Argon Ion Laser Excitation Research Articles

The cell-wall structure variation in Mycobacterium tuberculosis with different drug sensitivity using Raman spectroscopy in the high-wavenumber region

Over the past decade Raman spectroscopy has been successfully used to study the cell structure of various pathogens, including M. tuberculosis (MbT). It is especially important due to the high ability of pathogen to become resistant to anti-tuberculosis drugs usually leading to a lethal outcome. In the present study we investigate deactivated MbT strains with different drug sensitivity (extra-, multi- and sensitive drug resistance) of the Beijing family. Samples have been obtained from patients suffering from pulmonary and extra-pulmonary forms of tuberculosis. Samples have been investigated by Raman spectrometry using a diode-pumped solid-state argon (514 nm) laser excitation source in the high-wavenumber region. As a result, protein and lipid vibrational bands for strain discrimination with various drug resistances have been determined. The obtained results can be used as fast diagnostic tool development for screening MbT strains in clinical medical practice.

Water-soluble, lignin-derived carbon dots with high fluorescent emissions and their applications in bioimaging

We report the synthesis of water-soluble and environmentally friendly fluorescent carbon dots (CDs) using the renewable resource lignin as a precursor, which is a waste emitted from pulp and paper industries and lignocellulosic biofuel production, using a compressed liquid CO2 antisolvent method followed by carbonization and chemical oxidation with an acid mixture (H2SO4:HNO3=1:3, v/v) at 90°C for 8, 16, and 24h. The as-synthesized CDs emitted bright yellow fluorescence (FL) under argon laser excitation at 458nm with excitation-wavelength-independent and excitation-wavelength-dependent FL emission characteristics. Moreover, the CDs exhibited excellent stability in a highly concentrated NaCl solution, photostability with pH-dependent FL emission activities, and cellular imaging capability with very low cytotoxicity. Notably, the FL emission intensities of the as-synthesized CDs increased with longer reaction times, indicating high degree of oxidation on the CD surface and consequently, high quantum yield of 13% was achieved. Therefore, the as-synthesized CDs can be used as promising fluorescent probes for bioimaging applications in vitro and in vivo.

Bench-scale evaluation of water disinfection by visible-to-UVC upconversion under high-intensity irradiation

The feasibility of applying visible-to-UVC upconversion (UC) luminescence to enhance the kinetics of solar water disinfection was evaluated using Lu7O5F9:Pr3+ ceramics incorporated into a solar reactor containing E. coli suspensions. Inactivation was assessed in batch conditions using both laser and lens-concentrated sunlight excitation conditions. Under 840-mW argon laser excitation, the UC efficiency was estimated to be 1 order of magnitude greater than previously reported under lamp excitation and UVC emitted by the reactors resulted in 3.6-log inactivation in 20min. However, experiments using ~1500mW of concentrated natural sunlight showed no additional inactivation that could be attributed to UC within the timescale studied. Due to the fundamental and practical limitations of solar focusing, the optical concentration ratio employed herein prevented the excitation beam from achieving the power densities required to attain UC efficiencies comparable to the laser experiments. We also observed that the high intensity of both the laser and sunlight induced rapid photoreactivation by the bacteria, which detracted from net disinfection performance. The results suggest that current UC materials perform inadequately for environmental application; nonetheless, valuable qualitative and quantitative insight was gained that more explicitly defines materials development goals and considerations for application of UC to environmental technology.

Visible photoluminescence of color centers in LiF crystals for absorbed dose evaluation in clinical dosimetry

Among insulating materials, lithium fluoride (LiF) has been successfully used as ionizing radiation dosemeter for more than 60 years. Thermoluminescence (TL) has been the most commonly used reading technique to evaluate the absorbed dose. Lately, optically stimulated luminescence (OSL) of visible emitting color centers (CCs) has also been explored in pure and doped LiF. This work focuses on the experimental behaviour of nominally pure LiF crystals dosemeters for 6 MV x rays at low doses based on photoluminescence (PL) of radiation induced CCs. Polished LiF crystals were irradiated using 6 MV x rays produced by a clinical linear accelerator. The doses (absorbed dose to water) covered the 1-100 Gy range. Optical absorption spectra show stable formation of primary F defects up to a maximum concentration of 2×1016 cm−3, while no significant M absorption band at around 450 nm was detected. On the other hand, under Argon laser excitation at 458 nm, PL spectra of the irradiated LiF crystals clearly exhibited the characteristic F2 and F+3 visible broad emission bands. Their sum intensity is linearly proportional to the absorbed dose in the investigated range. PL integrated intensity was also measured using a conventional fluorescence optical microscope under blue lamp illumination. The relationship between the absorbed dose and the integrated F2 and F+3 PL intensities, represented by the net average pixel number in the optical fluorescence images, is also fairly linear. Even at the low point defect densities obtained at the investigated doses, these preliminary experimental results are encouraging for further investigation of CCs PL in LiF crystals for clinical dosimetry.

Micro-Raman spectroscopic analysis of TiO2 phases on the root surfaces of commercial dental implants

ObjectivesTo identify the TiO2 phases of the root surface of commercially available titanium dental implants, subjected to various surface treatments. MethodsThe titanium implants studied were: Allfit (ALF), Ice (ICE), IMZ TPS (TPS), Laser Lok (LLK), Prima Connex (PRC), Ospol (OSP), Osseospeed TX (OSS), Osseotite Full (OTF), Replace Select (RPS), SLA (SLA) and Trilobe (TRB). The root parts of the implants (n:2) were analyzed by Raman microspectroscopy employing argon ion laser excitation (514.5nm wavelength) and a 100μm×100μm sampling area at two randomly selected sites. ResultsThe spectra of OSP and RPS showed the characteristic peaks of anatase, with traces of rutile (RPS). Complex phases composed of anatase, rutile and amorphous TiO2 were identified in ALF, ICE and LLK. Rutile and amorphous TiO2 were found in PRC, OSS, OTF, TPS and TRB, whereas rutile and possibly brookite were traced in SLA. In all implants, except OSP and RPS, peaks assigned to organic impurities (CH2, CH3) and carbonates were recorded. Ti2O3 was identified in OTF, PRC and Al2O3 in TRB. SignificanceGreat variations in the TiO2 polymorphs were registered among the implant root surfaces tested. Considering the important differences in the biological activity of these polymorphs, it can be concluded that provision of information regarding the TiO2 state on implant surfaces should be a mandatory task for implant manufacturers.

Time-dependent photoluminescence fatigue-recovery phenomena in germanium sulfide glasses

Photoluminescence (PL) fatigue-recovery phenomena in germanium sulfide were investigated, and the temperature dependence of the time-dependent PL intensity for several compositions (Ge33.3S66.7, Ge20S80 and Ge10S90) was analyzed. Side-bands were observed and two bands out of them for argon-ion laser excitation, one at 2.25eV for all compositions and the other extended from 2.15 to 2.45 for Ge10S90 especially at the lower temperatures, were used to fully describe the time-dependence.The functional form which is similar to those in the previous report [T. Nakanishi et al., J. Non-Cryst. Solids 354 (2008) 1627] was used and it is extended to account for the effect of side-band at below 30K for Ge20S80 and monotonous decrease of background for Ge10S90.

Characterization of Few-Layer Graphene on Stretchable Substrate Using Thermally-Treated Exfoliation

thin layer of gluelike PMMA, onto a stretchable substrate. It obtained a large, high-quality layer of graphene printed on the flexible substrate by using our proposed thermallytreated exfoliation technique. Aluminum (Al) film with a thickness of 100 nm was thermally deposited over the transparent PEN substrate using a metal mask in order to gather accurate signals of Raman spectra. We measured the Raman spectra to identify the presence of FLG. The Raman data were obtained using 514-nm argon-ion laser excitation at room temperature. Figure 2 shows the comparison of the 514 nm Raman spectra between the FLG and PMMA layer. The most prominent features in the Raman spectra as were also seen in other studies, 8,15,16 i.e., G band at ~1580 cm �1 and a band 2D band at ~2700 cm �1 : the 2D peak of the FLG formed by a single Lorentzian peak. The two peaks are enough data to prove the presence of the FLG. The PMMA did not show a peak in the Raman spectra. Prior to obtaining the Raman spectra above, we tried to get the Raman data of the graphene on PMMA layer, but without Al film, meaning the sample was entirely transparent. The Raman spectra we captured were out of the range that can be analyzed. The main cause is that the argon laser goes through the sample without any scattering. Thus, the layer to block the laser is needed to get the Raman spectra of the graphene. In addition to the Raman spectra analysis, the atomic force microscopic (AFM) measurement was performed in order to confirm the thickness of the FLG on the PMMA layer, since the number of the graphene layer can be estimated by the thickness.

Cooperative emission study in ytterbium doped NdVO4

The infrared and visible cooperative emissions of ytterbium ions are studied in Yb-doped NdVO4 single crystals. The absorption of optical phonons allows the emissions at room temperature when a Nd:YAG laser is used. Low temperature emissions are observed due to the Nd3+→Yb3+ energy transfer following an argon ion laser excitation of the Nd3+ ions. Analysis of the cooperative emission at low doping concentration (1%) indicates that it is generated by distant pair forming Yb3+ ions while at high doping concentration (≥ 5%) close ions magnetically coupled and superexchange mechanisms prevail in the emitting process.

Melatonin and IP3-induced Ca2+ Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

Growth of DLD-1 Colon Cancer Cells on Variotis™ Scaffolds of Controlled Porosity: A Preliminary Study

Tissue engineering will play an increasingly vital role in cancer research. Provision of biomimetic microenvironment systems for in vitro cancer models can be addressed in part by utilizing thick 3D scaffolds with high interconnective porosity . This approach gives rise to new analytical challenges and opportunities. In this preliminary study, Variotis™ synthetic scaffolds of high interconnected porosity and hierarchical structure were used. An effective macroscopic porosity of 94.3 ±1.74 vol% was attained by using microCT and finite element methods. The actual porosity was determined to be 94.6±0.29 vol%. Scaffolds were compressed in a customized jig to thicknesses of 99.5 mm, 74.6 mm, 46.3 mm (±0.5% tolerance) and then annealed to set respective porosities of 94.3 vol%, 93.2 vol%, 89.5 vol% (±1.5% tolerance). Scaffolds were then sectioned to 2mm thickness. DLD-1 colon cancer cells were grown on 3D scaffolds of three specified porosities for varying periods of time then imaged using confocal and scanning electron microscopy methods. Hoechst staining resulted with minimal scaffold autofluoresence while autofluoresence exceeded useful limits when used in conjunction with Alexa488-phalloidin under argon laser excitation in confocal microscopy. Using Hoechst staining, DLD-1 cells (nuclei) were observed to readily attach and proliferate on Variotis™ scaffolds. Normal DLD-1 cell morphologies were evident using scanning electron microscopy. The high interconnected porosity of the scaffolds allowed cells to be observed deep within scaffolds. Scaffolds remained structurally stable and unified throughout all culture experiments and provided ease of handling during cell culture and microscopy.

Quantification of luminally released serotonin in rat proximal colon by capillary electrophoresis with laser-induced fluorescence detection

Serotonin (5-hydroxytryptamine, 5-HT) plays vital roles in regulating gastrointestinal functions. Thus, the detection of 5-HT in the gastrointestinal tract is of great importance for biomedical research, medical diagnosis, and pharmaceutical therapy. This paper presents a simple, sensitive, and fast method for the quantification of luminally released serotonin in the feces and tissues of the rat proximal colon by means of capillary electrophoresis with laser-induced fluorescence detection. 5-Carboxyfluorescein N-succinimidyl ester was used for precolumn derivatization of serotonin. The optimal separation and detection conditions were obtained with an electrophoretic buffer containing 60 mM borate (pH 8.90) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). The serotonin concentrations in the feces and tissues of proximal colons were analyzed with this method, and the average values of serotonin in the feces samples were 1.951 +/- 0.446 ng/mg (male) and 2.095 +/- 0.533 ng/mg (female) and 1.397 +/- 0.267 ng/mg in rat proximal colon tissues. The results demonstrate that this method can accurately determine luminally released 5-HT in rats.

Excitation-time-dependent increment in the luminescence of fundus autofluorescence

Aim:To investigate the excitation-time-dependent increment in luminescence of fundus autofluorescence.Methods:Fundus autofluorescence (FAF) images were obtained with a confocal scanning laser ophthalmoscope. Consecutive 60 images of each eye were taken at...

Le pigment maculaire et sa mesure in vivo

Interest in macular pigment has been growing since it was first identified 200 years ago. Because of its antioxidant properties and putative protection against age-related macular degeneration (AMD), clinical, experimental, and epidemiologic studies have attempted to show a positive correlation between the high density of macular pigment and low risk for macular degeneration. Since the two main components of macular pigment, lutein and zeaxanthin, cannot be endogenously synthesized, oral supplementation may be an efficient means to increase macular pigment density. However, the ideal dosage for such supplementation remains to be determined, since absorption varies greatly from one subject to another. Hence, reliable quantitative measurement of macular pigment density is necessary before any beneficial effects of oral supplementation on AMD occurrence can be demonstrated; then at-risk subjects who would require such supplementation can be targeted. Several techniques can be used to measure macular pigment density noninvasively in vivo. Psychophysical methods are the most commonly used. They include heterochromatic flicker photometry and Raman spectroscopy. Heterochromatic flicker is the most widely used method and seems to be the easiest and least costly. However, because it requires very good cooperation from patients and a good grasp of the instructions, it is probably the least reliable and reproducible method. Raman spectroscopy, on the other hand, requires less cooperation on the part of the patient. Subjects are asked to overlap a blue disc and a red polka-dot pattern to ensure proper alignment of the eye. Since these instructions are simpler than those for heterochromatic flicker photometry, this technique is more reliable. Overall, the data obtained are more objective because they are based on the analysis of a signal emitted after an argon laser excitation of the macular pigment. Nevertheless, data are closely related to pupil diameter. Finally Raman spectroscopy requires expensive equipment. Physical methods are based on retinal excitation by an incident light and the analysis of the reflected signal obtained. These methods are mainly represented by autofluorescence measurement and fundus reflectance. These two techniques differ in the wavelength used in the incident light, because the reflected signal is itself related to the amount of incident light absorbed by retinal structures. Both techniques allow rapid and objective measurement of spatial distribution of macular pigment. The software of the modified Scanning Laser Ophthalmoscope (Heidelberg, Germany) further creates a density map image after digital subtraction of the log reflectance data obtained at two different wavelengths. Physical methods require little cooperation from patients and could be considered the most objective and high-performance technique available today. However, the instruments are still expensive and therefore cannot be widely used in clinical practice.

Capillary electrophoresis of aminoglycosides with argon-ion laser-induced fluorescence detection

A new analytical method for aminoglycosides (kanamycin, bekanamycin, paromomycin and tobramycin) based on capillary electrophoretic separation and argon-ion laser-induced fluorescence detection has been developed. 6-Carboxyfluorescein succinimidyl ester (CFSE) was used for precolumn derivatization of the non-fluorescent aminoglycosides. Optimal separation and detection were obtained with an electrophoretic buffer of 30 mM sodium borate (pH 9.0) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). The concentration limits of detection in aqueous solution were 3.9–8.2 nM. Combined with a simple cleanup procedure, this method can be applied to the determination of aminoglycosides in human plasma. A calibration curve ranging from 0.15 to 30 μM was shown to be linear. The limits of detection of aminoglycosides in human plasma were between 14.4 and 24.0 nM. Recoveries of spiked aminoglycosides in human plasma were between 92 and 105%.

Molecular Perspective of Antigen-mediated Mast Cell Signaling

Antigen-mediated cross-linking of the high affinity receptor for IgE (Fc epsilon RI), in the plasma membrane of mast cells, is the first step in the allergic immune response. This event triggers the phosphorylation of specific tyrosines in the cytoplasmic segments of the beta and gamma subunits of Fc epsilon RI by the Src tyrosine kinase Lyn, which is anchored to the inner leaflet of the plasma membrane. Lyn-induced phosphorylation of Fc epsilon RI occurs in a cholesterol-dependent manner, leading to the hypothesis that cholesterol-rich domains, or "lipid rafts," may act as functional platforms for IgE receptor signaling. Testing this hypothesis under physiological conditions remains challenging because of the notion that these functional domains are likely transient and much smaller than the diffraction limit of optical microscopy. Here we use ultrafast fluorescence dynamics to investigate the correlation between nanostructural changes in the plasma membrane (labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (diI-C18)) and IgE-Fc epsilon RI cross-linking in adherent RBL mast cells stimulated with multivalent antigen. Time-dependent two-photon fluorescence lifetime imaging microscopy of diI-C18 shows changes in lifetime that agree with the kinetics of stimulated tyrosine phosphorylation of Fc epsilon RI, the first identifiable biochemical step of the allergic response, under the same conditions. In addition, two-photon fluorescence lifetime imaging microscopy of Alexa Fluor 488-labeled IgE indicates that Förster resonance energy transfer occurs with diI-C18 in the plasma membrane. Our live cell studies provide direct evidence for the association of IgE-Fc epsilon RI with specialized cholesterol-rich domains within approximately 4-nm proximity and with an energy transfer efficiency of 0.22 +/- 0.01 at maximal association during IgE receptor signaling.

Opposing Roles of Syndecan-1 and Syndecan-2 in Polyethyleneimine-mediated Gene Delivery

Polyethyleneimines (PEIs) are efficient non-viral vectors for gene transfer. Heparan sulfate proteoglycans have been proposed to be the cell-surface receptors for PEI.DNA complexes (polyplexes). Here, we investigated if syndecan-1 (SDC1) and syndecan-2 (SDC2) are involved in PEI-mediated transfection. Following addition of polyplexes to HEK293 cells, green fluorescent protein-tagged SDCs rapidly formed clusters with PEI that were dependent of lipid raft integrity. However, although SDC1 overexpression slightly enhanced PEI-mediated gene expression, SDC2 dramatically inhibited it. Confocal microscopy analysis showed that SDC1.polyplex endocytosis occurred within minutes after addition of polyplexes, whereas SDC2.polyplex endocytosis took hours. Expression of SDC1 cytoplasmic deletion mutants revealed that the SDC1 cytoplasmic tail is required for gene expression, but not for clustering or endocytosis, whereas overexpression of SDC1/SDC2 chimeras showed that the SDC2 ectodomain is responsible for the inhibitory effect on gene transfer. This study provides evidence that SDCs may have opposing effects on PEI-mediated transfection.

Novel Allele Quantification Method To Classify Childhood Leukemia.

Background: Leukemia is the most common pediatric malignancy, accounting for nearly 40% of all new childhood cancer. The cure rates for pediatric ALL have increased to more than 80% and the cure rates for pediatric AML now approach 50%. Much of this progress can be attributed to cytogenetic and molecular risk stratification with subsequent randomized control trials. Genomic instability events may also serve as relevant prognostic biomarkers. A novel high-throughput genomic technology called Molecular Inversion Probes (MIPs) quantifies genomic instability, gene copy number and allelic imbalances at the highest genomic resolution. MIPs can analyze genetic target sequences in parallel with high specificity and sensitivity. Further classifying leukemic blasts using more precise molecular techniques could prove useful in further risk stratifying and developing new treatment strategies.Objective: To adapt MIPs to characterize and define unique molecular subtypes of childhood leukemia.Methods: DNA was extracted from AML and ALL clinical samples obtained at diagnosis. 400 ng of leukemic DNA was mixed with a previously synthesized MIP cancer probe set (667 probes representing all human chromosomes from exon sequences of 298 cancer genes); each probe contains two unique recognition sequences to targeted genomic DNA and a unique barcode (tag) sequence. A thermostable polymerase and ligase were added, and the mixture was denatured. Probe homology sequences were then annealed to complementary genome sites. Standard PCR reagants were added including one fluorescent-labeled primer and unlabeled primer. The reaction was thermocycled and probes circularized in the nucleotide specific “gap fill” reaction were amplified. The PCR mixture was hybridized to a barcode microarray overnight. The chip was stained and washed, and then interrogated by an Affymetrix scanner via argon laser excitation at 488-nm. ‘Cluster along Chromosomes' analysis was performed to detect significant amplifications or deletions.Results: Childhood ALL and AML samples had unique patterns of multiple gene deletions. Additionally, overlap was found in several of these deleted genes. Significantly deleted genes in common between ALL and AML samples included: RAP1GA1, HYAL1, HSPA9B, CSF1R, TNF, NOTCH4, FANCE, CCND3, BAG2, PLAGL1, MAD1L1, POLM, POLD2, IGFBP3, PSD, SUFU, CYP17A1, MXI1, HRAS, IGF2, RRM1, BRCA2, TP53, ERBB2, ERCC2, and POLD1.Conclusions: MIPs represent a novel technology for highly sensitive and specific gene copy analysis of childhood leukemia. Unique and distinguishing signatures of allelic imbalance can be determined between ALL and AML clinical samples using MIP technology. The unexpected overlap of deleted genes in both ALL and AML may represent a common molecular mechanism that requires further investigation. We currently are adapting MIPs for an expanded cancer probe set and are exploring new techniques to determine loss of heterozygosity (LOH) in pediatric leukemia.

Nuclear Transportation of Diacylglycerol Kinase γ and Its Possible Function in the Nucleus

Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to phosphatidic acid, and both lipids are known to play important roles in lipid signal transduction. Thereby, DGKs are considered to be a one of the key players in lipid signaling, but its physiological function remains to be solved. In an effort to investigate one of nine subtypes, we found that DGKgamma came to be localized in the nucleus with time in all cell lines tested while seen only in the cytoplasm at the early stage of culture, indicating that DGKgamma is transported from the cytoplasm to the nucleus. The nuclear transportation of DGKgamma didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGKgamma acts as a nuclear transport signal. Furthermore, to address the function of DGKgamma in the nucleus, we produced stable cell lines of wild-type DGKgamma and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGKgamma were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGKgamma regulates cell cycle.

Microscopic autofluorescence study of cardiac cancer and normal gastric tissues

To investigate the microscopic autofluorescent characteristics of cardiac cancer and autofluorescence distribution in different layers of gastric tissues. A double-channel laser scanning confocal microscopy with Argon ion laser (excitation wavelength 488 nm) and Helium-Neon laser (excitation wavelength 543 nm) were used to detect the autofluorescence emitted from 16 surgical specimens of cardiac cancer and corresponding normal gastric tissue. The autofluorescence image was analyzed between the cardiac cancer tissue and normal gastric tissue. Autofluorescence was detected successfully in cardiac carcinoma and corresponding normal gastric corpus tissues of all 16 cases. In different layers of gastric tissue, fluorescence presented the strongest signal in submucosa,the second strong in luminal propria with fluorescence mostly distributed in the glands, fluorescence signal from gastric cancer was significantly decreased compared with those in the different layers of normal tissues (P< 0.01). There are significant differences in the shape, color, distribution and fluorescence intensity of microscopic autofluorescence between cardiac cancer tissues and normal gastric corpus tissues.

Mutant Protein Kinase Cγ Found in Spinocerebellar Ataxia Type 14 Is Susceptible to Aggregation and Causes Cell Death

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease characterized by various symptoms including cerebellar ataxia. Recently, several missense mutations in the protein kinase Cgamma (gammaPKC) gene have been found in different SCA14 families. To elucidate how the mutant gammaPKC causes SCA14, we examined the molecular properties of seven mutant (H101Y, G118D, S119P, S119F, Q127R, G128D, and F643L) gammaPKCs fused with green fluorescent protein (gammaPKC-GFP). Wild-type gammaPKC-GFP was expressed ubiquitously in the cytoplasm of CHO cells, whereas mutant gammaPKC-GFP tended to aggregate in the cytoplasm. The insolubility of mutant gammaPKC-GFP to Triton X-100 was increased and correlated with the extent of aggregation. gammaPKC-GFP in the Triton-insoluble fraction was rarely phosphorylated at Thr(514), whereas gammaPKC-GFP in the Triton-soluble fraction was phosphorylated. Furthermore, the stimulation of the P2Y receptor triggered the rapid aggregation of mutant gammaPKC-GFP within 10 min after transient translocation to the plasma membrane. Overexpression of the mutant gammaPKC-GFP caused cell death that was more prominent than wild type. The cytotoxicity was exacerbated in parallel with the expression level of the mutant. These results indicate that SCA14 mutations make gammaPKC form cytoplasmic aggregates, suggesting the involvement of this property in the etiology of SCA14.