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1.
Breast Cancer ; 25(4): 407-415, 2018 Jul.
Article in English | MEDLINE | ID: mdl-29445928

ABSTRACT

BACKGROUND: Dual blockade of HER2 promises increased pathological complete response (pCR) rate compared with single blockade in the presence of chemotherapy for HER2-positive (+) primary breast cancer. Many questions remain regarding optimal duration of treatment and combination impact of endocrine therapy for luminal HER2 disease. METHODS: We designed a randomised phase II, five-arm study to evaluate the efficacy and safety of lapatinib and trastuzumab (6 weeks) followed by lapatinib and trastuzumab plus weekly paclitaxel (12 weeks) with/without prolongation of anti-HER2 therapy prior to chemotherapy (18 vs. 6 weeks), and with/without endocrine therapy in patients with HER2+ and/or oestrogen receptor (ER)+ disease. The primary endpoint was comprehensive pCR (CpCR) rate. Among the secondary endpoints, pCR (yT0-isyN0) rate, safety, and clinical response were evaluated. RESULTS: In total, 215 patients were enrolled; 212 were included in the full analysis set (median age 53.0 years; tumour size = T2, 65%; and tumour spread = N0, 55%). CpCR was achieved in 101 (47.9%) patients and was significantly higher in ER- patients than in ER+ patients (ER- 63.0%, ER+ 36.1%; P = 0.0034). pCR with pN0 was achieved in 42.2% of patients (ER- 57.6%, ER+ 30.3%). No significant difference was observed in pCR rate between prolonged exposure groups and standard groups. Better clinical response outcomes were obtained in the prolongation phase of the anti-HER2 therapy. No surplus was detected in pCR rate by adding endocrine treatment. No major safety concern was recognised by prolonging the anti-HER2 treatment or adding endocrine therapy. CONCLUSIONS: This study confirmed the therapeutic impact of lapatinib, trastuzumab, and paclitaxel therapy for each ER- and ER+ subgroup of HER2+ patients. Development of further strategies and tools is required, particularly for luminal HER2 disease.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Breast Neoplasms/metabolism , Female , Humans , Lapatinib , Middle Aged , Neoadjuvant Therapy , Paclitaxel/administration & dosage , Quinazolines/administration & dosage , Receptor, ErbB-2/metabolism , Trastuzumab/administration & dosage , Treatment Outcome
2.
Breast Cancer Res Treat ; 145(1): 143-53, 2014 May.
Article in English | MEDLINE | ID: mdl-24682674

ABSTRACT

We investigated the disease-free survival (DFS) of HER2-positive primary breast cancer patients treated with neoadjuvant chemotherapy plus trastuzumab, as well as predictive factors for DFS and pathologic response. Data from 829 female patients treated between 2001 and 2010 were collected from 38 institutions in Japan. Predictive factors were evaluated using multivariate analyses. The 3-year DFS rate was 87 % [95 % confidence interval (CI) 85-90]. The pathologic complete response (pCR: ypT0/is + ypN0) rate was 51 %. The pCR rate was higher in the ER/PgR-negative patients than in the ER/PgR-positive patients (64 vs. 36 %, P < 0.001). Patients with pCR showed a higher DFS rate than patients without pCR (93 vs. 82 %, P < 0.001). Multivariate analysis revealed three independent predictors for poorer DFS: advanced nodal stage [hazard ratio (HR) 2.63, 95 % CI 1.36-5.21, P = 0.004 for cN2-3 vs. cN0], histological/nuclear grade 3 (HR 1.81, 95 % CI 1.15-2.91, P = 0.011), and non-pCR (HR 1.98, 95 % CI 1.22-3.24, P = 0.005). In the ER/PgR-negative dataset, non-pCR (HR 2.63, 95 % CI 1.43-4.90, P = 0.002) and clinical tumor stage (HR 2.20, 95 % CI 1.16-4.20, P = 0.017 for cT3-4 vs. cT1-2) were independent predictors for DFS, and in the ER/PgR-positive dataset, histological grade of 3 (HR 3.09, 95 % CI 1.48-6.62, P = 0.003), clinical nodal stage (HR 4.26, 95 % CI 1.53-13.14, P = 0.005 for cN2-3 vs. cN0), and young age (HR 2.40, 95 % CI 1.12-4.94, P = 0.026 for ≤40 vs. >40) were negative predictors for DFS. Strict pCR (ypT0 + ypN0) was an independent predictor for DFS in both the ER/PgR-negative and -positive datasets (HR 2.66, 95 % CI 1.31-5.97, P = 0.006 and HR 3.86, 95 % CI 1.13-24.21, P = 0.029, respectively). These results may help assure a more accurate prognosis and personalized treatment for HER2-positive breast cancer patients.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/mortality , Neoadjuvant Therapy , Receptor, ErbB-2/metabolism , Antibodies, Monoclonal, Humanized/therapeutic use , Disease-Free Survival , Female , Humans , Prognosis , Retrospective Studies , Trastuzumab
3.
Med Oncol ; 20(2): 117-26, 2003.
Article in English | MEDLINE | ID: mdl-12835514

ABSTRACT

There are still many questions to be elucidated concerning the relationship between telomerase activity and various factors associated with cancer. Whether or not the level of telomerase activity could be a prognostic factor in breast cancer was investigated through 5-yr follow-up observation. Telomerase activity was quantified by the fluorescence-based telomeric repeat amplification protocol assay in 54 patients with breast cancer and its relationship with patient prognosis was examined. Telomerase activity was detected in 92.6% of breast cancer patients, with a median of 65.4 TPG (total product generated) (Min 0-Max 446.2). The follow-up observation for 5 yr demonstrated that among background factors examined, recurrence was the only factor that showed a significant association with the level of telomerase activity when a cutoff at 100 TPG was adopted. This suggested the possibility of 100 TPG telomerase activity being a prognostic factor for recurrence. Prospective studies will be necessary to clarify this matter.


Subject(s)
Breast Neoplasms/enzymology , Telomerase/metabolism , Aged , Antineoplastic Combined Chemotherapy Protocols , Breast Neoplasms/therapy , Chemotherapy, Adjuvant , Female , Humans , Middle Aged , Prognosis , Regression Analysis , Survival Analysis
4.
Breast Cancer ; 8(3): 202-5, 2001.
Article in English | MEDLINE | ID: mdl-11668241

ABSTRACT

BACKGROUND: The tendency for breast cancer to form multiple lesions is important to consider when planning breast-conserving surgery. However, many unknowns remain regarding the pathology and prognosis of multiple breast cancer, and therefore it is clinically significant to investigate its clinicopathological properties. METHODS: Over the past 25 years, in the period between April 1972 and March 1997, we investigated the clinicopathological findings including the 5-year and 10-year survival rates of 66 patients treated for unilateral multiple breast cancer. RESULTS: Of the total of 1,334 female patients with unilateral breast cancer who underwent curative surgery at our hospital, we identified 66 (5.0%) patients with unilateral multiple cancer. The incidence of such cancer has been higher in recent years. Of the 66 patients, 50 (75.8%) were premenopausal, and the remaining patients were postmenopausal, but multiple cancer among postmenopausal women is a recent phenomenon. The ER positivity rate of the main lesion in patients with multiple breast cancer was 69.2% and that of PgR was 50.0%. The 5-and 10-year overall survival rate in all 66 patients with multiple breast cancer was 90.8% and 79.7%, respectively. CONCLUSION: In the past, multiple breast cancer was frequently identified in premenopausal women. However, the current findings indicate that its incidence among postmenopausal women has increased in recent years. In addition, prognoses were comparable for patients with multiple or solitary breast cancer, a relevant finding in the planning of breast-conserving surgery.


Subject(s)
Breast Neoplasms/epidemiology , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/epidemiology , Carcinoma, Ductal, Breast/pathology , Adult , Age Distribution , Age Factors , Aged , Aged, 80 and over , Breast Neoplasms/mortality , Carcinoma, Ductal, Breast/mortality , Female , Humans , Incidence , Japan/epidemiology , Menopause , Middle Aged , Neoplasms, Hormone-Dependent/epidemiology , Survival Analysis
5.
J Biol Chem ; 276(52): 49459-65, 2001 Dec 28.
Article in English | MEDLINE | ID: mdl-11591721

ABSTRACT

When co-expressed with a receptor activity-modifying protein (RAMP) accessory protein, calcitonin receptor-like receptor (CRLR) can function as a calcitonin gene-related peptide receptor (CRLR-RAMP1) or an adrenomedullin (AM) receptor (CRLR-RAMP2/3). Here we report on the structural domain(s) involved in selective AM binding that were examined using various RAMP chimeras and deletion mutants. Co-expression of chimeric RAMPs and CRLR in HEK293 cells revealed that residues 77-101, situated in the extracellular N-terminal domain of human RAMP2 (hRAMP2), were crucial for selective AM-evoked cAMP production. More detailed analysis showed that deletion of hRAMP2 residues 86-92 significantly attenuated high-affinity (125)I-AM binding and AM-evoked cAMP production despite full cell surface expression of the receptor heterodimer and that deletion of hRAMP3 residues 59-65 had a similar effect. There is little sequence identity between hRAMP3 residues 59-65 and hRAMP2 residues 86-92; moreover, substituting alanine for Trp(86) (Ala(87)), Met(88), Ile(89), Ser(90), Arg(91), or Pro(92) of hRAMP2 had no effect on AM-evoked cAMP production. It thus seems unlikely that any one amino acid residue is responsible for determining selective AM binding or that AM binds directly to these peptide segments. Instead these findings suggest that the respective seven-amino acid sequences confer selectivity either by directly contributing to the structure of ligand binding pocket or by allosteric modulation of the conformation of CRLR.


Subject(s)
Membrane Proteins/metabolism , Receptors, Calcitonin Gene-Related Peptide/metabolism , Receptors, Peptide/metabolism , Adrenomedullin , Amino Acid Sequence , Animals , Cell Line , Cell Separation , Cyclic AMP/metabolism , Flow Cytometry , Humans , Intracellular Signaling Peptides and Proteins , Membrane Proteins/chemistry , Membrane Proteins/genetics , Molecular Sequence Data , Mutation , Peptides/metabolism , Protein Binding , Protein Structure, Tertiary , Radioligand Assay , Receptor Activity-Modifying Protein 1 , Receptor Activity-Modifying Protein 2 , Receptor Activity-Modifying Protein 3 , Receptor Activity-Modifying Proteins , Receptors, Adrenomedullin , Receptors, Calcitonin Gene-Related Peptide/genetics , Recombinant Fusion Proteins/metabolism
6.
Gan To Kagaku Ryoho ; 27(11): 1703-8, 2000 Oct.
Article in Japanese | MEDLINE | ID: mdl-11057321

ABSTRACT

A phase II trial has demonstrated that paclitaxel (210 mg/m2/3 hr) showed a 33.3% response rate among anthracycline-resistant breast cancer patients in Japan. Recently, weekly dosing of paclitaxel has been demonstrated to be a well-tolerated, feasible and effective administration schedule. Standard premedication is commonly administered prior to treatment with paclitaxel. However, this regimen requires dexamethasone administration beginning at 12 to 14 hours prior to paclitaxel, which would not be convenient for outpatients. In this study, paclitaxel was administered by 1 hour intravenous infusion at a dose of 80 mg/m2 every week. Administration was continued for 3 weeks with a 1 week rest. A short course premedication schedule consisted of dexamethasone 20 mg i.v. (diluted in 50 ml normal saline, 1 hour prior to paclitaxel), and oral diphenhydramine 50 mg, H2-antagonist and anti-emetic agent i.v. (diluted in 50 ml normal saline, 30 minutes prior to paclitaxel). A total of 14 outpatients were enrolled in the study. There were 10 partial responders and no complete responders, and the overall response rate was 71.4%. No hypersensitivity reactions were observed, and grade 3/4 leukopenia occurred in 43% (6/14). Allopecia was observed in 4 patients, and peripheral neuropathy in 1 patient (both grade 1). Weekly administration of paclitaxel is effective and well-tolerated in patients with advanced or metastatic breast cancer, with a minimum of peripheral neuropathy. In addition to the above, no hypersensitive reaction in the short course premedication schedule suggests that this administration schedule is feasible for outpatients.


Subject(s)
Breast Neoplasms/drug therapy , Neoplasm Recurrence, Local/drug therapy , Paclitaxel/administration & dosage , Adult , Aged , Ambulatory Care , Antineoplastic Agents, Hormonal/administration & dosage , Antineoplastic Agents, Phytogenic/administration & dosage , Dexamethasone/administration & dosage , Drug Administration Schedule , Feasibility Studies , Female , Humans , Infusions, Intravenous , Middle Aged
7.
Int J Oncol ; 17(6): 1125-9, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11078797

ABSTRACT

Loss of heterozygosity (LOH) on chromosome 6q is often observed in breast cancer, suggesting the existence of a putative tumor suppressor. Recently, a human homolog of the Drosophila warts tumor suppressor gene, h-warts/LATS1, was identified and mapped at chromosome 6q24-25.1. Mutation analysis of the h-warts/LATS1 was performed using 25 breast cancer tissues by RT-PCR SSCP analysis. Although LOH of the h-warts/LATS1 was found in one patient, no mutations were found. Two polymorphisms were found, but neither of them caused amino acid substitutions. Further investigations are necessary to elucidate the role of the h-warts/LATS1 gene in the carcinogenesis of breast cancer.


Subject(s)
Breast Neoplasms/genetics , Chromosomes, Human, Pair 6/genetics , Drosophila Proteins , Genes, Tumor Suppressor , Neoplasm Proteins/genetics , Protein Kinases , Protein Serine-Threonine Kinases/genetics , Breast Neoplasms/chemistry , Breast Neoplasms/ultrastructure , DNA Mutational Analysis , DNA, Complementary/genetics , DNA, Neoplasm/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Loss of Heterozygosity , Neoplasm Proteins/analysis , Polymorphism, Genetic , Polymorphism, Single-Stranded Conformational , Protein Serine-Threonine Kinases/analysis , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Reverse Transcriptase Polymerase Chain Reaction , Subcellular Fractions/chemistry
8.
Masui ; 49(2): 191-4, 2000 Feb.
Article in Japanese | MEDLINE | ID: mdl-10707527

ABSTRACT

A 49-year-old male with amyotrophic lateral sclerosis (ALS) was scheduled for gastrectomy. Anesthetic management was performed under general anesthesia with sevoflurane and epidural anesthesia with lidocaine. He showed increased response to vecuronium under monitoring of neuromuscular block. But he responded favorably to anticholineesterase. He had little pain and showed no progress in neurological symptoms in the postoperative period. Neuromuscular monitoring is essential in administrating non-depolarizing neuromuscular blocking agents to patients with ALS, and epidural anesthesia may be useful for perioperative management of patients with ALS.


Subject(s)
Amyotrophic Lateral Sclerosis , Anesthesia, Epidural , Anesthesia, General , Amyotrophic Lateral Sclerosis/complications , Gastrectomy , Humans , Male , Middle Aged , Monitoring, Intraoperative , Perioperative Care , Stomach Neoplasms/complications , Stomach Neoplasms/surgery
10.
Arch Med Res ; 30(6): 514-21, 1999.
Article in English | MEDLINE | ID: mdl-10714366

ABSTRACT

Hyperfunctioning thyroid adenomas are benign tumors characterized by their autonomous growth and functional activity, which frequently cause clinical hyperthyroidism and show a predominant radioactive iodine uptake in the nodule. Activating mutations in the gene encoding the alpha subunit of the stimulatory G protein (Gs alpha), as well as activating mutations in the gene encoding thyrotropin receptor in hyperfunctioning thyroid adenomas, have been reported. The mutations in Gs alpha involved the replacement of either arginine 201 with cysteine or histidine, or glutamine 227 with arginine or leucine. These residues are involved in GDP/GTP binding of Gs alpha and these mutations inhibit intrinsic GTPase activity that results in constitutive activation of adenylyl cyclase. The pathophysiological roles of these mutations in the formation of hyperfunctioning thyroid adenoma have been suggested.


Subject(s)
Adenoma/genetics , Adenoma/physiopathology , GTP-Binding Protein alpha Subunits, Gs/genetics , Mutation , Oncogene Proteins/genetics , Thyroid Neoplasms/genetics , Thyroid Neoplasms/physiopathology , Animals , Humans
12.
Mol Cell Endocrinol ; 138(1-2): 137-42, 1998 Mar 16.
Article in English | MEDLINE | ID: mdl-9685222

ABSTRACT

We analyzed cultured cells from hyperfunctioning thyroid adenoma and its surrounding thyroid tissue from a Japanese woman and determined the nucleotide sequences of genes encoding the alpha subunit of the stimulatory G-protein 1 (G alphas) and thyrotropin (TSH) receptor in its tumor tissue. Primary culture of cells from hyperfunctioning thyroid adenoma and its surrounding thyroid tissue revealed that cAMP production was constitutively activated while intracellular Ca2+ concentration was suppressed both at the basal level and in the response to TSH stimulation in the cells from tumor tissue compared with those from non-tumor tissue. Nucleotide sequence analysis demonstrated the somatic missense mutation at codon 201 (CGT(Arg)-CAT(His)) of G alphas gene in tumor tissue but not in its surrounding tissue. No mutation was observed in the transmembrane region of TSH receptor. These results suggest that cAMP regulatory cascade is constitutively activated while phospholipase C-Ca2+ signaling cascade is suppressed in hyperfunctioning thyroid adenoma with an activating mutation of G alphas gene in the present case.


Subject(s)
Adenoma/genetics , Adenoma/pathology , GTP-Binding Protein alpha Subunits, Gs/genetics , Point Mutation , Receptors, Thyrotropin/genetics , Thyroid Gland/metabolism , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathology , Adenoma/metabolism , Adenoma/surgery , Amino Acid Sequence , Arginine , Base Sequence , Calcium/metabolism , Cell Culture Techniques/methods , Cyclic AMP/metabolism , Female , GTP-Binding Protein alpha Subunits, Gs/biosynthesis , Histidine , Humans , Kinetics , Middle Aged , Polymerase Chain Reaction , Receptors, Thyrotropin/biosynthesis , Thyroid Gland/pathology , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/surgery , Thyrotropin/pharmacology , Tumor Cells, Cultured
13.
Oncol Rep ; 5(2): 367-71, 1998.
Article in English | MEDLINE | ID: mdl-9468559

ABSTRACT

The transforming growth factor-beta (TGFbeta) binds the type II TGFbeta growth factor receptor (TGFbetaRII) to inhibit the growth of most epithelial tissues. Most human colon and gastric cancers with microsatellite instability (MI) have frameshift mutations in polynucleotide repeats within the TGFbetaRII coding region; these mutations truncate the receptor protein and disable the serine/threonine kinase to produce TGF-beta resistance. To further investigate the type, frequency and tissue distribution of TGFbetaRII gene mutations, in this study, we examined 36 sporadic breast cancers. We previously produced eight intron based primer pairs for mutational analysis of the entire coding region of the TGFbetaRII gene. Using these primers, we developed protocols for polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of PCR products from genomic DNA samples of 36 breast cancer patients and we tested them for microsatellite instability (MI) at eight microsatellite loci. One case demonstrated MI (2.8%) and we found no mutations. These and other recent data indicate that TGFbetaRII mutations are essentially confined to colon and gastric cancers with MI. The narrow spectrum of tissues containing RII mutations illustrates the complexity of genetic checkpoints in human carcinogenesis.


Subject(s)
Breast Neoplasms/genetics , Carcinoma/genetics , Mutation , Receptors, Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/genetics , Base Sequence , Breast Neoplasms/pathology , Carcinoma/pathology , DNA Mutational Analysis , DNA Primers/chemistry , DNA, Neoplasm/analysis , DNA, Satellite/genetics , Female , Humans , Microsatellite Repeats , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Protein Serine-Threonine Kinases , Receptor, Transforming Growth Factor-beta Type II
14.
Surg Endosc ; 11(9): 961-2, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9294283

ABSTRACT

The present paper introduces a new fine probe for electric cautery (1.65 mm in diameter, 22 cm long) that can be connected to a conventional cylindrical hand-controlled cautery holder, which is monopolar and widely used in general surgery. When cautery was required, a 14-gauge intravenous catheter was inserted at an appropriate site under the guidance of a videoscope. After removing the steel inner needle, the extra tube was used as the fine surgical port for the cautery probe. The position of insertion could be altered according to the operating field. Cautery was performed by conventional methods. There was no bleeding or air leakage at the site of puncture during or after surgery. The puncture wound was closed without any sutures. Based on these results, the new fine probe for cautery can reduce the number of surgical ports required for instruments during video-assisted surgery, thus improving the ease and safety of endoscopic surgery.


Subject(s)
Electrocoagulation/instrumentation , Endoscopy/methods , Electrocoagulation/methods , Endoscopes , Equipment Design , Humans
15.
Mol Cell Endocrinol ; 118(1-2): 47-56, 1996 Apr 19.
Article in English | MEDLINE | ID: mdl-8735590

ABSTRACT

Digital video imaging indicated that about 80% of fura-2-loaded single human thyroid cells responded to TSH, resulting in an increase in intracellular Ca2+ concentration ([Ca2+]i). Most of the TSH-sensitive cells further responded to N6-(L-2-phenylisopropyl)-adenosine (PIA) showing a transient [Ca2+]i rise in a PIA dose-dependent manner. Addition of PIA prior to TSH administration had no effect or showed only a slight [Ca2+]i increase, but in about 80% of the cells, regardless of the response to PIA, the addition of TSH after PIA resulted in a higher transient [Ca2+]i response than that in the absence of PIA. Inactivation of Gi/G(o) by pertussis toxin (PTX) treatment markedly reduced the effect of PIA on TSH action to the level induced by PIA alone. Immunoglobulin fractions obtained from two Graves' patients with high TSAb (antibody activity measured by cAMP response) activity induced [Ca2+]i increase and cooperated with PIA. Under the same conditions, TSH-dependent cAMP accumulation was inhibited by PIA. These results suggest that adenosine Ai receptor is expressed in human thyroid cells in primary culture as well as in FRTL-5 rat thyroid cells, and that in the presence of adenosine. TSH or Graves' IgG signal tends to be directed to the Ca2+ pathway in the human thyroid.


Subject(s)
Calcium/metabolism , Graves Disease/immunology , Immunoglobulin G/pharmacology , Phenylisopropyladenosine/pharmacology , Thyroid Gland/drug effects , Thyrotropin/pharmacology , Cells, Cultured , Cyclic AMP/metabolism , Humans , Thyroid Gland/cytology , Thyroid Gland/metabolism
16.
Immunol Lett ; 31(2): 137-42, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1531474

ABSTRACT

T cells obtained from normal mouse spleen cells showed significant cytotoxic activity against Fc receptor positive tumor cells in the presence of anti-CD3 monoclonal antibody (mAb). This activity was designated as natural cytotoxic T cell (NCT) activity and compared with natural killer (NK) activity. Considerable levels of NCT activity were detected in mouse strains with both high and low NK activity. NCT cells were distributed in both lower and higher density fractions of Percoll discontinuous density gradients, while NK cells were enriched in the lower density fraction of Percoll gradients. Moreover, NCT activity was resistant to in vivo anti-asialo GM1 treatment, in contrast to NK cells. These results indicate that NCT cells, which have different characteristics from NK cells, are present in normal, nonimmunized mouse spleen cells. Unexpectedly, CD4+ T cells sorted from normal mouse spleen T cells revealed significant NCT activity, as did CD8+ T cells. It was also demonstrated that NCT cells require the LFA-1 molecule to lyse tumor cells in the presence of anti-CD3 mAb.


Subject(s)
Immunity, Innate , T-Lymphocytes, Cytotoxic/immunology , Animals , Antibodies, Monoclonal , Antigens, Differentiation, T-Lymphocyte , CD3 Complex , Cytotoxicity, Immunologic , In Vitro Techniques , Killer Cells, Natural/immunology , Lymphocyte Function-Associated Antigen-1/immunology , Male , Mice , Mice, Inbred Strains , Receptors, Antigen, T-Cell , T-Lymphocyte Subsets/immunology
17.
Biochim Biophys Acta ; 977(3): 329-34, 1989 Dec 07.
Article in English | MEDLINE | ID: mdl-2556182

ABSTRACT

A small-sized c-type cytochrome, designated cytochrome c-551, was prepared from membrane fraction of the thermophilic bacterium PS3 grown under air-limited conditions by extraction with cholate, precipitation with polyethylene glycol, and successive chromatographies with DEAE-cellulose and Sephacryl S-200 in the presence of a detergent. The purified sample contained approximately 1 mol of heme c per 10,000 g protein; it showed absorption bands at 551, 522 and 416 nm upon reduction, and a Soret peak at 409 nm upon oxidation. This cytochrome showed a single band of 10 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulfate. The isoelectric point of this cytochrome c-551 was pH 4.0. Cytochrome c-551 was suggested to play an important role in the respiratory chain with a terminal oxidase cytochrome o, which is produced under air-limited conditions, since cytochrome c-551 could mediate electron transfer between cytochrome bc1(b6f) complex and cytochrome o, showing quinol oxidase activity.


Subject(s)
Bacterial Proteins , Cytochrome b Group , Cytochrome c Group/analysis , Escherichia coli Proteins , Gram-Positive Bacteria/analysis , Cytochrome c Group/isolation & purification , Cytochrome c Group/metabolism , Cytochromes/metabolism , Electron Transport , Electrophoresis, Polyacrylamide Gel , Gram-Positive Bacteria/metabolism , Hot Temperature , Isoelectric Point , Kinetics , Molecular Weight , Oxidation-Reduction , Spectrum Analysis
18.
Proc Natl Acad Sci U S A ; 84(4): 925-9, 1987 Feb.
Article in English | MEDLINE | ID: mdl-3547402

ABSTRACT

The purification and properties of a protein serine kinase (PK-P) extracted with Triton X-100 from membranes of bakers' yeast are described. The enzyme is virtually inactive unless either a histone or a heat-stable polypeptide from yeast membranes and Mg2+ are added. Other divalent cations substitute for Mg2+ poorly or not at all; most of them, including Mn2+, inhibit when added in the presence of 5 mM Mg2+. The enzyme is unstable but can be stabilized by addition of 0.1% Triton X-100 and 20% glycerol. The final preparation shows, on silver-stained electrophoresis gels, two major bands (Mr 41,000 and 35,000). According to gel filtration the molecular weight of the active protein is about 75,000. Of the two subunits, only the smaller one appears to be autophosphorylated. In addition to casein, the enzyme phosphorylates several proteins including the H+-ATPase (Mr 100,000) in the yeast plasma membrane. In order to demonstrate the phosphorylation of the ATPase (up to 0.9 equivalents), exposure of the latter to an acid phosphatase was required. Other phosphorylated proteins include mRNA cap-binding protein from mammalian erythrocytes and yeast, a glucocorticoid receptor protein, and a preparation of the guanine nucleotide-binding proteins Gi and Go from brain. A partial purification of a natural activator from yeast plasma membranes is described.


Subject(s)
Peptides/physiology , Protein Kinases/isolation & purification , Protein Serine-Threonine Kinases , Saccharomyces cerevisiae/enzymology , Chemical Phenomena , Chemistry
19.
J Biol Chem ; 259(3): 1405-8, 1984 Feb 10.
Article in English | MEDLINE | ID: mdl-6319397

ABSTRACT

The stoichiometry of vectorial H+ ejection, coupled to ferrocytochrome c oxidation by a three-subunit bacterial cytochrome c oxidase (EC 1.9.3.1) from the thermophilic bacterium PS3, was measured. Three methods of measuring the H+/e- ratio were applied to proteoliposomes containing a relatively small amount of PS3 cytochrome oxidase, which showed a relatively low oxidation rate and a very low H+ leakage, as follows: (a) simultaneous measurements of H+ ejection and cytochrome c oxidation upon addition of a yeast ferrocytochrome c pulse, which enable us to calculate the H+/e- ratio as H+ ejected per cytochrome c oxidized; (b) computer simulations to find out the fit for the pH meter trace by changing the H+/e- ratio and the velocity constant of leakage; and (c) two successive measurements of initial rates of H+ movement in the absence and presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The H+/e- ratios obtained were 1.39, the 10-s value after ferrocytochrome c addition in (a), 1.35 in (b), and 1.33 in (c). This high H+/e- stoichiometry observed, exceeding 1 and as high as 1.4, is discussed with respect to the controversy of the H+/e- ratio at the cytochrome oxidase site.


Subject(s)
Bacteria/enzymology , Electron Transport Complex IV/metabolism , Liposomes , Proteolipids/metabolism , Hot Temperature , Hydrogen-Ion Concentration , Kinetics
20.
Biochem Biophys Res Commun ; 113(2): 575-80, 1983 Jun 15.
Article in English | MEDLINE | ID: mdl-6307293

ABSTRACT

A proton-pumping heme aa3-type cytochrome oxidase purified from the thermophilic bacterium PS3 was treated with trypsin, thermolysin, chymotrypsin, subtilisin, or pronase. The cleavage of the oxidase subunits and the effects of their cleavage on the oxidase activity and proton-pumping in reconstituted vesicles were studied. Trypsin and thermolysin cleaved some of the oxidase subunits without affecting the proton-pumping, but subtilisin and pronase cleaved all the subunits resulting in partial decrease in both activities. Chymotrypsin had an intermediate effect. Subunit II of this enzyme contains heme c which is also cleaved by proteases.


Subject(s)
Bacteria/enzymology , Electron Transport Complex IV/isolation & purification , Oxidoreductases/isolation & purification , Bacterial Proteins/isolation & purification , Catalysis , Chemical Phenomena , Chemistry , Chymotrypsin , Oxidoreductases/physiology , Peptide Hydrolases , Protons , Thermolysin , Trypsin
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