Synthesis of hydrogels with extremely low surface friction.
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Biocompatible Materials↗
Biomedical subjects
M Kinjo
Publications and source records attributed to M Kinjo.
Monitoring of in vitro and in vivo translation of green fluorescent protein and its fusion proteins by fluorescence correlation spectroscopy.
BACKGROUND: Because the process of protein translation is an event of sparse molecules, the measurement requires high sensitivity. One of the candidates for studying the molecules is fluorescence correlation spectroscopy (FCS), which gleans quantitative information from fluctuating fluorescence signals in a diluted solution. METHODS: Using FCS, the translation products of expression plasmid for green fluorescent protein (GFP) and its fusion proteins were measured in vitro and in vivo. RESULTS: In in vitro translation, the number of products increased linearly for 90 min upon concentration of the plasmid. The autocorrelation function for GFP was fitted with a one-component model with a diffusion time of 0.18 ms, which was identical to the value expected from the molecular weight. In the cases of GFP- tagged hypoxia-inducible factor-1 alpha and glucocorticoid receptor, each fitting result was significantly improved with a two-component model. The slow component with a diffusion time of 6 ms appeared to be related to the ribosome or polysome. In response to the addition of dexamethasone, the nuclear translocation from cytosol clearly induced the decrease in number of molecules in the focal point. CONCLUSIONS: FCS permits monitoring of the number of molecules translated in vitro and in vivo, the translation rate, and the molecular weight.
Animals↗
Microenvironment of endosomal aqueous phase investigated by the mobility of microparticles using fluorescence correlation spectroscopy.
Temporal observation of the dynamic behavior of molecules in cells gives information about the physiological environment at the region of interest. Here we report the direct measurement of the mobility of rhodamine-labeled microparticles (14 and 35 nm in diameter) ingested in endosomes of cultured bovine aortic endothelial cells using fluorescence correlation spectroscopy (FCS). The fluctuation of fluorescent signals from microparticles were measured by FCS. Obtained autocorrelation functions (FAFs) were analyzed by the 2-D multicomponent model according to an evaluation procedure we newly developed. It was found that microparticles moved freely in endosomes with average diffusion coefficients of 4.3 x 10(-8) and 2.7 x 10(-8) cm2 s(-1) for 14 and 35 nm, which were 45% slower than in water. This result implies that the endosomal aqueous phase is homogeneous with the viscosity about 2.2 times of water. Our study also proposes the new use of FCS for investigation of the internal space of organelles.
Animals↗
Two putative tumor suppressor genes on chromosome arm 8p may play different roles in prostate cancer.
Although loss of heterozygosity (LOH) on the short arm of chromosome 8 has been frequently observed in human prostate cancer, the relationship between LOH and clinical background is poorly understood. Fluorescence in situ hybridization (FISH) was employed to evaluate the chromosomal deletion on 8p in 42 prostate cancers using a centromeric probe for chromosome 8, in combination with 4 cosmid probes spanning 8p12 to 8p22. Deletions for at least one locus on the 8p were observed in 29 (69.0%) tumors. The most frequently deleted regions were 8p22 (54.8%) and 8p21.3 (52.4%), in almost the same frequency. The second most frequently deleted region was 8p21.1-p21.2 (38.1%). Deletions of 8p22 and 8p21.3 significantly correlated with tumor grade (P=0.0034, Fisher's exact probability test). A significantly higher frequency of the deletion on 8p21.1-p21.2 was observed in advanced prostate cancer (beyond capsular penetration or positive nodal metastases) than in localized prostate cancer (P=0.0033). In particular, deletion of 8p21.1-p21.2 was more frequently observed in the cases with lymph node metastases than without them (P=0.0029). No clinicopathological parameters had significant relation to deletions on 8p12. These results suggest that deletions on 8p22-p21.3 play an important role in tumor differentiation, while an 8p21.1-p21.2 deletion plays a role in the progression of prostate cancer.
Aged↗
[Comparisons of factors affecting voiding disorders between patients with benign prostate hyperplasia and volunteers].
PURPOSE: The prostate size and motivation to visit clinics were investigated in patients with prostate hyperplasia. OBJECTS AND METHODS: One hundred ninety-five patients who had urinary symptoms and visited our outpatient clinic between September 1994 and October 1999 and 268 age-matched volunteers in Mitaka City who underwent a medical examination of the prostate in June 1997 were compared. International Prostate Symptom Score (IPSS), Quality Of Life Score (QOL score), residual urine volume, prostate volume and urinary flow rate were measured. RESULTS: The prostate volume of the volunteers was 20-25 cm3 irrespective of the age. The prostate size of the outpatients was larger than that of the volunteers for every age group. IPSS and QOL score were significantly higher in the outpatients than in the volunteers. Diurnal urinary frequency and sense of residual urine contributed to the discrimination index of the two groups more significantly than the other scores. There was a significant correlation between prostate volume and residual urine volume. The score of weak urinary steam was inversely and significantly correlated with peak urinary flow rate. CONCLUSIONS: There was no age-related enlargement of the prostate gland. The prostate gland was significantly larger in the patients than in the volunteers even in those in their fifties. Urinary frequency and sense of residual urine are important factors for men to seek and receive medical care.
Adult↗
Oligomeric tubulin in large transporting complex is transported via kinesin in squid giant axons.
Slow axonal transport depends on an active mechanism that conveys cytosolic proteins. To investigate its molecular mechanism, we now constructed an in vitro experimental system for observation of tubulin transport, using squid giant axons. After injecting fluorescence-labeled tubulin into the axons, we monitored the movement of fluorescence by confocal laser scanning microscopy and fluorescence correlation spectroscopy. Here, from the pharmacological experiments and the functional blocking of kinesin motor protein by anti-kinesin antibody, we show that the directional movement of fluorescent profile was dependent on kinesin motor function. The fluorescent correlation function and estimated translational diffusion time revealed that tubulin molecule was transported in a unique form of large transporting complex distinct from those of stable polymers or other cytosolic protein.
Actomyosin↗
c-fos expression and redox state of cytochrome oxidase of rat brain in hypoxia.
Hypoxic induction of c-fos was studied in rat brains as a function of the cerebral oxygenation state using near-infrared spectroscopy by which the hemoglobin oxygenation state and redox state of mitochondrial cytochrome oxidase could be monitored noninvasively. Following reoxygenation after hypoxia, the expression of c-fos and MAP2 mRNAs was followed by reverse transcription-coupled PCR. The expression of MAP2 remained unchanged throughout all the conditions from 21 to 8% FiO2. Under mildly hypoxia conditions, c-fos mRNA was not induced. Hemoglobin was partially deoxygenated but cytochrome oxidase remained fully oxidized. Severe hypoxia, where cytochrome oxidase was reduced, caused a significant induction of c-fos mRNA At this stage, the oxygen concentration in cerebral tissue fell to < 10(-7) M. These data suggest that the decline in oxidative phosphorylation might be a trigger for the induction of c-fos mRNA.
Animals↗
Effect of electrostatic interactions on the binding of charged substrate to GroEL studied by highly sensitive fluorescence correlation spectroscopy.
The binding processes of GroEL with apo cytochrome c (apo-cyt c) and disulfide-reduced apo alpha-lactalbumin (rLA) in homogeneous solution at low concentration were analyzed by fluorescence correlation spectroscopy (FCS) with extremely high sensitivity. Although apo-cyt c, a positively charged substrate, was tightly bound to GroEL in both the absence and the presence of 200 mM KCl, the strength of the binding was changed with varying salt concentration. Results from experiments when two different salts (KCl or MgCl(2)) were titrated into a sample solution containing GroEL and apo-cyt c clearly showed that the binding strength decreased with increasing salt concentration. On the other hand, the binding affinity of GroEL for rLA, a negatively charged substrate, increased by adding of 200 mM KCl. These results indicate that electrostatic interactions substantially contribute to the binding interactions by manipulating the binding affinity of charged substrates.
Animals↗
Familial amyloidotic polyneuropathy (ATTR Ser50Ile): the first autopsy case report.
We report an autopsy case of a pedigree of familial amyloidotic polyneuropathy (FAP) with a mutation of isoleucine-50 transthyretin (ATTR Ser50Ile). A 47-year-old man started developing severe diarrhea and weight loss at age 41 years, followed by urinary incontinence, autonomic-nervous-system abnormalities and serious heart failure; the diagnosis of FAP (ATTR Ser50Ile) was made on the basis of genetic, histochemical and immunohistochemical analysis. Six years after the initial symptoms, he died of septic shock. Autopsy revealed suppurative peritonitis, perforation of the sigmoid colon and marked systemic amyloid deposition. The total amount of amyloid deposited in the heart was greatly increased and was much lower in the thyroid gland and kidneys compared with amyloid deposits in ordinary FAP (ATTR Val30Met). Amyloid deposition in peripheral vessel walls was prominent, particularly in lymphatics and veins. His elder sister, 54 years old, started to develop orthostatic hypotension at age 49 years, followed by dysesthesia, diarrhea and severe congestive heart failure. Endomyocardial biopsy revealed severe TTR-amyloid deposition; ultrastructural examination demonstrated that amyloid fibrils were deposited disproportionately and extended radially around microvessels.
Aged↗
The significance of the free-to-complexed prostate-specific antigen (PSA) ratio in prostate cancer detection in patients with a PSA level of 4.1-10.0 ng/mL.
OBJECTIVE: To compare the ratio of free prostate specific antigen (fPSA), total PSA (tPSA) and complexed PSA (cPSA, measured using a novel immunoassay) with other variables used to detect prostate cancer in patients with intermediate serum PSA levels of 4.1-10.0 ng/mL. PATIENTS AND METHODS: From July 1997 to August 1998, 140 consecutive patients were assessed; all had intermediate serum PSA levels and/or abnormal findings on a digital rectal examination. All patients underwent transrectal ultrasonography (TRUS)-guided biopsy, and the prostate and transition zone volumes were determined by TRUS. Free and tPSA were measured using the Tandem-R assay (Hybritech Corp., San Diego, CA). PSA complexed with alpha1-antichymotrypsin (cPSA) was measured using an appropriate assay. The ability of cPSA, free-to-total PSA ratio (f/tPSA), free-to-complexed PSA ratio (f/cPSA), tPSA density of the whole prostate (PSAD), of the transition zone (tPSATZ), and cPSA density of the whole prostate (cPSAD) and of the transition zone (cPSATZ) to improve the power of PSA in detecting prostate cancer was evaluated using receiver operating characteristic (ROC) curves. Results Of the 140 patients, 126 had histologically confirmed benign disease and 14 had prostate cancer. The cPSA alone had better specificity for detecting prostate cancer than had tPSA alone but the difference was not significant. The area under the ROC curve for f/cPSA was larger than those for all other variables. With a 93% sensitivity for detecting prostate cancer, a f/cPSA threshold of 25% would result in fewer unnecessary biopsies (40% f/cPSA specificity) than with all other PSA variables. The difference in the resolution was significant between f/cPSA and tPSA, cPSA, tPSAD and tPSATZ, but not with f/tPSA, cPSAD or cPSATZ. In patients with a prostate volume of < 30 mL, the cPSATZ showed better specificity for prostate cancer than tPSA alone. CONCLUSION: Measuring the level of cPSA and its derivatives may provide better differentiation of prostate cancer and benign disease than tPSA alone in patients with a tPSA level of 4.1-10.0 ng/mL.
Aged↗
Analysis of interaction between chaperonin GroEL and its substrate using fluorescence correlation spectroscopy.
Fluorescence correlation spectroscopy (FCS) provides information about translational diffusion properties of fluorescent molecules in tiny detection volume and allows the analysis of binding processes of biomolecules in homogeneous solution. In this study, FCS was used to measure equilibrium binding constants of disulfide-reduced apo-alpha-lactalbumin (rLA), denatured pepsin, and apo-cytochrome c (apo-cyt c) bound by chaperonin GroEL at different salt concentrations. The results indicate that apo-cyt-c has a much stronger affinity to GroEL than denatured pepsin and rLA have. Titration experiments of GroEL to each substrate with various concentrations of four kinds of salts (K+, Na+, Ca2+, and Mg2+) show that the binding constant of denatured pepsin and rLA to GroEL depends on the salt concentration. The dependence of denatured pepsin binding to GroEL on salt concentration is much stronger than that of rLA. However, the interaction of positively charged apo-cyt c with GroEL is not affected by the salt concentration. Furthermore, the divalent cation promotes the binding of GroEL to denatured pepsin and rLA more strongly than does the monovalent cation.
Animals↗
Acute pan-costochondritis demonstrated by gallium scintigraphy.
Costochondral inflammation is a rare clinical finding. A case is presented, in which acute chostochondritis is demonstrated by gallium scinitigraphy.
Acute Disease↗
Multiple extra-bone accumulations of technetium-99m-HMDP.
Bone scintigraphy was performed on a woman 2 y and 10 mo after surgery for rectal cancer. Intense extra-bone accumulations of 99mTc-HMDP were visible in an aortic atherosclerosis lesion and in a metastatic liver tumor. Uptake in the metastatic lesion was confirmed by x-ray CT. Uptake in the aortic lesion was shown as typical calcification by x-ray CT.
Aged↗
[Single molecule detection using fluorescence correlation spectroscopy].
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DNA↗
Hot spots observed on pulmonary perfusion imaging: a case report.
A case of hot spots observed on perfusion lung images is presented. This artifact is well known, however, it is rarely seen in our experience. Although this artifact can occur if a faulty injection technique is used, the artifact also may be caused by embolization of the MAA in the upper extremity venous blood after injection. This cause of the artifact is beyond the control of the nuclear medicine professional.
Artifacts↗
Fluorescence correlation spectroscopy of enzymatic DNA polymerization.
We show that fluorescence correlation spectroscopy (FCS) can be used as a reliable, simple, and fast tool for detecting products of the polymerase chain reaction (PCR). By use of autocorrelation experiments, it is demonstrated that fluorescent 217-bp DNA fragments can be detected at very low initial ss M13mp18(+) DNA and tetramethylrhodamine-4-dUTP concentrations and that these polymers are cleaved by the chosen restriction enzymes. A FCS calibration curve is presented, where the translational diffusion times of different size DNA fragments are plotted versus the number of base pairs they contain. At zero and very low template concentrations a large "background" species emerges, which is a reflection of the experimental conditions chosen and the extremely high sensitivity of FCS. The relative amount of nonspecific product formation is less than 1%. The ease by which a FCS measurement can be performed (a few minutes at most) also enables the technique to be used as an effective screening method.
Base Composition↗
Single-molecule analysis of restriction DNA fragments using fluorescence correlation spectroscopy.
The cleavage of fluorescence-labeled M13DNA (7250 bp) using HaeIII, HgaI, BsmAI, and BspMI was analyzed by fluorescence correlation spectroscopy (FCS) in a small volume (1.5 x 10(-15) liters). The digestion process can be monitored by the decrease in amplitude of the fluorescence correlation function while the original DNA molecule is divided into several fragments by the enzymes. To analyze this reaction by FCS, we derived a practical equation for estimating the number of molecules in the FCS measurements. Under standard enzymatic conditions, HaeIII and BsmAI digested fluorescence-labeled DNA to completion in the range of 8 h, whereas HgaI and BspMI digested the DNA after 40 h. The comparison of recognition sequences suggested that some tagged nucleotides could be inserted between the recognition site and the cleavage site of the slow enzyme group. The decrease in amplitude in the fluorescence correlation function quantitatively monitors the hydrolysis of DNA during the digestion process.
Base Sequence↗
Detection of asymmetric PCR products in homogeneous solution by fluorescence correlation spectroscopy.
The yield of the double-stranded DNA product (500 bp) of asymmetric PCR with a rhodamine-labeled primer (Rho-primer) was determined in a homogeneous solution using fluorescence correlation spectroscopy (FCS). FCS provides the average number of molecules in a focused volume and the diffusion constant that relates the molecular weight. Since FCS measures the fluctuation of fluorescence intensity in a very small sample volume, the reaction mixture was directly placed on the FCS optical field without any purification procedure after amplification. The result of changing the initial number of templates suggested that elongation of the Rho-primer could be detected by FCS in a PCR mixture containing a single copy of the target gene in the initial condition. Possible scientific applications and perspectives of the proposed approach are discussed.
DNA Primers↗