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Biomedical subjects

T Kamitani

Publications and source records attributed to T Kamitani.

At least 55 records · Page 3Linked to original sources
PubMed2000

[High-dose chemotherapy with autologous peripheral blood stem cell transplantation for treatment of non-Hodgkin's lymphoma].

Findings for 41 patients with non-Hodgkin's lymphoma (NHL) treated with high-dose chemotherapy (HDC) and/or autologous peripheral blood stem cell transplantation (PBSCT) are reported. Two of the 41 patients were treated with HDC alone without PBSCT. At transplant, 20 patients were in complete remission, while 19 had resistant NHL and had failed to achieve a complete remission (CR) after several courses of conventional chemotherapy. The conditioning regimens used were mainly ACE (cytarabine, cyclophosphamide, etoposide) and MEAC (MCNU, etoposide, cytarabine, cyclophosphamide). The treatment-related mortality rate was 4.9%. Two patients treated with MEAC died from intractable congestive heart failure. Nine of the 19 patients with resistant NHL achieved CR, and at a median follow-up of 26 months (range, 3 to 93 months) the estimated two-year disease-free survival rate for these patients was 44.4%. Four patients in CR at present were in partial remission before HDC and PBSCT. Fifteen of the 20 patients in CR before HDC were transplanted in first CR and 5 in 2nd CR. At a median follow-up of 49 months (range, 3 to 96 months), the estimated 3-year DFS for the group of all patients was 73.7%. Five relapses occurred between 5 and 35 months post-transplantation. In conclusion, HDC and PBSCT as induction therapy was only effective for patients with resistant NHL who responded to conventional chemotherapy, and may improve the survival of patients in CR as consolidation therapy.

Adolescent↗
PubMed1999

Identification of novel isoforms of human RAD52.

In yeast, RAD52 has been shown to be essential for homologous recombination of DNA and to be involved in the repair of double-stranded DNA breaks. Recently, the human homologue of yeast RAD52, a 418-amino-acid protein, has been identified. In this study, we report three different isoforms of human RAD52 isolated from brain and testis cDNA libraries. cDNAs of these isoforms contain distinct insertions and encode truncated proteins due to translational frame-shifts. The three isoforms consist of 226-, 139-, and 118-amino-acid residues, and are designated as RAD52beta, gamma, and delta, respectively. The original RAD52 is termed as RAD52alpha in this paper. Messages of these isoforms have been detected in various human tissues. We found that the RAD52 isoforms were unable to interact with RAD52alpha because of partial defect of the self-interaction domain. Furthermore, like RAD52alpha, the isoforms have been shown to bind to both single-stranded and double-stranded DNA. These results suggest that RAD52beta, gamma, and delta might affect RAD52alpha function through their DNA-binding property and their inability to bind to RAD52alpha. Thus, these isoforms might act as dominant negative mutants or negative regulators of RAD52alpha.

Alternative Splicing↗
PubMed1999

The von Hippel-Lindau tumor suppressor gene product promotes, but is not essential for, NEDD8 conjugation to cullin-2.

We have previously shown that human cullin-2 (Cul-2) is covalently modified at Lys-689 by NEDD8 (Wada, H., Yeh, E. T. H., and Kamitani, T. (1999) Biochem. Biophys. Res. Commun. 257, 100-105). Cul-2 has also been reported to form a multiprotein complex, Cul-2.VBC, with the von Hippel-Lindau tumor suppressor gene product (pVHL) and elongins B and C. In this study, using an in vivo coexpression system in COS cells, we show that NEDD8 conjugation to Cul-2 is promoted by coexpression with wild-type pVHL and elongins B and C. Interestingly, tumorigenic mutants and deletion mutants of pVHL, which are unable to form a Cul-2.VBC complex, do not have the activity to promote NEDD8 conjugation to Cul-2. These results suggest that the complex formation is required for NEDD8 conjugation to Cul-2. Furthermore, we used a pVHL-deficient cell line, 786-0, to show that Cul-2 is poorly but clearly conjugated by NEDD8, indicating that pVHL is not the only molecule that promotes NEDD8 conjugation to Cul-2. Taken together, the VBC complex appears to have ligase activity in the conjugation of NEDD8 to Cul-2.

Amino Acid Substitution↗
PubMed1999

PML is critical for ND10 formation and recruits the PML-interacting protein daxx to this nuclear structure when modified by SUMO-1.

Nuclear domain 10 (ND10), also referred to as nuclear bodies, are discrete interchromosomal accumulations of several proteins including promyelocytic leukemia protein (PML) and Sp100. In this study, we investigated the mechanism of ND10 assembly by identifying proteins that are essential for this process using cells lines that lack individual ND10-associated proteins. We identified the adapter protein Daxx and BML, the RecQ helicase missing in Bloom syndrome, as new ND10-associated proteins. PML, but not BLM or Sp100, was found to be responsible for the proper localization of all other ND10-associated proteins since they are dispersed in PML-/- cells. Introducing PML into this cell line by transient expression or fusion with PML-producing cells recruited ND10-associated proteins into de novo formed ND10 attesting to PMLs essential nature in ND10 formation. In the absence of PML, Daxx is highly enriched in condensed chromatin. Its recruitment to ND10 from condensed chromatin requires a small ubiquitin-related modifier (SUMO-1) modification of PML and reflects the interaction between the COOH-terminal domain of Daxx and PML. The segregation of Daxx from condensed chromatin in the absence of PML to ND10 by increased accumulation of SUMO-1-modified PML suggests the presence of a variable equilibrium between these two nuclear sites. Our findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Identification of NEDD8-conjugation site in human cullin-2.

NEDD8 is a novel ubiquitin-like protein that has been shown to conjugate to nuclear proteins in a manner analogous to ubiquitination and sentrinization. Recently, human cullin-4A was reported to be conjugated by a single molecule of NEDD8. Here, we show that human cullin-2 is also conjugated by a single molecule of the NEDD8. The C-terminal 171-amino-acid residues in human cullin-2 are sufficient for NEDD8-conjugation. In addition, the equivalent C-terminal fragments of other cullins have been shown to be conjugated by NEDD8. Mapping of the NEDD8-conjugation site revealed that Lys-689 in human cullin-2 is conjugated by NEDD8. Interestingly, the Lys residue at position 689 in cullin-2 is conserved in all cullin family members, including human cullin-1, -2, -3, -4A, -4B, and -5 and yeast cullin (Cdc53), suggesting the possibility that other cullin family members are conjugated by NEDD8/Rub1 at a Lys residue of equivalent position.

Amino Acid Sequence↗
PubMed1999

Visual event-related potential changes at two different tasks in nondemented Parkinson's disease.

A visual oddball paradigm and an S1-S2 paradigm were employed to evoke event-related potentials (ERPs) in 38 nondemented Parkinson's disease (PD) patients and 24 healthy elderly subjects. Delayed N200 and reduced P300 amplitude in the whole PD sample were only found in the S1-S2 paradigm. Delayed N200 and reaction time in PD with short duration of illness were found only after the S1-S2 paradigm, which might be an early sign of cognitive changes in PD. This is the first study to apply an S1-S2 paradigm for a visual P300 test in PD and proved the value of this paradigm for detecting minor cognitive abnormalities. ERP changes were correlated with clinical features. Reduced P300 amplitude for the S1-S2 paradigm was significantly correlated with WAIS-R scores and gait disturbance. The correlation between P300 amplitude and clinical scores has rarely been discussed before. P300 latency during the oddball paradigm in PD was influenced by age at test, age at onset, and duration of illness. This may explain why P300 results in nondemented PD have varied among previous authors.

Age of Onset↗
PubMed1999

A novel Sac I RFLP in the 3' untranslated region of the myotonin protein kinase gene.

We found a novel Sac I polymorphism downstream of CTG repeats in the 3' untranslated region of the myotonin protein kinase (MT-PK) gene. A C to G transition at nucleotide 13,590 in the gene was revealed by Southern blotting and confirmed by sequencing analyses. The allelic frequency of the C:G polymorphism in 63 unrelated Japanese individuals was estimated to be 0.98:0.02. When Southern blotting is employed in the analysis of the CTG repeat length in the MT-PK gene, this Sac I polymorphism should be taken into consideration.

3' Untranslated Regions↗
PubMed1999

Effect of interstimulus interval on visual P300 in Parkinson's disease.

OBJECTIVE: Visual event related potentials (ERPs) were studied during an oddball paradigm, to testify whether cognitive slowing in Parkinson's disease exists and to investigate whether cognitive information processing can be influenced by different interstimulus intervals (ISIs) of an oddball task in patients with Parkinson's disease and normal subjects. METHODS: ERPs and reaction time were measured in 38 non-demented patients with Parkinson's disease and 24 healthy elderly subjects. A visual oddball paradigm was employed to evoke ERPs, at three different interstimulus (ISI) intervals: ISI(S), 1600 ms; ISI(M), 3100 ms; and ISI(L), 5100 ms. The effect of ISIs on ERPs and reaction time was investigated. RESULTS: Compared with the normal subjects, P300 latency at Cz and Pz was significantly delayed after rare target stimuli in patients with Parkinson's disease only at ISI(L). Reaction time was prolonged in patients at all the three ISIs, compared with the normal controls. There was also significantly delayed N200 and reduced P300 amplitude at Cz and/or Pz to rare non-target stimuli in patients at the three ISIs, compared with the normal controls. During rare target visual stimulation, P300 latency and reaction time in the patients with Parkinson's disease and reaction time in the normal subjects were gradually prolonged as the ISI increased. CONCLUSION: Prolonged N200 latency to rare non-target stimuli might indicate that automatic cognitive processing was slowed in Parkinson's disease. Cognitive processing reflected by P300 latency to rare target stimuli was influenced by longer ISI in patients with Parkinson's disease.

Adult↗
PubMed1999

[Improved detection of fill-in using sublingual nitroglycerin in technetium-99m tetrofosmin exercise/rest single photon emission computed tomography one day protocol for old myocardial infarction].

This study assessed the effect of sublingual nitroglycerin administered before rest imaging in the technetium-99m (99mTc) tetrofosmin exercise and rest same day protocols to determine whether the use of nitroglycerin improved the detection of fill-in the rest single photon emission computed tomography (SPECT) images 3 hours after peak exercise. Twenty-one patients with old myocardial infarction underwent repeated 99mTc-tetrofosmin exercise/rest same day protocols with and without the administration of sublingual nitroglycerin (0.3 mg) 5 min before the second injection of 99mTc-tetrofosmin for rest SPECT imaging. Twelve of these patients also underwent ordinary exercise/redistribution 201T1 SPECT imaging. The control study protocol images showed decreased uptake of 99mTc-tetrofosmin on exercise in 157 of 420 segments and the presence of fill-in at rest in 58 segments. Images obtained with administration of nitroglycerin showed decreased uptake of 99mTc-tetrofosmin on exercise in 163 of 420 segments and fill-in in 74 segments at rest. The frequency of fill-in was greater in the nitroglycerin protocol than in the control protocol (p < 0.05). The segments were scored as different grades according to 99mTc-tetrofosmin uptake between 2 protocols. Fill-in was only present or more remarkable in 31 segments in the nitroglycerin protocol than in the control protocol. Fill-in was only present or more remarkable in 10 segments in the control protocol than in the nitroglycerin protocol. In the nitroglycerin protocol, the mean defect score of the exercise images, calculated from the bull's eye image automatically, was higher than that of the rest images (121 +/- 40 vs 94 +/- 53, p < 0.01). The mean severity score of the exercise images, also calculated from the bull's eye image automatically, was likewise higher than that of the rest images (691 +/- 382 vs 524 +/- 431, p < 0.01), whereas the mean severity score of the stress images and rest images in the control protocol was not significantly different. Moreover, the mean defect score and severity score of the rest images from the nitroglycerin protocol were significantly lower than those obtained from the control protocol (p < 0.05). Sublingual nitroglycerin administration before the injection of 99mTc-tetrofosmin at the rest study in the one day exercise/rest studies enhanced fill-in, so may enhance the detection of viable myocardium, allowing more informed decisions regarding cardiac revascularization in patients with chronic coronary artery disease.

Administration, Sublingual↗
PubMed1998

Cleavage of the C-terminus of NEDD8 by UCH-L3.

NEDD8 is a novel ubiquitin-like protein that has been shown to conjugate to nuclear proteins in a manner analogous to ubiquitination and sentrinization. To identify proteins that are involved in the NEDD8-conjugation and de-conjugation pathway, the yeast two-hybrid system was used to screen a human heart cDNA library using NEDD8 as a bait. Seven strongly positive clones were found to contain a cDNA insert encoding the ubiquitin C-terminal hydrolase, UCH-L3. In vitro GST pull-down assay demonstrated that UCH-L3 bound to both NEDD8 and ubiquitin. In contrast, UCH-L3 did not bind to sentrin-1, sentrin-2, or sentrin-3. Recombinant UCH-L3, but not UCH-L1, was able to cleave the C-terminus of NEDD8. Thus, UCH-L3 can function as a C-terminal hydrolase for both NEDD8 and ubiquitin. UCH-L3 may play a physiologically significant role in the cleavage of the C-terminus of NEDD8, which is required for NEDD8 to conjugate to target proteins.

Down-Regulation↗
PubMed1998

Identification of three major sentrinization sites in PML.

Acute promyelocytic leukemia arises following a reciprocal chromosome translocation t(15;17), which generates PML-retinoic acid receptor alpha fusion proteins (PML-RARalpha). We have shown previously that wild type PML, but not PML-RARalpha, is covalently modified by the sentrin family of ubiquitin-like proteins (Kamitani, T., Nguyen, H. P., Kito, K., Fukuda-Kamitani, T., and Yeh, E. T. H. (1998) J. Biol. Chem. 273, 3117-3120). To understand the mechanisms underlying the differential sentrinization of PML versus PML-RARalpha, extensive mutational analysis was carried out to determine which Lys residues are sentrinized. We show that Lys65 in the RING finger domain, Lys160 in the B1 Box, and Lys490 in the nuclear localization signal contributes three major sentrinization sites. The PML mutant with Lys to Arg substitutions in all three sites is expressed normally, but cannot be sentrinized. Furthermore, the triple substitution mutant is localized predominantly to the nucleoplasm, in contrast to wild type PML, which is localized to the nuclear bodies. Thus, sentrinization of PML, in the context of the RING finger and the B1 box, regulates nuclear body formation. Furthermore, we showed that sentrinization of PML-RARalpha could be restored by overexpression of sentrin, but not by retinoic acid treatment. These studies provide novel insight into the pathobiochemistry of acute promyelocytic leukemia and the sentrinization pathway.

Animals↗
PubMed1998

Characterization of a second member of the sentrin family of ubiquitin-like proteins.

Sentrin is a novel ubiquitin-like protein that can be conjugated to other proteins in a manner analogous to ubiquitination. Two additional cDNA sequences that encode proteins highly homologous to sentrin have been reported to GenBankTM. It is not known whether these sentrin-like proteins could also function as protein modifiers. In this report, a second member of the sentrin family was characterized in detail. Sentrin-2 is a 95-amino acid polypeptide that is 46% identical and 66% homologous to sentrin-1. Northern blot analysis showed that the sentrin-2 message was expressed in all tissues, but was barely detectable in the liver and placenta. The ability of sentrin-2 to conjugate to other proteins was tested by expressing hemagglutinin epitope-tagged sentrin-2 in COS cells. Western blot analysis showed that sentrin-2 could be transferred to other proteins in a pattern similar to that of sentrin-1 conjugation and had similar C-terminal processing. We further showed that both sentrin-1 and sentrin-2 could covalently modify RanGAP1, a Ran GTPase-activating protein critically involved in nuclear transport. Immunocytochemical analysis showed that sentrin-2 derivatives were highly enriched in the nucleus. Taken together, our results demonstrate that sentrin-2 is another protein modifier for the sentrinization pathway.

Amino Acid Sequence↗
PubMed1998

Covalent modification of PML by the sentrin family of ubiquitin-like proteins.

PML, a RING finger protein with tumor suppressor activity, has been implicated in the pathogenesis of acute promyelocytic leukemia that arises following a reciprocal chromosomal translocation that fuses the PML gene with the retinoic acid receptor alpha (RARalpha) gene. Immunocytochemical analysis has demonstrated that PML is co-localized with a novel ubiquitin-like protein in the nuclear bodies, which could be disrupted by the PML-RARalpha fusion protein. The physical nature of this co-localization is unknown. Using a COS cell expression system, we show that PML is covalently modified by all three members of the sentrin family of ubiquitin-like proteins. Covalent modification of PML requires the conserved Gly residue near the C termini of sentrin proteins. Sentrinization of PML is highly specific because neither NEDD8 nor ubiquitin could modify PML. Similar specificity is also observed for the covalent modification of RanGAP1 by the sentrin member of ubiquitin-like proteins. These observations highlight the fine substrate specificity of the sentrinization pathway. In acute promyelocytic leukemia, two forms of PML-RARalpha fusion proteins have been reported. Remarkably, both forms of PML-RARalpha fusion proteins could not be sentrinized. Thus differential sentrinization of PML and PML-RARalpha could play an important role in regulating the biological function of PML and in the pathogenesis of acute promyelocytic leukemia.

Amino Acid Sequence↗
PubMed1998

Mechanisms of non-drowsiness after oral administration of TMK688, a novel antiallergic drug.

The mechanisms of non-drowsiness after oral administration of TMK688 (1- [[5'-(3"-methoxy-4"-ethoxycarbonyloxyphenyl)-2',4'-pentadienoyl ] aminoethyl]-4-diphenylmethoxypiperidine, CAS 110501-66-1) were investigated using mice. TMK688 inhibited the histamine-induced vascular permeability at oral doses of 3.2-10 mg/kg with an ID50 value of 5.4 mg/kg. More than 100 times higher doses were needed to prolong the hexobarbital-induced sleeping. Pyrilamine, a typical antihistamine agent, showed little difference among these doses and antiallergic drugs having antihistamine activity, i.e., terfenadine, azelastine and ketotifen, had effects between TMK688 and pyrilamine. The inhibitory activity of orally administered TMK688 against ex vivo [3H]-pyrilamine binding to mouse cerebral histamine receptors appeared at the same doses as its potentiating activity against hexobarbital-induced sleeping. When given orally, TMK688 was hydrolyzed to TMK777 (CAS 101619-11-8), then conjugated with glucuronic acid to TMK777-glucuronide. No TMK688 was detected in the blood. The main metabolite TMK777-glucuronide could hardly penetrate the blood-brain barrier because of its polarity. Although the plasma concentrations of TMK777 were far lower than those of TMK777-glucuronide, TMK777 was penetrable into the brain and the cerebral concentrations of TMK777 increased in parallel with the plasma concentrations of the drug. Since intracerebroventricularly-injected TMK777 prolonged the sleeping time, and since the threshold concentration of TMK777 in the cerebral cortex to potentiate the hexobarbital-induced sleeping was consistent despite different administration routes, the drowsiness elicited by markedly high doses of TMK688 is though to be caused by intracerebral TMK777. In other words, TMK688 does not seem to cause drowsiness at effective doses because of the poor prenetrability of its main metabolites into the brain.

Administration, Oral↗
PubMed1997

Preferential interaction of sentrin with a ubiquitin-conjugating enzyme, Ubc9.

Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1. We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H. P., and Yeh, E. T. H. (1997) J. Biol. Chem. 272, 14001-14004). Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur. To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait. A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9. The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin. This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF. In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase. A beta-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay. Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate. Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway.

Binding Sites↗
PubMed1997

Characterization of NEDD8, a developmentally down-regulated ubiquitin-like protein.

NEDD8 is a novel 81 amino acid polypeptide which is 60% identical and 80% homologous to ubiquitin. Northern blot analysis showed that the NEDD8 message was developmentally down-regulated. In adult tissues, NEDD8 expression was mostly restricted to the heart and skeletal muscle. Antiserum specific for NEDD8 detected a 6-kDa monomer in SK-N-SH, BJAB, and HL60 cell lysates. A 14-kDa band was also detected in BJAB, HL60, and SK-MEL28 but not in SK-N-SH and K562 cell lysates. An approximately 90-kDa band was detected in all cell lines tested. Thus, NEDD8 is likely to be conjugated to other proteins in a manner analogous to ubiquitination. However, the conjugation pattern of NEDD8 is entirely different from that of ubiquitin in all cell lines tested. To study NEDD8 conjugation in more detail, hemagglutinin-epitope-tagged NEDD8 was expressed in COS cells. Western blot analysis revealed an NEDD8 monomer and a series of higher molecular weight NEDD8-conjugated proteins or NEDD8 multimers. Immunocytochemical analysis showed that NEDD8 expression was highly enriched in the nucleus and was much weaker in the cytosol. In contrast, ubiquitin expression was detectable equally well in the nucleus and cytosol. Mutational analysis showed that the C terminus of NEDD8 was efficiently cleaved and that Gly-76 was required for conjugation of NEDD8 to other proteins. Taken together, NEDD8 provides another substrate for covalent protein modification and may play a unique role during development.

Amino Acid Sequence↗
PubMed1997

Activation-induced aggregation and processing of the human Fas antigen. Detection with cytoplasmic domain-specific antibodies.

Fas (APO1/CD95) is a type 1 transmembrane protein critically involved in receptor-mediated apoptosis. Previous studies have shown that Fas exists in monomeric form in resting cells and aggregates upon cross-linking to form a complex that serves to recruit additional signaling molecules to the cell membrane. To study the molecular fate of the Fas antigen following receptor activation, a monoclonal antibody specific for the cell death domain of Fas has been generated. This monoclonal antibody (3D5) could be used in Western blot analysis using total cell lysates to identify different forms of Fas antigens without immunoprecipitation. High molecular mass (>200 kDa), SDS- and beta-mercaptoethanol-resistant Fas aggregates were formed immediately following receptor cross-linking, and a 97-kDa band (p97) was detected about 2 h later. p97 could be detected by antibodies against either the death domain or the C terminus. However, p97 could not be precipitated by antiextracellular domain antibodies. Thus, p97 most likely represents a processed form of the high molecular weight Fas aggregates. Although p97 generation followed a similar time course as CPP32 activation and poly(ADP-ribose) polymerase cleavage, it could not be inhibited by cysteine protease, calpain, or proteasome inhibitors.

Antibodies↗