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Toshiki Tanaka

Publications and source records attributed to Toshiki Tanaka.

30 records · Page 2Linked to original sources
PubMed2004

Two-metal ion, Ni(II) and Cu(II), binding alpha-helical coiled coil peptide.

Metalloproteins are an attractive target for de novo design. Usually, natural proteins incorporate two or more (hetero- or homo-) metal ions into their frameworks to perform their functions, but the design of multiple metal-binding sites is usually difficult to achieve. Here, we undertook the de novo engineering of heterometal-binding sites, Ni(II) and Cu(II), into a designed coiled coil structure based on an isoleucine zipper (IZ) peptide. Previously, we described two peptides, IZ-3adH and IZ-3aH. The former has two His residues and forms a triple-stranded coiled coil after binding Ni(II), Zn(II), or Cu(II). The latter has one His residue, which allowed binding with Cu(II) and Zn(II), but not with Ni(II). On the basis of these properties, we newly designed IZ(5)-2a3adH as a heterometal-binding peptide. This peptide can bind Cu(II) and Ni(II) simultaneously in the hydrophobic core of the triple-stranded coiled coil. The first metal ion binding induced the folding of the peptide into the triple-stranded coiled coil, thereby promoting the second metal ion binding. This is the first example of a peptide that can bind two different metal ions. This construction should provide valuable insights for the de novo design of metalloproteins.

Amino Acid Sequence↗
PubMed2004

Increased BBB permeability by parasympathetic sphenopalatine ganglion stimulation in dogs.

The blood-brain barrier (BBB) is a major obstacle for movement of large molecules to and from the brain. Stimulation of the sphenopalatine ganglion (SPG), the major source of parasympathetic innervation to brain vasculature, is known to vasodilate brain vessels, and has recently been shown to also increase the permeability of the BBB in the rat. In this work, we studied the effect of SPG stimulation on BBB permeability in larger animals--Beagle dogs. Left SPG was exposed by lateral approach in five Beagle dogs, and stimulated at 10 Hz. FITC labeled 10 kDa dextran was continuously infused to the left atrium during stimulation, and cerebral angiography was periodically obtained via the vertebral artery. Three control dogs received labeled dextran, without SPG exposure or stimulation. Brains were perfused with saline thoroughly at the end of stimulation, and samples from various regions were taken for fluorescence reading of tissue homogenates. Cerebral vasodilatation was evidenced in all but one dog, whose fluorescence results were consequently excluded from analysis, assuming that its SPG had been damaged by surgery. Fluorescence was significantly higher in the four stimulated compared to the three non-stimulated animals; e.g. mean FITC-dextran concentration in the anterior brain regions was 0.98+/-0.12 ug (mean+/-S.D.) FITC/g brain for experimental animals, and 0.40+/-0.02 for controls (p<0.01). No effect was seen in the pons and cerebellum (0.68+/-0.22 vs. 0.60+/-0.03, NS) whose vascular innervation is supplied by the otic rather than the SPG ganglion. SPG stimulation appears to be an effective way to increase BBB permeability, allowing introduction of large molecules to the brain. This could be a therapeutic method for a wide variety of brain disorders, including tumors and neurodegenerative diseases.

Animals↗
PubMed2004

Selective formation of AAB- and ABC-type heterotrimeric alpha-helical coiled coils.

The alpha-helical coiled coils have a representative amino acid sequence of (abcdefg)(n) heptad repeats. We previously reported that two peptides named IZ-2A and IZ-2W formed an (IZ-2A)(2)/IZ-2W heterotrimer with an Ala-Ala-Trp interaction in the hydrophobic core. In this paper, we describe the selective formation of AAB- and ABC-type heterotrimers. To increase the selectivity of the AAB-type heterotrimeric formation, Lys residues at the f position were mutated to either an Ala or a Gln residue to form IZ-2A(fA) or IZ-2W(fQ). Separately, both IZ-2A(fA) and IZ-2W(fQ) have a random structure at pH 7 and 20 degrees C. However, together IZ-2A(fA) and IZ-2W(fQ) form a 2:1 complex with a thermal transition midpoint (Tm) of 48 degrees C. This procedure was applied to prepare the ABC-type heterotrimer, in which two sets of Ala-Ala-Trp interactions were designed in the hydrophobic core. Interhelical interaction between the e and g positions and the alpha-helical propensity of the amino acid at the f position were also considered in the design. The resultant three peptides selectively formed the ABC-type heterotrimer with a Tm of 51 degrees C. Other peptide combinations had random coil properties.

Amino Acid Sequence↗
PubMed2004

Single chain antibodies that recognize the N-glycosylation site.

We aimed to identify antibodies that can recognize the Asn-Xaa-Ser/Thr(NXS/T) N-glycosylation site that guides oligosaccharyltransferase (OT) activity. We used synthetic Asn-Cys-Ser/Thr(NCS/T) tripeptides conjugated to bovine serum albumin to isolate single chain antibody fragments of a variable region (scFv) from the Griffin 1 phage antibody library. Although Ser and Thr have different side chains, the scFv proteins thus isolated bound to both NCS and NCT with Kd values of the order of 10(-6) M and accepted the substitution of the Cys residue with various amino acids, including Ala, Gly, and Val. However, these proteins recognized neither Asn-Pro-Ser/Thr nor non-NXS/T tripeptides. The scFv proteins recognized NCS/T and N-glycosylation site of mutant yeast protein disulfide isomerase when they were in their native but not denatured state. These results indicate that antibody recognition of the NXS/T motif is conformation dependent and suggest that NXS/T spontaneously adopts a specific conformation that is necessary for antibody recognition. These features are likely to correlate with the known binding specificity of OT.

Amino Acid Sequence↗
PubMed2004

Radioisotope lymph node mapping in nonsmall cell lung cancer: can it be applicable for sentinel node biopsy?

BACKGROUND: Previous studies on intrathoracic lymph node mapping have focused on the validity of a sentinel node concept, but not on the usefulness for sentinel node biopsy. METHODS: The subjects were 15 patients clinically diagnosed with N0 nonsmall cell lung cancer. Technetium-99m tin colloid was injected into the peritumoral area 1 day preoperatively and a time course of tracer migration was monitored by scintigraphy. A hand-held gamma probe counter was used to count the intrathoracic lymph node stations. Resected nodes were also counted to assess the accuracy of the intrathoracic counting. RESULTS: Serial scintigraphies showed that the tracer migrated through airways and the appearance resembled hot nodes. On intrathoracic counting, 50% of the nodal stations appeared positive; however, only 23% of these apparently positive nodal stations were ultimately shown to be truly radioactive. The true positive and true negative rates of detecting intrathoracic hot nodes were 100% and 56%, respectively. Because the counts of the nodal stations could include the counts from the hot primary tumor ("shine-through") or airway radioactivity, legitimate hot nodes were identified after dissecting all the apparently positive nodal stations. Two of the 9 patients in whom hot nodes were identified had nodal metastatic disease and actually had tumor cells within the hot nodes. The only complication related to the preoperative injection of technetium-99m was a minor pneumothorax. CONCLUSIONS: Although radioisotope intrathoracic lymph node mapping is safe, it appears to be unsuitable for sentinel node biopsy because shine-through and the airway-migrated radioactive tracer complicated the intrathoracic counting. Only serial scintigraphy could distinguish hot nodes from airway migration.

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PubMed2003

Independent predictive value of the overall number of metastatic N1 and N2 stations in lung cancer.

OBJECTIVE: The number of metastatic N2 stations is a known prognostic factor in patients with non-small-cell lung cancer (NSCLC). However, involvement of N1 stations as well as that of N2 stations seems to be important in the prognosis of these patients. We therefore attempt to clarify the significance of the total number of metastatic stations in pathologic N1 and N2 NSCLCs. METHODS: Patients with either pathologic N1 (n = 51) or N2 (n = 96) NSCLC who had undergone major pulmonary resection with complete mediastinal dissection were included in this retrospective study. All positive nodes were characterized by location according to the TNM classification system. The hilar station was included with the N2 stations. RESULTS: The total number of metastatic stations in patients with N2 disease ranged from 1 to 8 (average 2.5), whereas that in patients with N1 disease ranged from 1 to 3 (average 1.2). The incidence of multiple-station metastasis (> or = 3 metastatic stations) among N2 patients (35%) was significantly higher than that among N1 patients (2%) (p < 0.001). Multivariate analysis of survival showed pathologic N1 status (relative risk = 0.443, p = 0.013) and < or = 2 metastatic stations (relative risk = 0.515, p = 0.020) to be significant and independent prognostic factors. Age, sex, cell type, resected lobe, and pathological T status were statistically insignificant determinates of survival. CONCLUSIONS: The total number of metastatic stations (< or = 2 vs > or = 3) is an independent prognostic indicator in patients with completely resected pathologic N1 or N2 NSCLC. The number of metastatic stations will be useful as a stratification factor in prospective clinical trials of these patients.

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PubMed2003

Obstacles for shortening hospitalization after video-assisted pulmonary resection for lung cancer.

BACKGROUND: Video-assisted thoracic surgery for lung cancer facilitates early postoperative recovery when patients are treated by critical pathway management. Thus, we developed an original programmed regimen for postoperative management, evaluated the validity of this regimen, and analyzed clinical factors influencing postoperative recovery. METHODS: Forty consecutive patients with suspicious lung cancer undergoing anatomic pulmonary resection with video-assisted thoracic surgery were enrolled in this prospective study. After surgery, all patients who underwent anatomic resection were managed using our programmed regimen; a patient was considered recovered when the regimen had been completed. RESULTS: On final pathologic examination, 37 cases were determined to have lung cancer and underwent anatomic resection. The mean number of resected segments was 3.6. There were no complications caused by postoperative management. The mean day of postoperative recovery was 3.7 days and median, 3 days. Significant preoperative factors related to recovery were age, breathlessness, performance status, radiologic emphysema, partial pressure of arterial oxygen, and predictive postoperative forced expiratory volume in 1 second. The overall number of these risk factors was specifically related to postoperative recovery (p < 0.01): the rate of recovery on postoperative day 3 was 100% in patients with no risk, 68% in those with one to three risks, and 22% in those with four to six risks. CONCLUSIONS: Our original regimen is useful as a critical pathway for the management of lung cancer patients undergoing video-assisted thoracic surgery. Furthermore, we created specific criteria to identify risk factors related to postoperative recovery that may be useful in planning hospitalization for patients undergoing video-assisted thoracic surgery.

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PubMed2003

Protection by hypothermia of hypoxia-induced inhibition of neurogenic vasodilation in porcine cerebral arteries.

Porcine cerebral arterial strips denuded of the endothelium responded to transmural electrical stimulation (5 Hz for 40 s) with a relaxation, which was abolished by tetrodotoxin and N (G)-nitro-L-arginine, a NO synthase inhibitor. Lowering the temperature of the bathing media from 37 degrees C to 33 degrees C or 25 degrees C potentiated the response to nerve stimulation, but did not affect relaxations induced by NO applied exogenously. Hypoxia suppressed the stimulation-induced relaxation at 37 degrees C, but hypothermia blunted the inhibitory effect of hypoxia in a temperature-dependent manner. It is concluded that hypothermia augments vasodilatation associated with nitroxidergic (nitrergic) nerve activation possibly by increasing the production of NO from L-arginine and, in addition, prevents impairment of NO production by hypoxia. These mechanisms likely explain how hypothermia protects nerve cells against hypoxia. Inhibitions of cyclic GMP phosphodiesterase and of superoxide production by hypoxia do not seem to participate in the action of hypothermia. Mechanisms underlying its protective action remain to be ascertained.

Animals↗
PubMed2003

pH-induced conformational change in an alpha-helical coiled-coil is controlled by His residues in the hydrophobic core.

An alpha-helical coiled-coil structure is one of the basic structural units in proteins. Hydrophilic residues at the hydrophobic positions in the coiled-coil structure play important roles in structures and functions of natural proteins. We reported here a peptide that formed a triple stranded alpha-helical coiled-coil showing the pH-dependent structural change. The peptide was designed to have two His residues at the hydrophobic positions of the center of the coiled-coil structure. The peptide folded into a triple stranded coiled-coil at neutral pH, while it unfolded at acidic pH. This construct is useful to create a protein that the structure or function is controlled by pH.

Amino Acid Sequence↗
PubMed2002

HSP90, HSP70, and GAPDH directly interact with the cytoplasmic domain of macrophage scavenger receptors.

The macrophage scavenger receptor (MSR) is a trimeric membrane protein which binds to modified low-density lipoprotein (LDL) and has been indicated in the development of atherosclerosis. It has recently been demonstrated that the N-terminal cytoplasmic domain of MSR has an important role in the efficient internalization and cell-surface expression of the receptor. This study shows that the N-terminal cytoplasmic domain in bovine was constructed using a peptide architecture technique in which the peptide chain was bundled at their C-terminus to yield a trimeric form and that this did not form an ordered structure. Furthermore, the binding proteins to the cytoplasmic domain of MSR were determined for the first time using a peptide affinity column. Sequence analyses of the specific binding proteins in bovine revealed that heat shock protein 90 (HSP90), heat shock protein 70 (HSP70), leucine aminopeptidase (LAP), adenocylhomocysteinase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were included. GST-pull-down assay and immunoprecipitation analyses on HSP90, HSP70, and GAPDH showed that all these proteins could bind to the cytoplasmic domain of MSR in vitro and in vivo. These proteins interact with the cytoplasmic domain directly and may have an effect on the functions of MSR such as internalization, cell-surface expression, and signal transduction.

Animals↗
PubMed2002

Diclofenac (Voltaren)-induced pneumonitis after chest operation.

We report a rare case of nonsteroidal anti-inflammatory drug-induced pneumonitis in a 72-year-old man taking diclofenac for wound pain after pulmonary resection. The pneumonitis and pleural effusion were predominant on the operative side and resolved rapidly after the diclofenac was discontinued. The diagnosis of drug-induced pneumonitis was based on a lymphocyte stimulation test that was positive for diclofenac sodium and negative for other drugs. This case report demonstrated that surgeons should be aware of the possibility of pneumonitis induced by a nonsteroidal anti-inflammatory drug.

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