     

TIMP-1 inhibits microvascular endothelial cell migration by MMP-dependent and MMP-independent mechanisms

It was reported over a decade ago that tissue inhibitor of metalloproteinases-1 (TIMP-1) suppresses angiogenesis in experimental models but the mechanism is still incompletely understood. This in vitro study focused on the molecular basis of TIMP-1-mediated inhibition of endothelial cell (EC) migration, a key step in the angiogenic process. Both recombinant human TIMP-1 and the synthetic MMP inhibitors, GM6001 and MMP-2-MMP-9 Inhibitor III, suppressed migration of human dermal microvascular endothelial cells (HDMVEC) in a dose-dependent fashion. The MMP-dependent inhibition of migration was associated with increased expression of the junctional adhesion proteins, VE-cadherin and PECAM-1, and VE-cadherin accumulation at cell-cell junctions. TIMP-1 also caused MMP-independent dephosphorylation of focal adhesion kinase (FAK) (pY397) and paxillin, which was associated with reduced number of F-actin stress fibers and focal adhesions. Moreover, TIMP-1 stimulated expression of PTEN that has been shown to reduce phosphorylation of FAK and inhibit cell migration. Our data suggest that TIMP-1 inhibits HDMVEC migration through MMP-dependent stimulation of VE-cadherin and MMP-independent stimulation of PTEN with subsequent dephosphorylation of FAK and cytoskeletal remodeling.     

Detection and localization of a chloroplast-encoded HU-like protein that organizes chloroplast nucleoids

Chloroplast DNA (cpDNA) is packed into discrete structures called chloroplast nucleoids (cp-nucleoids). The structure of cpDNA is thought to be important for its maintenance and regulation. In bacteria and mitochondria, histone-like proteins (such as HU and Abf2, respectively) are abundant and play important roles in DNA organization. However, a primary structural protein has yet to be found in cp-nucleoids. Here, we identified an abundant DNA binding protein from isolated cp-nucleoids of the primitive red alga Cyanidioschyzon merolae. The purified protein had sequence homology with the bacterial histone-like protein HU, and it complemented HU-lacking Escherichia coli mutants. The protein, called HC (histone-like protein of chloroplast), was encoded by a single gene (CmhupA) in the C. merolae chloroplast genome. Using immunofluorescence and immunoelectron microscopy, we demonstrated that HC was distributed uniformly throughout the entire cp-nucleoid. The protein was expressed constitutively throughout the cell and the chloroplast division cycle, and it was able to condense DNA. These results indicate that HC, a bacteria-derived histone-like protein, primarily organizes cpDNA into the nucleoid.     

Bounds on direct effects in the presence of confounded intermediate variables

Summary .   This article considers the problem of estimating the average controlled direct effect (ACDE) of a treatment on an outcome, in the presence of unmeasured confounders between an intermediate variable and the outcome. Such confounders render the direct effect unidentifiable even in cases where the total effect is unconfounded (hence identifiable). Kaufman et al. (2005, Statistics in Medicine 24, 1683–1702) applied a linear programming software to find the minimum and maximum possible values of the ACDE for specific numerical data. In this article, we apply the symbolic Balke–Pearl (1997, Journal of the American Statistical Association 92, 1171–1176) linear programming method to derive closed-form formulas for the upper and lower bounds on the ACDE under various assumptions of monotonicity. These universal bounds enable clinical experimenters to assess the direct effect of treatment from observed data with minimum computational effort, and they further shed light on the sign of the direct effect and the accuracy of the assessments.     

The application of independent component analysis to the multi-channel surface electromyographic signals for separation of motor unit action potential trains: part II-modelling interpretation.

The purpose of this article was to investigate whether or not FastICA can separate identical motor unit action potential trains (MUAPTs) of the 8-channel surface electromyographic (sEMG) signals constructed by an sEMG model into the independent components. Firstly, we have examined how much the increase of motor units (MUs) in the simulated sEMG signals influenced the performance on the separation of MUAPTs by kurtosis. The decreased trend of mean kurtosis on both sEMG signals and their independent components were observed as MUs were increased. These data suggested that the separation performance decayed when MUs were increased. Secondary, the differences between the independent components and the principal components have been also applied to the simulated sEMG signals with or without time delay between the sEMG channels. FastICA could successfully separate identical MUAPTs with no time delay but principal component analysis (PCA) could not do so. Against it, both FastICA and PCA could not separate MUAPTs with some time delay. In conclusion, our results suggested that FastICA could separate identical MUAPTs with no time delay into the independent components by FastICA, which might offer a new technique for the separation of interfered MUAP waveforms based on statistical properties of sEMG signal distributions.     

Contribution of the neural cell recognition molecule NB‐3 to synapse formation between parallel fibers and Purkinje cells in mouse

The neural cell recognition molecule NB‐3, also referred to as contactin‐6, is expressed prominently in the developing nervous system after birth and its deficiency has been shown to cause impairment in motor coordination. Here, we investigated the contribution of NB‐3 to cerebellar development, focusing on lobule 3 where NB‐3 was expressed in granule cells but not in Purkinje cells. In the developing molecular layer, the neural cell recognition molecules TAG‐1, L1, and NB‐3 formed distinct expression zones from the external granule cell layer to the internal granule cell layer (IGL), respectively. The NB‐3‐immunoreactive zone did not overlap with TAG‐1‐immunoreactive zone. By contrast, the L1‐immunoreactive zone overlapped with both the TAG‐1‐ and NB‐3‐immunoreactive zones. NB‐3‐positive puncta overlapped with vesicular glutamate transporter 1, a presynaptic marker and were apposed close to metabotropic glutamate receptor 1A, a postsynaptic marker, indicating that NB‐3 is localized presynaptically at glutamatergic synapses between parallel fibers and Purkinje cells. In NB‐3 knockout mice, L1 immunoreactive signals were increased in the IGL at postnatal day (P) 5, suggesting the increase in the number of immature granule cells of the IGL. In addition, the density of parallel fiber synaptic terminals was reduced in NB‐3 knockout mice relative to wild‐type mice at P5 to P10. In parallel with these findings, caspase‐dependent cell death was significantly increased in the NB‐ 3‐deficient cerebellum at P15. Collectively, our results indicate that NB‐3 deficiency affects synapse formation during postnatal cerebellar development. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Differential distribution of gangliosides in adult rat ovary during the oestrous cycle

Gangliosides are ubiquitous membrane components in mammaliancells and are suggested to play important roles in various cellfunctions, such as cell-cell recognition, differentiation andtransmembrane signalling. Rat ovary contained GM3, GD3 and GD1aas major gangliosides, and GM1 as a minor one. In order to studytheir distribution in the rat ovary and its possible changesduring the oestrous cycle, frozen sections were stained withspecific monoclonal antibodies against 11 ganglio-series gangliosidesincluding those mentioned above. GM3, GM1 and GD1a were expressedin a spatiotemporally different manner during the oestrous cycle,but GD3 and other gangliosides were not immunohistochemicallydetected. In primary and secondary follicles, GM3, GM1 and GDlawere expressed in theca cells, but not in granulosa cells. Theoocyte in primary, but not secondary, follicles was positiveto the anti-GD1a antibody. In Graafian follicles, GM1 and GD1awere similarly expressed as in secondary follicles, however,the expression of GM3 spread gradually from theca cells to granulosacells. In early Graafian follicles, only GM3 was expressed toa detectable extent from the outer part of the granulosa layerto the inner part Shortly before ovulation, all granulosa cellsand cumulus cells became positive to anti-GM3 antibody. Afterovulation, differential distribution of GM3, GM1 and GD1a wasalso observed in luteal cells. GD1a was localized in thread-likestructures, while GM3 was distributed throughout the cytoplasm,but not in the nucleus. GM1 was localized only in the plasmamembrane and/or its close vicinity. Other ganglio-series gangliosides,including GD3, were not detected to an appreciable extent inthe ovaries by immunohistochemistry ganglioside oestrous cycle rat ovary     

Modified sympathetic nerve system activity with overexpression of the voltage-dependent calcium channel beta3 subunit

N-type voltage-dependent calcium channels (VDCCs) play determining roles in calcium entry at sympathetic nerve terminals and trigger the release of the neurotransmitter norepinephrine. The accessory beta3 subunit of these channels preferentially forms N-type channels with a pore-forming CaV2.2 subunit. To examine its role in sympathetic nerve regulation, we established a beta3-overexpressing transgenic (beta3-Tg) mouse line. In these mice, we analyzed cardiovascular functions such as electrocardiography, blood pressure, echocardiography, and isovolumic contraction of the left ventricle with a Langendorff apparatus. Furthermore, we compared the cardiac function with that of beta3-null and CaV2.2 (alpha1B)-null mice. The beta3-Tg mice showed increased expression of the beta3 subunit, resulting in increased amounts of CaV2.2 in supracervical ganglion (SCG) neurons. The beta3-Tg mice had increased heart rate and enhanced sensitivity to N-type channel-specific blockers in electrocardiography, blood pressure, and echocardiography. In contrast, cardiac atria of the beta3-Tg mice revealed normal contractility to isoproterenol. Furthermore, their cardiac myocytes showed normal calcium channel currents, indicating unchanged calcium influx through VDCCs. Langendorff heart perfusion analysis revealed enhanced sensitivity to electric field stimulation in the beta3-Tg mice, whereas beta3-null and Cav2.2-null showed decreased responsiveness. The plasma epinephrine and norepinephrine levels in the beta3-Tg mice were significantly increased in the basal state, indicating enhanced sympathetic tone. Electrophysiological analysis in SCG neurons of beta3-Tg mice revealed increased calcium channel currents, especially N- and L-type currents. These results identify a determining role for the beta3 subunit in the N-type channel population in SCG and a major role in sympathetic nerve regulation.