Biomedical subjects

H Kamada

Publications and source records attributed to H Kamada.

At least 55 records · Page 3Linked to original sources

PubMed2000

In vivo electron spin resonance spectroscopy on signal decay of intrastriatal nitroxide radical after acute administration of haloperidol in rats.

Sequential changes in the electron spin resonance (ESR) signal intensity of nitroxide radical perfused in the striatum of rats treated with haloperidol (HPD) were evaluated using a 700-MHz ESR spectrometer. Nitroxide radical was perfused in the striatum by in vivo microdialysis. Nitroxide used was 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl. Following 6-h perfusion of the nitroxide radical by dialysis at the rate of 2 microl/min through the radical introducer that had been stereotaxically implanted in the rat's striatum, HPD or saline was injected intraperitoneally into the rats in the resonator. The sequential changes in the ESR spectrum of the nitroxide radical were then evaluated. Spectra were successively observed in all animals. The half-life, which was estimated on the basis of the exponential decay in signal intensity, was used as a parameter of decay rate of the ESR signal intensity of nitroxide radical. The half-life in the rats injected with HPD was significantly longer than that in controls. This finding suggests that the reducing ability of the striatal extracellular space of a rat acutely treated with HPD was decreased in comparison with that of the control.

Animals↗

PubMed2000

In vitro remodeling of tumor vascular endothelial cells using conditioned medium from various tumor cells and their sensitivity to TNF-alpha.

Prevention of tumor-associated blood vessel formation (angiogenesis) is a potentially powerful strategy to treat cancer. We found that tumor vascular endothelial cells were rearranged in vitro with conditioned culture medium derived from tumor cells and compared the sensitivity to the effects of TNF-alpha between normal and tumor endothelial cells. Incubation with tumor (Meth-A, Colon26)-derived conditioned medium showed that no effect was observed on cell growth. Tumor cells (Meth-A, Colon26, and B16BL6) only showed no sensitivity to TNF-alpha. Normal and control endothelial cells in culture showed little cytotoxicity in response to TNF-alpha treatment, but marked cytotoxicity of TNF-alpha was observed in endothelial cells cultured with tumor-derived conditioned medium. Sensitivity to TNF-alpha was different depending on the type of tumor from which the conditioned medium was derived. This difference in sensitivity was assumed to be due to the in vivo sensitivity to TNF-alpha. The results of this study suggested that the sensitivity of tumors to TNF-alpha is controlled by the sensitivity of tumor vasculature.

Animals↗

PubMed2000

Three-nucleon force and the A(y) puzzle in intermediate energy p--> + d and d--> + p elastic scattering

New vector analyzing-power data on p-->+d elastic scattering at E(p) = 150 and 190 MeV have been measured. These are presented together with existing data and with recent d-->+p vector and tensor analyzing power data at E(d) = 270 MeV. The strong negative extremum of both vector analyzing powers A(p)(y) and A(d)(y) at straight theta(c.m.) approximately 80 degrees -120 degrees is underestimated by Faddeev calculations using modern NN forces. Inclusion of the Tucson-Melbourne 3N force shifts the minima upwards, but with conflicting results for A(p)(y), and leading to a good description for A(d)(y). An A(p)(y) puzzle, previously thought to exist at energies E(N)</=30 MeV only, appears to exist also at intermediate energies.

Journal Article↗

PubMed2000

Proteome analysis of Oncorhynchus species during embryogenesis.

To understand the molecular mechanisms underlying normal and abnormal development of two salmonids, masu salmon (Oncorhynchus masou) and rainbow trout (O. mykiss), we used two-dimensional (2-D) electrophoresis to construct a series of 2-D maps during the embryonic period. We identified all visible protein spots on the 2-D map by assigning numbers for masu salmon and rainbow trout, and we determined N-terminal sequences of proteins for one hundred of the spots, that appear at very high concentrations in the whole embryos of masu salmon and rainbow trout. We also characterized embryonic stages according to the periods of appearance of spots. Most of the N-terminal sequences were identical or at least highly similar to partial sequences reported for vitellogenin (Vtg) of O. mykiss. A potential proteolytic processing of Vtg for rainbow trout is discussed in relation to the time of appearance and relative position of Vtg fragments within the complete protein sequence.

Amino Acid Sequence↗

PubMed2000

Onset of pulsatile waves in the heart walls at end-systole.

We have previously developed a novel ultrasonic method, namely, the phased tracking method, for accurately tracking the movement of the heart wall based on both the phase and magnitude of the demodulated signals to determine the instantaneous position of an object. With this method, it is possible to accurately detect small-amplitude velocity signals of less than a few micrometers of the heart wall that are superimposed on the motion of the heart wall due to the heart beat. There are several remarkable pulsatile waves during one cardiac cycle in the resultant velocity signals, some of them being commonly obtained for both healthy subjects and patients. These pulsatile waves cannot be recognized in standard echocardiography M-mode images. In this paper, by focusing on one pulsatile wave that occurs around the end-systole, the physiological meaning of these is considered based on various in-vivo experiments. The pulsatile wave measured by this novel ultrasonic method will offer potential for a quantitative assessment of myocardial viability.

Adult↗

PubMed2000

In vivo EPR imaging by using an acyl-protected hydroxylamine to analyze intracerebral oxidative stress in rats after epileptic seizures.

EPR imaging by using an acyl-protected hydroxylamine, 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP), in the head of a living rat after kainic acid (KA)-induced epileptic seizures was performed. ACP is a stable non-radical compound, but is easily deprotected with intracellular esterase to yield a hydroxylamine, which is oxidized by intracellular oxidative stress to yield an EPR-detectable nitroxide radical. From in vivo image data, the average values of EPR signal intensity from the hippocampus, striatum, and cerebral cortex were computed. There was no significant difference in cortical signal intensity between the control and KA-treated rats. The signal intensities from the hippocampus and striatum for the KA-treated rats were significantly higher than those for the control. The in vitro study showed that almost the same quantity of ACP moved into all regions of the brain of the control and KA-treated rats. These findings indicate that following a KA-induced seizure, the oxidative stress in the hippocampus and striatum is enhanced, but not so in the cerebral cortex.

Animals↗

PubMed2000

Ex vivo measurement of tissue distribution of a nitroxide radical after intravenous injection and its in vivo imaging using a rapid scan ESR-CT system.

To establish the usefulness of ESR-CT imaging with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) in living animals, we investigated the tissue distribution of carbamoyl-PROXYL after i. v. injection. Ten minutes after injection of carbamoyl-PROXYL, its concentrations in the liver, spleen, kidney, and plasma were higher than those in the small intestine and stomach. However, the inter-organ differences in concentrations were not striking. We selected the liver as a representative organ and attempted to measure the concentration of carbamoyl-PROXYL in it after washing out all of the blood by in situ perfusion with saline. The ESR spectrum of the liver homogenate after complete blood washout revealed that the concentration of carbamoyl-PROXYL was significantly reduced. Thus, at this time, carbamoyl-PROXYL was distributed predominantly in the plasma and/or loosely attached to the surfaces of cells. We obtained high-quality ESR-CT images of the murine abdomen at a measurement time of 40 s and found that a high-intensity area of carbamoyl-PROXYL appeared in the liver and kidneys, indicating an abundant blood circulation. Although the organ specificity of carbamoyl-PROXYL was weak, we consider that ESR-CT imaging with carbamoyl-PROXYL will be a powerful new tool for non-invasive anatomic analysis of the liver and the kidneys.

Animals↗

PubMed2000

In vivo evaluation of hippocampal anti-oxidant ability of zonisamide in rats.

We evaluated the anti-oxidant property of zonisamide (ZNS) in the rat brain under freely moving conditions by means of in vivo microdialysis of two exogenous nitroxide radicals, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) and 3-methoxy carbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM). Time-dependent changes in the signal intensities of these exogenous nitroxide radicals obtained from the hippocampal perfusates were observed using an X-band ESR spectrometer at 20-min intervals. The ESR signal intensities of nitroxide radicals decreased exponentially in all animals, which indicates that their half-life could be used as a parameter to estimate the decay rate of nitroxide radicals. Nitroxide radicals lose their paramagnetism when exposed to reductants in a biological system. Thus, half-life reflects the in vivo reducing ability. Although the half-life of carbamoyl-PROXYL, which could not pass the blood-brain barrier (BBB), was not changed when compared with the controls, pre-treatment with ZNS significantly shortened the half-life of PCAM, which could pass through the BBB. These findings suggest that the ZNS-induced increase in reducing ability did not occur within the extracellular space, but rather mainly at the neural cell membrane. This study is the first in vivo evaluation of the reducing ability of ZNS in freely moving animals.

Animals↗

PubMed2000

Site-specific accumulation of a LINE-like retrotransposon in a sex chromosome of the dioecious plant Cannabis sativa.

Male-associated DNA sequences were analysed in hemp (Cannabis sativa L.), a dioecious plant with heteromorphic sex chromosomes. A male-associated DNA sequence in C. sativa (MADC1) and its flanking sequence encoded a reverse transcriptase that was strongly homologous to those of LINE-like retrotransposons from various plants and other organisms, as well as another open reading frame (ORF). Fluorescence in situ hybridization (FISH) with MADC1 as probe, which yielded strong signals specific for male genomic DNA in gel blot analysis, generated a clear doublet signal at the end of the long arm of the Y chromosome. FISH using pachytene chromosomes of pollen mother cells at meiotic prophase I revealed that pairing of X and Y chromosomes occurred at the short arm of the Y chromosome where MADC1 was not present. Furthermore, FISH using extended DNA fibers, with MADC1 and its flanking DNA as probes, revealed that 100 to 200 copies of the retrotransposon were located in tandem on the Y chromosome. These results support the hypothesis that accumulation of a specific LINE-like retrotransposon at the terminal region of the long arm of the Y chromosome might be one cause of heteromorphism of sex chromosomes.

Amino Acid Sequence↗

PubMed2000

Role of malate synthesis mediated by phosphoenolpyruvate carboxylase in guard cells in the regulation of stomatal movement.

To clarify the pathway and role of malate synthesis in guard cells, epidermal strips isolated from Vicia faba L. leaflets were treated with 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP), a specific inhibitor of phosphoenolpyruvate carboxylase (PEPC). When dark-closed stomata were illuminated, malate accumulated in guard cells and stomata opened; these were inhibited by 60% and 30%, respectively, by 5 mM DCDP treatment. When light-opened stomata were treated with DCDP, both malate level in guard cells and stomatal aperture decreased. Treatment with 5 mM DCDP partially inhibited CO2 incorporation into malate in guard cells. Treatment with mannitol at 0.4 M (osmotic stress) in the light increased malate level in guard cells and closed stomata. DCDP treatment decreased both malate level and stomatal aperture under stressed condition. These results show that malate synthesis in the light under both non-stressed and stressed conditions is dependent on PEPC activity. The extent of the decrease in malate level by DCDP treatment was larger under stressed condition than under nonstressed condition, suggesting that osmotic stress may enhance the activity of this pathway of malate synthesis which is induced by light. Role of malate synthesis in guard cells is discussed.

Acrylates↗

PubMed2000

A secreted peptide growth factor, phytosulfokine, acting as a stimulatory factor of carrot somatic embryo formation.

Somatic embryogenesis of the carrot (Daucus carota L.) depends on a set of factors, some of which accumulate in culture medium (conditioned medium, CM). When embryogenic cell clusters were transferred to an embryo-inducing medium, addition of CM derived from somatic embryo culture markedly stimulated somatic embryo formation. The active principles were purified using a simple bioassay system and identified to be phytosulfokines (PSKs), sulfated oligopeptide growth factors originally isolated from a CM derived from asparagus (Asparagus officinalis L.) mesophyll culture. Quantification studies using a competition ELISA system employing an anti-PSK-alpha polyclonal antibody showed that PSK production might be related to growth of cells, rather than development of somatic embryos. Thus the stimulatory effect of PSK on somatic embryo formation might be due to promotion of cell proliferation.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2000

Purification from conditioned medium and chemical identification of a factor that inhibits somatic embryogenesis in carrot.

Somatic embryogenesis is strongly inhibited in cultures of carrot (Daucus carota L.) cells when the cell density is high. The inhibition is caused by factors that are released by cells into the medium of such cultures. In this study, we purified and identified one of the inhibitory factors found in the medium of high-cell-density cultures of carrot cells. The inhibitory factor with the strongest apparent activity was purified by fractionation with ethylacetate, chromatography on an octadecylsilyl (ODS) silica gel-column and HPLC. The inhibitory factor had a single peak of absorbance at 280 nm and was identified as 4-hydroxybenzyl alcohol by mass spectrometry and 1H- and 13C-NMR spectroscopy. Authentic 4-hydroxybenzyl alcohol strongly inhibited the formation of somatic embryos at a concentration equal to that in high-cell-density cultures. These results suggest that 4-hydroxybenzyl alcohol is a major factor that accumulates in high-cell-density cultures of carrot cells and inhibits somatic embryogenesis.

Benzyl Alcohols↗

PubMed2000

Facile synthesis of a chitosan hybrid of a laminin-related peptide and its antimetastatic effect in mice.

Laminin, a cell adhesion protein, consists of three peptide chains (alpha-1, beta-1 and gamma-1). The beta-1 chain contains a Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence that has been found to inhibit experimental metastasis in mice. We have prepared a hybrid of a water-soluble chitosan and a laminin-related peptide, and have examined its inhibitory effect on experimental metastasis in mice. A laminin-related peptide, acetyl-Tyr-Ile-Gly-Ser-Arg-betaAla-OH (Ac-YIGSRbetaA-OH), was prepared by a solid-phase method. Ac-YIGSRbetaA-OH was then reacted with a water-soluble chitosan. BetaAla is a spacer and was placed to avoid racemization of the Arg residue when the peptide was coupled with chitosan. Although chitosan has amino groups, they did not react with the peptide. Four methods were tried to achieve a coupling reaction, the diphenylphosphoryl azide method, the diisopropylcarbodiimide/1-hydroxybenzotriazole method, the water-soluble carbodiimide (WSC), and the 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) method, but all four methods were unsuccessful. Therefore, a small spacer, tert-butyloxycarbonyl-Gly, was intercalated in chitosan, by the TBTU method, to facilitate its coupling with the peptide. After removal of the protecting group, the Gly-chitosan was coupled with Ac-YIGSRbetaA-OH by the water-soluble carbodiimide method to give Ac-YIGSRbetaAG-chitosan. Conjugation of the peptide with the larger chitosan molecule did not reduce the inhibitory effect of the peptide on experimental metastasis in mice, it actually potentiated the antimetastatic effect, demonstrating that chitosan may be effective as a drug carrier for peptides.

Animals↗

PubMed2000

In vivo imaging of increased oxidative stress in the liver by electron spin resonance-computed tomography.

The aim of this study was to investigate whether increased hepatic oxidative stress could be visualised in living animals before the onset of obvious liver injury. Acute hepatic injury was induced in mice by priming with heat-killed Corynebacterium parvum followed by injection of a low dose of lipopolysaccharide (LPS). Low frequency band electron spin resonance-computed tomography (ESR-CT) with 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) was used to visualize hepatic oxidative stress. Biochemical and histological investigations performed 3 h after injection of LPS revealed no obvious injury to the liver. Conversely, significant hepatic oxidative stress could be detected at this time. Nitroxides such as carbamoyl-PROXYL are rapidly reduced to the corresponding hydroxylamine in vivo. resulting in the disappearance of their ESR signals. The kinetic clearance of carbamoyl-PROXYL after intravenous administration was delayed significantly in mice that had received LPS, due to impairment of the reduction system by hepatic oxidative stress. ESR-CT of the murine abdomen revealed a high intensity area of carbamoyl-PROXYL which consisted mainly of the liver and enlarged spleen. Time-course observations with ESR-CT using carbamoyl-PROXYL showed that the high intensity area in the liver disappeared rapidly due to reduction of carbamoyl-PROXYL. Three hours after LPS injection into the same mouse, ESR-CT images were obtained again by intravenous injection of carbamoyl-PROXYL. The ESR-CT images of the mouse with hepatic oxidative stress clearly showed that the high intensity area of carbamoyl-PROXYL in the liver persisted for a long period of time. This study is the first report to describe the use of in vivo ESR-CT for visualizing the state of increased oxidative stress in the liver before the onset of obvious hepatic injury.

Animals↗

PubMed1999

Bioconjugation of laminin-related peptide YIGSR with polyvinyl pyrrolidone increases its antimetastatic effect due to a longer plasma half-life.

Polyvinyl pyrrolidone (PVP) which can be radically synthesized and have a long blood residency was used to modify the laminin-related peptide YIGSR, and its inhibitory effect on experimental lung metastasis of B16-BL6 melanoma cells was examined. The antimetastatic effect of PVP-conjugated YIGSR (PVP-YIGSR) was more than 100-fold greater than that of native YIGSR. When injected intravenously, PVP-YIGSR showed more than a 15-fold longer plasma half-life relative to native YIGSR. In addition, the stability of YIGSR in plasma was increased by conjugation with PVP. These findings suggest that PVP is a useful polymeric modifier for increasing the antimetastatic activity of YIGSR.

Animals↗

PubMed1999

Molecular design of conjugated tumor necrosis factor-alpha: synthesis and characteristics of polyvinyl pyrrolidone modified tumor necrosis factor-alpha.

We conjugated tumor necrosis factor-alpha (TNF-alpha) with the synthetic polymeric modifier polyvinyl pyrrolidone (PVP) to facilitate its clinical use for anti-tumor therapy. TNF-alpha was chemically conjugated with the terminal carboxyl-bearing PVP at one end of its main chain, which was radically polymerized via the formation of an amide bond between the lysine amino groups of TNF-alpha and carboxyl group of PVP. In vitro specific bioactivity of PVP-conjugated TNF-alpha (PVP-TNF-alpha) relative to that of native TNF-alpha gradually decreased with increases in the degree of PVP attachment. In contrast, PVP-TNF-alpha in which 40% of TNF-alpha lysine residues were coupled with PVP (MPVP-TNF-alpha) exhibited the highest anti-tumor activity among the conjugated derivatives examined. MPVP-TNF-alpha had more than 200-fold higher anti-tumor efficacy than native TNF-alpha, and the anti-tumor activity of MPVP- TNF-alpha was more than 5-fold stronger than that MPEG- TNF-alpha which had the highest anti-tumor activity among PEG-conjugated TNF-alphas examined. Additionally, a high dose of native TNF-alpha induced toxic side-effects such as body weight reduction, piloerection and tissue inflammation, while no side effects were observed following i.v. administration of MPVP-TNF-alpha. The plasma half-life of MPVP-TNF-alpha (360 min) was about 80 and 3-fold longer than those of native TNF-alpha (4.6 min) and MPEG-TNF-alpha (122 min), respectively. These results suggested that PVP is a useful polymeric modifier for increasing the anti-tumor activity of PVP.

Animals↗

PubMed1999

Bioconjugation of laminin peptide YIGSR with poly(styrene co-maleic acid) increases its antimetastatic effect on lung metastasis of B16-BL6 melanoma cells.

A comb-shaped polymeric modifier, SMA [poly(styrene comaleic anhydride)], which binds to plasma albumin in blood was used to modify the synthetic cell-adhesive laminin peptide YIGSR, and its inhibitory effect on experimental lung metastasis of B16-BL6 melanoma cells was examined. YIGSR was chemically conjugated with SMA via formation of an amide bond between the N-terminal amino group of YIGSR and the carboxyl anhydride of SMA. The antimetastatic effect of SMA-conjugated YIGSR was approximately 50-fold greater than that of native YIGSR. When injected intravenously, SMA-YIGSR showed a 10-fold longer plasma half-life than native YIGSR in vivo. In addition, SMA-YIGSR had the same binding affinity to plasma albumin as SMA, while native YIGSR did not bind to albumin. These findings suggested that the enhanced antimetastatic effect of SMA-YIGSR may be due to its prolonged plasma half-life by binding to plasma albumin, and that bioconjugation of in vivo unstable peptides with SMA may facilitate their therapeutic use.

Animals↗

PubMed1999

Electrically detected magnetic resonance signal intensity at resonant frequencies from 300 to 900 MHz in a constant microwave field.

A method for electrically detected magnetic resonance (EDMR) measurement at different ESR frequencies under a constant alternating magnetic field has been established wherein the accurate relationship between EDMR signal intensity (from a photoexcited silicon crystal and a silicon diode) and a resonant frequency of 300 to 900 MHz (UHF band) was systematically clarified. EDMR signal intensity from a photoexcited silicon crystal against a resonant frequency fitted the curve of y = a(1 - e(-bx)) well, which approached a constant value at higher frequencies. The increase in the EDMR signal intensity from the silicon diode at higher resonant frequencies was smaller than that from the photoexcited silicon crystal. The difference can be explained by the influence of the skin effect; i.e., the microwaves do not penetrate deep into a highly conductive sample at higher frequencies. EDMR signal intensities of samples vs microwave power were measured at 890 MHz. The EDMR signal intensity from the silicon diode continued to increase as the microwave power was increased, while the signal intensity from the photoexcited silicon crystal saturated within the range. The difference can be similarly explained: due to the skin effect, the microwaves gradually penetrate into the silicon diode as the power increases, so that even when saturation has been reached outside, the microwave field inside the diode does not reach the saturation level.

Electricity↗