Browsing by Author "Yamamoto, Tatsuyuki"

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Imaging phospholipid conformational disorder and packing in giant multilamellar liposome by confocal Raman microspectroscopy
(2017-12-05)
Noothalapati, Hemanth
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Iwasaki, Keita
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Yoshimoto, Chikako
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Yoshikiyo, Keisuke
;
Nishikawa, Tomoe
Liposomes are closed phospholipid bilayer systems that have profound applications in fundamental cell biology, pharmaceutics and medicine. Depending on the composition (pure or mixture of phospholipids, presence of cholesterol) and preparation protocol; intra- and inter-chain molecular interactions vary leading to changes in the quality (order and packing) of liposomes. So far it is not possible to image conformational disorders and packing densities within a liposome in a straightforward manner. In this study, we utilized confocal Raman microspectroscopy to visualize structural disorders and packingefficiency within a giant multilamellar liposome model by focusing mainly on three regions in the vibrational spectrum (C-C stretching, C-H deformation and C-H stretching). We estimated properties such as trans/gauche isomers and lateral packing probability. Interestingly, our Raman imaging studies revealed gel phase rich domains and heterogeneous lateral packing within the giant multilamellar liposome. (C) 2017 Elsevier B.V. All rights reserved.
Label-free Chemical Imaging of Fungal Spore Walls by Raman Microscopy and Multivariate Curve Resolution Analysis
(2016-06-09)
Noothalapati, Hemanth
;
Sasaki, Takahiro
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Kaino, Tomohiro
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Kawamukai, Makoto
;
Ando, Masahiro
Fungal cell walls are medically important since they represent a drug target site for antifungal medication. So far there is no method to directly visualize structurally similar cell wall components such as alpha-glucan, beta-glucan and mannan with high specificity, especially in a label-free manner. In this study, we have developed a Raman spectroscopy based molecular imaging method and combined multivariate curve resolution analysis to enable detection and visualization of multiple polysaccharide components simultaneously at the single cell level. Our results show that vegetative cell and ascus walls are made up of both alpha- and beta-glucans while spore wall is exclusively made of alpha-glucan. Co-localization studies reveal the absence of mannans in ascus wall but are distributed primarily in spores. Such detailed picture is believed to further enhance our understanding of the dynamic spore wall architecture, eventually leading to advancements in drug discovery and development in the near future.
Organelle specific simultaneous Raman/green fluorescence protein microspectroscopy for living cell physicochemical studies
(1970-01-01)
Wattanavichean, Nungnit
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Nishida, Ikuhisa
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Ando, Masahiro
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Kawamukai, Makoto
;
Yamamoto, Tatsuyuki
We demonstrate a novel bio-spectroscopic technique, "simultaneous Raman/GFP microspectroscopy". It enables organelle specific Raman microspectroscopy of living cells. Fission yeast, Schizosaccharomyces pombe, whose mitochondria are green fluorescence protein (GFP) labeled, is used as a test model system. Raman excitation laser and GFP excitation light irradiate the sample yeast cells simultaneously. GFP signal is monitored in the anti-Stokes region where interference from Raman scattering is negligibly small. Of note, 13 568 Raman spectra measured from different points of 19 living yeast cells are categorized according to their GFP fluorescence intensities, with the use of a two-component multivariate curve resolution with alternate least squares (MCR-ALS) analysis in the anti-Stokes region. This categorization allows us to know whether or not Raman spectra are taken from mitochondria. Raman spectra specific to mitochondria are obtained by an MCR-ALS analysis in the Stokes region of 1389 strongly GFP positive spectra. Two mitochondria specific Raman spectra have been obtained. The first one is dominated by protein Raman bands and the second by lipid Raman bands, being consistent with the known molecular composition of mitochondria. In addition, the second spectrum shows a strong band of ergosterol at 1602 cm(-1), previously reported as "Raman spectroscopic signature of life of yeast."
Studying anti-oxidative properties of inclusion complexes of alpha-lipoic acid with gamma-cyclodextrin in single living fission yeast by confocal Raman microspectroscopy
(2018-05-15)
Noothalapati, Hemanth
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Ikarashi, Ryo
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Iwasaki, Keita
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Nishida, Tatsuro
;
Kaino, Tomohiro
alpha-lipoic acid (ALA) is an essential cofactor for many enzyme complexes in aerobic metabolism, especially in mitochondria of eukaryotic cells where respiration takes place. It also has excellent anti-oxidative properties. The acid has two stereo-isomers, R- and S-lipoic acid (R-LA and S-LA), but only the R-LA has biological significance and is exclusively produced in our body. A mutant strain of fission yeast, Delta dpsl, cannot synthesize coenzyme Q10, which is essential during yeast respiration, leading to oxidative stress. Therefore, it shows growth delay in the minimal medium. We studied anti-oxidant properties of ALA in its free form and their inclusion complexes with gamma-cyclodextrin using this mutant yeast model. Both free forms R- and S-LA as well as 1:1 inclusion complexes with gamma-cyclodexhin recovered growth of Delta dps1 depending on the concentration and form. However, it has no effect on the growth of wild type fission yeast strain at all. Raman microspectroscopy was employed to understand the anti-oxidant property at the molecular level. A sensitive Raman band at 1602 cm(-1) was monitored with and without addition of ALAs. It was found that 0.5 mM and 1.0 mM concentrations of ALAs had similar effect in both free and inclusion forms. At 2.5 mM ALAs, free forms inhibited the growth while inclusion complexes helped in recovered. 5.0 mM ALA showed inhibitory effect irrespective of form. Our results suggest that the Raman band at 1602 cm(-1) is a good measure of oxidative stress in fission yeast. (C) 2018 Elsevier B.V. All rights reserved.
The effect of coenzyme Q10 included by gamma-cyclodextrin on the growth of fission yeast studied by microscope Raman spectroscopy
(2013-09-24)
Nishida, Tatsuro
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Kaino, Tomohiro
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Ikarashi, Ryo
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Nakata, Daisuke
;
Terao, Keiji
The inclusion complex of coenzyme Q10 (CoQ10) by gamma-cyclodextrin (gamma-CD), CoQ10-CD complex, was recently developed. The addition of the CoQ10-CD complex recovered the growth of a fission yeast mutant strain, Delta dps1, which otherwise cannot grow well due to the lack of coenzyme Q producing ability. However, the oxygen consumption rate of this strain was not restored by the addition of the CoQ10-CD complex. The addition of two other anti-oxidative reagents, glutathione and ascorbic acid, also recovered the growth of the Delta dps1 strain as well. These results indicated that the recovery of the growth of Delta dps1 was brought about by the anti-oxidative property of CoQ10. The intensity of Raman spectra of Delta dps1 at 1602 cm(-1), which is prominently observed for the wild type of the fission yeast, was compared between before and after addition of the CoQ10-CD complex. The signal was very weakly observed for Delta dps1 and did not increase in intensity by the addition of the CoQ10-CD complex. These results suggested the recovery of the growth of Delta dps1 was brought about not by the restoration of respiration function of Delta dps1 but by the anti-oxidative property of CoQ10 to result in the decrease in the oxidative stress. (c) 2013 Elsevier B.V. All rights reserved.
Towards the development of a non-bioptic diagnostic technique for eosinophilic esophagitis using Raman spectroscopy
(2016-07)
Noothalapati, Hemanth
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Uemura, Suguru
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Ohshima, Naoki
;
Kinoshita, Yoshikazu
;
Ando, Masahiro
Eosinophilic esophagitis (EoE) is believed to be a rare pathological condition that is characterized by dense infiltration of eosinophils in esophageal epithelial layer. Occurrence of this disease worldwide has started increasing rapidly in the last decade. Routine endoscopic observations can help in diagnosis only if clear longitudinal furrows or multiple concentric rings are observed but does not give any definitive conclusion in the early stages. Hence esophageal tissue samples are collected from multiple sites by biopsy and the number of eosinophils is counted after staining. Such a procedure is time consuming and has an inherent risk of bleeding, eventually damaging esophagus. Hence we developed a resonance Raman spectroscopy based approach to detect eosinophils in esophagus using mouse models. Our results show the presence of eosinophils in mice esophageal tissues suffering from inflammation by administering interleukin-33 while there are none in control mice. We believe this method can be used in clinical application for diagnosing EoE in the near future. (C) 2016 Elsevier B.V. All rights reserved.