Large-scale analysis of proteins, which are functional biomolecules, has assumed an important role in the life sciences.
Mass spectrometry is one of the techniques used to identify ptoreins. Direct desorption and ionization of proteins from
polyacrylamide gel is expected to be a the high-throughput technique in proteomics, which will eliminate several
problems such as sample loss, adduct formation, and contamination. In this study, we performed direct ionization of a
protein, bovine insulin, in a polyacrylamide gel without matrix addition with a tunable MIR nanosecond pulsed laser.
Mass spectra of insulin in an acrylamide gel and an acrylamide solution were recorded in the wavelength range 5.8-5.9
μm and at a wavelength of 6.0 μm, respectively. This wavelength corresponds to the >C=O stretching vibration mode of acrylamide.
Large-scale analysis of proteins, which can be regarded as functional biomolecule, assumes an important role in the life
science. A MALDI using an ultraviolet laser (UV-MALDI) is one of ionization methods without fragmentation and has
achieved conformation analysis of proteins. Recently, protein analysis has shifted from conformation analysis to
functional and direct one that reserves posttranslational modifications such as the sugar chain addition and
phosphorylation. We have proposed a MALDI using a mid-infrared tunable laser (IR-MALDI) as a new ionization
method. IR-MALDI is promising because most biomolecules have a specific absorption in mid-infrared range, and IR-MALDI
is expected to offer; (1) use of various matrices, (2) use of biomolecules such as water and lipid as the matrix,
and (3) super-soft ionization. First, we evaluated the wavelength dependence of ionization of different matrices using a
difference frequency generation (DFG) laser, which can tune the wavelength within a range from 5.5 to 10.0 &mgr;m. As
results, ionization was specifically occurred at 5.8 &mgr;m which the C=O vibration stretching bond in matrix material and
mass spectrum was observed. Next, protein mass spectrum was observed in the culture cells, MIN6, which secrete
insulin, without the conventional cell-preparation processes. We demonstrate that the IR-MALDI has an advantage over
the conventional method (UV-MALDI) in direct analysis of intracellular proteins.
An absorption characteristic and a thermal relaxation time of a target biomedical tissue is an important parameter for
development of low-invasive treatment that considers of interaction between biomedical tissue and laser. Laser
irradiations with a wavelength corresponding to the absorption characteristics of tissue enable selective treatment.
Furthermore, the thermal effect to tissue can be controlled at the laser irradiation time which depends on the laser pulse
width and reception rate. A free electron laser (FEL) can continuously vary the wavelength in the mid-infrared region,
has a unique pulse structure; the structure at the Institute of the Free Electron Laser (iFEL) consist of train of
macropulses with a 15 &mgr;s pulse width, and each macropulse contained a train of 300-400 ultrashort micropulse with a 5
ps pulse width. In a previous report, we have proposed a novel laser treatment such as soft tissue cutting, dental
treatment and laser angioplasty using the tenability of the FEL. To investigate the thermal effect to the biomedical tissue,
we developed a FEL pulse control system using an acousto-optic modulator (AOM). The AOM commonly are used the
Q-switch for the pulse laser generation, has a high pulse control efficiency and good operationally. The system can
control the FEL macropulse width from 200 ns. This system should be a novel tool for investigating the interaction
between the FEL and biomedical tissue. In this report, the interaction between FEL pulse width and biomedical tissue
will be discussed.
The non-invasive methods of treatments have been studying for the improvement of quality of life (QOL) of patients undergoing treatment. A photodynamic therapy (PDT) is one of the non-invasive treatments. PDT is the methods of treatment using combination of a laser and a photosensitizer. PDT has few risks for patients. Furthermore, PDT enables function preservation of a disease part. PDT has been used for early cancer till now, but in late years it is applied for age-related macular degeneration (AMD). AMD is one of the causes of vision loss in older people. However, PDT for AMD does not produce the best improvement in visual acuity. The skin photosensivity by an absorption characteristic of a photosensitizer is avoided. We examined new PDT using combination of an ultra-short pulsed laser and indocyanine green (ICG).
Phosphorylation and dephosphorylation are considered to be important reactions that control the active and inactive factors of proteins. In regenerative medicine of the osteoconnective tissue (a tendon, a ligament), it has been reported that the biomaterial possessing phosphate groups promote formation of HAP, the main component of hard tissues. The noncontact measurement of phosphate groups and low-destructive controlling of phosphate groups allow for the accurate regeneration of the osteoconnective tissue, and the validation. Our objective is to propose the nondestructive controlling and measuring method of phosphorylation for regenerative medicine. In this study, as the indirect quantitative analysis of phosphate groups, we examine the correlation between the mid-infrared absorbance ratio and the ratio of phosphate groups introduction theoretically calculated from a colorimetric determination method. And the noncontact controlling method of the quantities of phosphate groups, we examine the selective and low-destructive bond cutting of phosphate groups in the phosphogelatin using a mid-infrared laser.
A new type of cell-cultivation system based on laser processing has been developed for the on-chip cultivation of living cells. We introduce a "laser cell-chip", on which migration of cells, such as stem cells, tumor cells or immunocompetent cells, can be observed. A sheet prepared from epoxy resin was processed by KrF excimer laser (248 nm, 1.6 J/cm<sup>2</sup>) for preparation of microgrooved surfaces with various groove width, spacing, and depth. A laser cell-chip can make kinetic studies of cell migration depending on the concentration gradient of a chemoattractant. In this study, megakaryocytes were used for the migration on a groove of laser cell-chip by the concentration gradient of the stromal cell derived factor 1 (SDF-1/CXCL12). SDF-1/CXCL12 plays an important and unique role in the regulation of stem/progenitor cell trafficking. A megakaryocyte was migrated on a groove of laser cell-chip depending on the optical concentration gradient of SDF-1/CXCL12. Since SDF-1/CXCL12-induced migration of mature megakaryocyte was known to increase the platelet production in the bone marrow extravascular space, the diagnosis of cell migration on laser cell-chip could
provide a new strategy to potentially reconstitute hematopoiesis and avoid life-threatening hemorrhage after myelosuppression or bone marrow failure.
We have proposed selective removal of cholesterol ester by infrared laser of wavelength with 5.75 μm irradiation; the wavelength of 5.75 μm correspond with the ester bond C=O stretching vibration. The flexible laser guiding line and a compact light source are required for our proposal. We used a compact mid-infrared tunable laser by difference frequency generation; DFG laser was developed for substitute light source of free electron laser. In the present work, first, we have developed hollow optical fiber with a diamond lens-tip to deliver DFG laser in the blood vessel and evaluated the transmission of DFG laser from 5.5 μm to 7.5 μm. The transmission of 5.75 μm is about 65%, the DFG beam was focused on the tip of fiber by diamond lens-tip. Secondly, we performed the selective removal experiment of cholesterol ester using the hollow optical fiber with diamond lens-tip and DFG laser. The sample used a two layer model, cholesterol oleate and gelatin. The cholesterol oleate was decomposed by 5.75 μm DFG irradiation with 3.8 W/cm<sup>2</sup>.
It is necessary to control enhancement of transdermal drug delivery with non-invasive. The present study was investigated to assess the effectivity of enhancing the drug delivery by irradiating 6-μm region mid infrared free electron laser (MIR-FEL). The enhancement of transdermal drug (lidocaine) delivery of the samples (hairless mouse skin) irradiated with lasers was examined for flux (μg/cm<sup>2</sup>/h) and total penetration amount (μg/cm<sup>2</sup>) of lidocaine by High performance Liquid Chromatography (HPLC). The flux and total amount penatration date was
enhanced 200-300 fold faster than the control date by the laser irradiation. FEL irradiating had the stratum corneum, and had the less thermal damage in epidermis. The effect of 6-μm region MIR-FEL has the enhancement of transdermal drug delivery without removing the stratum corneum because it has the less thermal damage. It leads to enhancement drug delivery system with non-invasive laser treatment.
Phosphorylation and dephosphorylation, which are the most remarkable posttranslational modifications, are considered to be important chemical reactions that control the activation of proteins. In regenerative medicine, it has been reported that phosphorylated proteins are instrumental in the calcification of osteoconnective tissue. We examine the phosphorylation analysis method by measuring the infrared absorption peak of phosphate group that observed at about 1070 cm<sup>-1</sup> (9.4 mm) with Fourier Transform Infrared Spectrometer (FT-IR). This result indicates that it is possible to identify a phosphorylation by measuring the infrared absorption peak of phosphate group observed at about 1070 cm<sup>-1</sup> with FT-IR method. And we examine laser-dephosphorylation using Free Electron Laser (FEL) as a novel dephosphorylation method. After irradiation of 9.4mm-FEL, infrared absorption peak of phosphate group is reduced. It is suspected that this lowering of the peak of phosphate group is the effect like dephosphorylation. These novel methods can be applied to quality control technology in regenerative medicine.
A cholesterol ester is selective dissociated by MIR-FEL irradiation with wavelength of 5.75 μm correspond to C=O stretching vibration of ester bond. To evaluate the optimum irradiation condition for cholesterol ester decomposition without normal endothelium cell damage, we perform a 5.75 μm-FEL irradiation to a two-layer vessel model which is cholesterol oleate as a model of atherosclerotic lesions and gelatin as a model of endothelial cells. The ester decomposition and gelatin damege depends on total power density of 5.75 μm-FEL provided the two-layer model. Exposure of the FEL with power density exceed 17.8 W/cm<sup>2</sup> decomposed cholesterol ester thorough gelain layer of 15 μm thickness. If FEL with power density of 25.0 W/cm<sup>2</sup> is exposed during 10 seconds, the gelatin is evaporized. Therefore, the optimum condition for ester decomposition without gelatin evaporation is between 75 J/cm<sup>2</sup> from 20 J/cm<sup>2</sup> in the case of 15 μm gelatin layer thickness. The maximum ester decomposition rate without gelatin vaporization is 71% on FEL of power density of 17.8 W/cm<sup>2</sup> for 10 seconds.
Phosphorylation and dephosphorylation, which are the most remarkable posttranslational modifications, are considered to be important chemical reactions that control the activation of proteins. We examine the phosphorylation analysis method by measuring the infrared absorption peak of phosphate group that observed at about 1070cm<sup>-1 </sup>(9.4μm) with Fourier Transform Infrared Spectrometer (FT-IR). This study indicates that it is possible to identify a phosphorylation by measuring the infrared absorption peak of phosphate group observed at about 1070 cm<sup>-1 </sup>with FT-IR method. As long as target peptides have the same amino acid sequence, it is possible to identify the phosphorylated sites (threonine, serine and tyrosine).