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Biomedical subjects

H Kamada

Publications and source records attributed to H Kamada.

At least 73 records · Page 4Linked to original sources
PubMed1999

New sensitive agents for detecting singlet oxygen by electron spin resonance spectroscopy.

Free radicals are well-established transient intermediates in chemical and biological processes. Singlet oxygen, though not a free radical, is also a fairly common reactive chemical species. It is rare that singlet oxygen is studied with the electron spin resonance (ESR) technique in biological systems, because there are few suitable detecting agents. We have recently researched some semiquinone radicals. Specifically, our focus has been on bipyrazole derivatives, which slowly convert to semiquinone radicals in DMSO solution in the presence of potassium tert-butoxide and oxygen. These bipyrazole derivatives are dimers of 3-methyl-1-phenyl-2-pyrazolin-5-one and have anti-ischemic activities and free radical scavenging properties. In this work, we synthesized a new bipyrazole derivative, 4,4'-bis(1p-carboxyphenyl-3-methyl-5-hydroxyl)-pyrazole, DRD156. The resulting semiquinone radical, formed by reaction with singlet oxygen, was characterized by ESR spectroscopy. DRD156 gave no ESR signals from hydroxyl radical, superoxide, and hydrogen peroxide. DRD156, though, gives an ESR response with hypochlorite. This agent, nevertheless, has a much higher ability to detect singlet oxygen than traditional agents with the ESR technique.

Dimethyl Sulfoxide↗
PubMed1999

EPR imaging for in vivo analysis of the half-life of a nitroxide radical in the hippocampus and cerebral cortex of rats after epileptic seizures.

Recently, we developed an in vivo temporal electron paramagnetic resonance (EPR) imaging technique to be applied to the brain of a rat, into which a blood-brain barrier (BBB)-permeable nitroxide radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM) was injected intraperitoneally. This imaging technique made it possible to measure decay rates of a nitroxide radical in multiple regions of the brain simultaneously. Using this technique, the half-life of PCAM was estimated from the exponential decay of the signal intensity derived from the temporal EPR images in the hippocampus and cerebral cortex of rats after a kainic acid (KA)-induced seizure. The hippocampal half-life of PCAM after KA-induced seizures was significantly prolonged (p < .01), whereas the prolongation of the cortical half-life was not significant. These findings suggest that following a KA-seizure, the intrahippocampal ability to reduce the nitroxide radical is impaired, but the ability is intact in the cerebral cortex. This is the first in vivo quantitative EPR imaging study that has a bearing on the pathogenesis of KA-induced seizures in the brain.

Animals↗
PubMed1999

Exogenous expression of p16INK4a is associated with decrease in telomerase activity.

In this study, gene transfection was used to determine whether the exogenous expression of p16INK4a modulated the biological characteristics of glioblastoma cells. The human glioblastoma cell line U87MG was doubly transfected with the plasmids pVgRXR and pIND harboring the wild-type p16 gene. The expression of p16INK4a in the resulting transfectants was regulated by the addition of the ecdysone homologue, muristerone A. When the cells expressed p16INK4a, their growth capacity was reduced and morphological changes such as an increase in cell size and cellular flattening were observed. The analysis of cell cycle regulation provided evidence that cells expressing p16INK4a were inhibited from entry into the cell cycle, as assessed by Ki-67 antigen expression. In addition, it was observed that the exogenous expression of p16INK4a was associated with decrease in telomerase activity.

Brain Neoplasms↗
PubMed1999

Alterations of tubulin function caused by chronic antidepressant treatment in rat brain.

1. Antidepressants have been used clinically for many years; however, the neurochemical mechanism for their therapeutic effect has not been clarified yet. Recent reports indicate that chronic antidepressant treatment directly affects the postsynaptic membrane to increase the coupling between the stimulatory GTP-binding (G) protein, Gs, and adenylyl cyclase. Tubulin, a cytoskeletal element, is involved in the stimulatory and inhibitory regulation of adenylyl cyclase in rat cerebral cortex via direct transfer of GTP to G proteins. In this study, we investigated whether the functional change of the adenylyl cyclase system caused by chronic antidepressant treatment involves an alteration of tubulin function in the regulation of adenylyl cyclase activity. 2. Male Sprague-Dawley rats were treated once daily with amitriptyline or saline by intraperitoneal injection (10 mg/kg) for 21 days, and their cerebral cortex membranes and GppNHp-liganded tubulin (tubulin-GppNHp) were prepared for what. 3. GppNHp-stimulated adenylyl cyclase activity in cortex membranes from amitriptyline-treated rats was significantly higher than that in control membranes. Furthermore, tubulin-GppNHp prepared from amitriptyline-treated rats was more potent than that from control rats in the stimulation of adenylyl cyclase activity in the cortex membranes of the controls. However, there was no significant difference in manganese-stimulated adenylyl cyclase activity between control and amitriptyline-treated rats. 4. The present results suggest that chronic antidepressant treatment enhances not only the coupling between Gs and the catalytic subunit of adenylyl cyclase but also tubulin interaction with Gs in the cerebral cortex of the rat.

Adenylyl Cyclases↗
PubMed1999

Mapping of 25 drought-inducible genes, RD and ERD, in Arabidopsis thaliana.

We mapped 25 Arabidopsis thaliana drought-inducible genes. Responsive to Dehydration (RD) and Early Responsive to Dehydration (ERD), to the Arabidopsis genome and compared map positions with those of mutants that show environmental stress response. We hybridized CIC yeast artificial chromosome (YAC) library filters with the cDNAs and determined the map positions of 18 corresponding genes. We screened the P1 library with 7 other clones and analyzed segregation of their restriction fragment length polymorphisms (RFLP) in recombinant inbred (RI) lines. One cDNA could be mapped because it had been sequenced by the Arabidopsis genome sequencing program.

Adaptation, Biological↗
PubMed1999

Regioselective conjugation of chitosan with a laminin-related peptide, Tyr-Ile-Gly-Ser-Arg, and evaluation of its inhibitory effect on experimental cancer metastasis.

A conjugate from the YIGSR peptide and chitosan has been prepared on the basis of a regioselective modification strategy of chitosan, and its antimetastatic activity has been assayed. Chitosan was converted to its organosoluble derivative, 6-O-trityl-chitosan, in 3 steps, and then coupled with the peptide portion containing a spacer amino acid, Ac-Tyr-Ile-Gly-Ser-Arg-beta Ala-OH [beta Ala; beta-alanine]. The product was treated with CHCl2CO2H to afford the desired conjugate, Ac-Tyr-Ile-Gly-Ser-Arg-beta Ala-chitosan, which proved to inhibit the experimental lung metastasis of B16BL6 melanoma cells in mice at lower doses than the parent peptide.

Animals↗
PubMed1999

Promotive effects of the peptidyl plant growth factor, phytosulfokine-alpha, on the growth and chlorophyll content of Arabidopsis seedlings under high night-time temperature conditions.

In order to investigate the function of the peptidyl plant growth factor, phytosulfokine-alpha (PSK-alpha), in plants, we examined the effect of PSK-alpha on the growth and chlorophyll content of Arabidopsis seedlings under high night-time temperature conditions. Although exposure to high night-time temperatures markedly reduced the fresh weight and chlorophyll content of the seedlings, these parameters in the plants supplied with PSK-alpha remained at the same levels as those of non-treated controls. These effects were not apparent when [2-5]PSK, Tyr-SO3H and kinetin were similarly supplied. The results suggest that PSK-alpha not only promotes cell proliferation, but may aid plants in their tolerance of heat stress.

Adenine↗
PubMed1999

Enhanced antitumor potency of polyethylene glycolylated tumor necrosis factor-alpha: a novel polymer-conjugation technique with a reversible amino-protective reagent.

We attempted to develop a novel method for the chemical modification of cytokines with synthetic polymers to increase in vivo therapeutic efficacy. A pH-reversible amino-protective reagent, dimethylmaleic anhydride (DMMAn), was used for polymer conjugation of tumor necrosis factor-alpha (TNF-alpha) with polyethylene glycol (PEG). The novel PEGylated TNF-alpha, PEG-TNF-alpha(+), which was pretreated with DMMAn before PEGylation, had 20% to 40% higher specific activity than PEG-TNF-alpha(-) (not treated with DMMAn) in vitro. Moreover, PEG-TNF-alpha(+) more potently caused tumor necrosis in Meth-A solid tumors in mice than did PEG-TNF-alpha(-). The middle fraction (M) of PEG-TNF-alpha(+), which was of the optimal degree of modification among PEG-TNF-alpha(+)s with different molecular weights, caused the highest degree of tumor hemorrhagic necrosis: 30-fold higher than native TNF-alpha and 2-fold higher than the most potent MPEG-TNF-alpha(-) that also had nearly the same molecular weight. Significantly, improvements in antitumor activity in vivo were more marked than were changes in specific activity. Furthermore, native TNF-alpha caused a dose-dependent body weight loss in mice, whereas no obvious side effects were observed in any PEG-TNF-alpha-treated mice. These results suggest that PEGylation using DMMAn is a useful for clinical cytokine delivery.

Animals↗
PubMed1998

Highly sensitive hepatitis B surface antigen detection by measuring stable nitroxide radical formation with ESR spectroscopy.

In areas where hepatitis B virus (HBV) is prevalent, HBV carriers negative for hepatitis B surface antigen (HbsAg) by enzyme-linked immunosorbent assay (ELISA) have been reported. Moreover, even after screening donor blood for HbsAg and hepatitis B core antibody (HBcAb), post-transfusion hepatitis B continues to occur, though with a decreasing frequency. Therefore, screening tests far more sensitive for detecting HBsAg than those currently available are needed. We developed a highly sensitive method for HBsAg detection. It is based on the recognition of peroxidase activity through measuring the formation of stable nitroxide radical with electron spin resonance (ESR) spectroscopy in the presence of hydrogen peroxide, p-acetamidophenol (p-AP), and 4-hydrazonomethyl-1-hydroxy-2,2,5,5,-tetramethyl-3-imidazoline-3-o xide (HHTIO). A cut-off value was established by testing of 186 healthy adults and 50 HBsAg-positive individuals. The signal to noise (S/N) ratio of less than 1.488 obtained by ESR spectroscopy was considered to be negative and more than 2.181, positive. The p-AP/HHTIO method was found to be 10 times more sensitive than the standard ELISA and reproducibility was excellent. Additional investigations were made on the HBsAg levels in the serum from 26 healthy subjects, in whom cut-off index levels on ELISA were negative but relatively high (range: 0.6 to 1.0); and on 15 patients with non B non C hepatitis. Three of 26 cases and 3 of 15 with non B non C hepatitis were judged to be HBsAg positive. Of these, 5 were found to be positive for HBV DNA by polymerase chain reaction (PCR). It was shown in this study that the p-AP/HHTIO method is practical and useful in screening HBV carriers because of the sensitivity in HBsAg detection, which is comparable to PCR analysis.

Acetaminophen↗
PubMed1998

Amino acids and peptides. XXXIII. A bifunctional poly(ethylene glycol) hybrid of laminin-related peptides.

A novel amino acid type poly(ethylene glycol) (aaPEG) was prepared and its application as a drug-carrier was examined. The peptides, Pro-Asp-Ser-Gly-Arg (PDSGR) and Tyr-Ile-Gly-Ser-Arg (YIGSR) which are active fragments of Laminin (a cell adhesion protein), were previously reported to be inhibitors of experimental metastasis. Both peptides were conjugated with aaPEG (average molecular weight, 3,000) to prepare a bifunctional peptide-PEG hybrid. The hybrid, PDSGR-aaPEG-YIGSR, was manually prepared by the solid-phase fluorenylmethyloxycarbonyl (Fmoc) strategy. The antimetastatic activity of the peptides in mice was not lost when conjugated to form a larger aaPEG molecule. YIGSR(375 nmol) and PDSGR (375 nmol and 750 nmol) did not demonstrate antimetastatic activity, but a mixture of PDSGR (187 nnmol) and YIGSR (187 nmol) exhibited an inhibitory effect. The inhibitory effect of the hybrid (187 nmol) was more potent than that of the mixture (PDSGR and YIGSR), indicating that the inhibitory effect of the peptides was potentiated by hybrid formation with aaPEG.

Animals↗
PubMed1998

Spatiotemporal measurement of free radical elimination in the abdomen using an in vivo ESR-CT imaging system.

Electron spin resonance (ESR) imaging can visualize the distribution of free radicals in living systems according to their concentrations. However, the application of ESR imaging to living animals has not been well established. Using a rapid field scan L-band ESR imaging system, we have successfully obtained two-dimensional ESR projection (xz-plane projection) and three-dimensional ESR-CT (trans-axial section along the y-axis) images of the abdomen of living mice after an injection of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) into the tail vein. The in vivo two-dimensional ESR projection imaging clearly visualized the carbamoyl-PROXYL distribution and the rapid decay process in the abdomen. Because among the viscera, the liver is most abundantly associated with a blood volume, the outline of the image can be composed mainly of this organ. We therefore attempted to find whether there will be a difference in spatiotemporal dynamics of carbamoyl-PROXYL in the abdomens between the control and the mice with liver damage by two-dimensional ESR projection. In the control mice, carbamoyl-PROXYL was almost completely eliminated from the abdomen within 5 minutes after administration. On the other hand, in mice with carbon tetrachloride-damaged livers, the decay of carbamoyl-PROXYL was markedly prolonged. Even at 5 min after administration, carbamoyl-PROXYL remained clearly visible in the abdomen. In vivo three-dimensional ESR-CT imaging showed an even distribution of carbamoyl-PROXYL throughout the whole liver, which corresponded well with the images of trans-axial sections of the murine abdomen. We have succeeded in displaying two-dimensional ESR projection and three-dimensional ESR-CT images of carbamoyl-PROXYL distribution and clearance in the abdomen of a living animal. The ESR-CT imaging technique is considered to be a powerful new tool for noninvasive investigations of the in vivo spatiotemporal dynamics of free radical distribution and elimination in the organs.

Abdomen↗
PubMed1998

A chromo box gene from carrot (Daucus carota l.): its cDNA structure and expression during somatic and zygotic embryogenesis.

A cDNA clone, designated DcDB1, was isolated from a cDNA library prepared from embryogenic cell clusters of carrot (Daucus carota L.) and characterized. The cDNA (1416 bp) encoded for a protein of 392 amino acid residues that contained a conserved chromo domain. The chromo domain is a 37 aa region found in both the Polycomo gene product, which is a repressor of homeotic genes, and a heterochromatin protein 1 of Drosophila. This domain is postulated to function in the binding of proteins to chromatin. Genomic blot hybridization experiments suggested that the number of DcCB1 genes in the carrot genome is low. The level of DcCB1 mRNAs was high in somatic embryos at globular and heart-shaped stages but low in torpedo-shaped somatic embryos. The level of DcCB1 transcripts decreased during the formation of seeds. The existence of both homeo and chromo box genes in plants suggests that regulatory mechanisms of developmental genes in plants may resemble those in Drosophila.

Amino Acid Sequence↗
PubMed1998

Alternatively spliced forms of cyclin D1 modulate entry into the cell cycle in an inverse manner.

Alternative splicing of cyclin D1 gene mRNA has recently been demonstrated. The novel transcript shows no splicing at the downstream exon 4 boundary and encodes a protein with an altered carboxyl-terminal domain that is a cyclin D1 variant; exon 5 is not included in the coding sequence which terminates downstream of exon 4. We here produced cells that exogenously express each form of cyclin D1 and analysed their cell cycle regulation. We found that (1) alternative splicing forms of cyclin D1 modulated entry into the cell cycle in an inverse manner; (2) both splicing forms suppressed cell growth; and (3) cells overexpressing form [a] were inhibited from entry into and completion of the S phase, although form [b]-expressing cells showed no reduction of G1- to S transition. We also found that overexpression of either cyclin D1 form upregulated Rb gene products, suggesting that this upregulation may be one of the causes of growth suppression in cyclin D1 overexpressing cells.

Alternative Splicing↗
PubMed1998

Antitumor activity of tumor necrosis factor alpha conjugated with divinyl ether and maleic anhydride copolymer on solid tumors in mice.

The purpose of this study is to further explore the usefulness of conjugation with functional polymeric modifiers for clinical application of bioactive proteins and to increase the therapeutic efficacy of tumor necrosis factor alpha (TNF-alpha) by conjugation in vivo. We synthesized TNF-alpha conjugated with the copolymer of divinyl ether and maleic anhydride (DIVEMA), which has intrinsic antitumor activity as a synthetic biological response modifier. The synthesis of DIVEMA-TNF-alpha could be controlled by the addition of 2,3-dimethylmaleic anhydride (DMMAn), which binds to or separates from amino groups when the pH is changed. The specific activity of DIVEMA-TNF-alpha (+) synthesized with DMMAn was hardly decreased in vitro. However, DIVEMA-TNF-alpha (-), which is conjugated without blocking by DMMAn, had a markedly diminished specific activity. DIVEMA-TNF-alpha (+) caused a dramatic hemorrhagic necrotic effect on the tumor when compared to native TNF-alpha 24 h after i.v. injection into mice bearing Sarcoma-180 solid tumors. In addition, DIVEMA-TNF-alpha (+) at a dose of only 100 Japan reference units per mouse revealed a dramatic antitumor effect that is approximately 100 times greater than native TNF-alpha and that could induce complete regression in all five mice bearing Meth-A solid tumors without any apparent side effects. Because neither DIVEMA alone nor a mixture of TNF-alpha and DIVEMA caused antitumor activity with i.v. administration, the increase in antitumor potency of TNF-alpha may be caused by the covalent conjugation with DIVEMA. DIVEMA-TNF-alpha at low dose revealed dramatic antitumor potency. Because TNF-alpha injected in vivo is extremely low-dose, concentration of intrinsic TNF-alpha in vivo is not influenced. Therefore, the cytokine network in vivo is not destroyed. These results suggest that DIVEMA is a useful polymeric modifier for conjugation of TNF-alpha to increase its antitumor activity.

Animals↗
PubMed1998

ESR spectroscopy for analysis of hippocampal elimination of a nitroxide radical during kainic acid-induced seizure in rats.

To analyze the balance between free radical production and elimination during seizure, techniques were developed to monitor the sequential changes in the level of an exogenous free radical by ESR in the brains of freely moving rats given kainic acid (KA) to induce seizures (n = 6) and control rats given saline (n = 6). The hippocampus of each rat was perfused with a nitroxide radical solution for 120 min by in vivo microdialysis. Then an intraperitoneal injection of KA or the physiological saline was given to each rat, and the perfusate was changed to Ringer's solution. ESR analysis of sequential samples of dialysate taken after injection revealed an exponential decay in signal amplitude. The median half-life of the nitroxide radical was significantly longer in the KA-treated group than in the control group (P < 0.01). This finding demonstrates a decreased capacity of the hippocampus to eliminate the nitroxide radical from the extracellular space during seizure and may help clarify the mechanism of neuronal cell vulnerability to free radicals during insult or degeneration.

Animals↗
PubMed1998

In vivo 300 MHz longitudinally detected ESR-CT imaging in the head of a rat treated with a nitroxide radical.

In vivo longitudinally detected ESR (LODESR)-CT system operating at 300 MHz was developed, based on the combination of a bridged loop-gap resonator and a pair of saddle-type pickup coils (STPCs). The sensitivity distribution of the STPCs was simulated on the basis of a magnetic dipole model. A LODESR-CT image of a phantom (a physiological saline solution of a nitroxide radical) was satisfactorily corrected by using the simulation result. LODESR-CT images of the head of a rats, in which nitroxide radical was injected intraperitonally, were also obtained through the correction in the same manner. This is the first in vivo CT (i.e. three-dimensional imaging) study with a 300 MHz ESR.

Animals↗
PubMed1998

Immunopharmacological studies on experimental allergic encephalomyelitis in DA rats.

The immunopharmacological profile of experimental allergic encephalomyelitis (EAE) in DA rats was compared to that in Lewis rats, which are commonly used for the study of EAE. DA rats showed higher susceptibility to EAE than Lewis rats. The immunological studies indicate that DA rats showed higher humoral and cellular immune response to myelin basic protein (MBP) than Lewis rats. This is probably due to the susceptible T cells to mitogen and autoreactive T cells to MBP in DA rats. In pharmacological studies, cyclosporin A, FK-506 and prednisolone suppressed the development of EAE in both strains in a similar manner. These results suggest the existence of two different types of T cells with respect to the onset of EAE in DA rats: one is highly reactive to mitogen and MBP; the other is regulated by cyclosporin A and FK-506.

Animals↗