Search PubMed⌕ Search

Biomedical subjects

Tomoko Tada

Publications and source records attributed to Tomoko Tada.

18 recordsLinked to original sources
PubMed2006

The roles of conserved amino acids on substrate binding and conformational integrity of ClpB N-terminal domain.

Escherichia coli heat shock protein ClpB disaggregates denatured protein in cooperation with the DnaK chaperone system. Several studies showed that the N-terminal domain is essential for the chaperone activity, but its role is still largely unknown. The N-terminal domain contains two structurally similar subdomains, and conserved amino acids Thr7 and Ser84 share the same position in two apparent sequence repeats. T7A and S84A substitutions affected chaperone activity of ClpB without significantly changing the native conformation [Liu, Z. et al. (2002) J. Mol. Biol. 321, 111-120]. In this study, we aimed to better understand the roles of several conserved amino acid residues, including Thr7 and Ser84, in the N-terminal domain. We investigated the effects of mutagenesis on substrate binding and conformational states of ClpB N-terminal domain fragment (ClpBN). Fluorescence polarization analysis showed that the T7A and S84A substitutions enhanced the interaction between ClpBN and protein aggregates. Interestingly, further analyses suggested that the mechanisms by which they do so are quite different. For T7A substitution, the increased substrate affinity could be due to a conformational change in the hydrophobic core as revealed by NMR spectroscopy. In contrast, for S84A, increased substrate binding would be explained by a unique conformational state of this mutant as revealed by pressure perturbation analysis. The thermal transition temperature of the S84A mutant, monitored by DSC, was 6.1 degrees C lower than that of wild-type. Our results revealed that conserved amino acids Thr7 and Ser84 both participated in maintaining the conformational integrity of the ClpB N-terminal domain.

Amino Acid Sequence↗
PubMed2006

Synergetic effects of pressure and chemical denaturant on protein unfolding: stability of a serine-type carboxyl protease, kumamolisin.

Kumamolisin, a serine carboxyl proteinase, is very stable and hardly denatured by single perturbation of a chemical denaturant (urea), pressure (<500 MPa) or temperature (<65 degrees C). In order to investigate the cooperative effects of these three denaturing agents, DSC, CD, intrinsic fluorescence, and fourth derivative UV absorbance were measured under various conditions. By application of pressure to kumamolisin in 8 M urea solution, substantial red-shift in the center of fluorescence emission spectral mass was observed, and the corresponding blue-shift was observed for two major peaks in fourth derivative UV absorbance, under the similar urea-containing conditions. The denaturation curves were analyzed on the basis of a simple two-state model in order to obtain thermodynamic parameters (DeltaV, DeltaG, and m values), and the combined effects of denaturing agents are discussed, with the special interest in the large cavity and neighboring Trp residue in kumamolisin.

Aspartic Acid Endopeptidases↗
PubMed2005

Molecular mechanisms of dendritic spine morphogenesis.

Excitatory synapses are formed on dendritic spines, postsynaptic structures that change during development and in response to synaptic activity. Once mature, however, spines can remain stable for many months. The molecular mechanisms that control the formation and elimination, motility and stability, and size and shape of dendritic spines are being revealed. Multiple signaling pathways, particularly those involving Rho and Ras family small GTPases, converge on the actin cytoskeleton to regulate spine morphology and dynamics bidirectionally. Numerous cell surface receptors, scaffold proteins and actin binding proteins are concentrated in spines and engaged in spine morphogenesis.

Animals↗
PubMed2005

Effect of the polypeptide binding on the thermodynamic stability of the substrate binding domain of the DnaK chaperone.

The effect of polypeptide binding on the stability of the substrate binding domain of the molecular chaperone DnaK has been studied by thermodynamic analysis. The calorimetric scan of the fragment of the substrate binding domain DnaK384-638, consisting of a beta-domain and an alpha-helical lid, showed two transitions centered at 56.2 and 76.0 degrees C. On the other hand, the thermal unfolding of the shorter fragment DnaK386-561, which lacks half of the alpha-helical lid, exhibited a single transition at 57.0 degrees C. Therefore, the transition of DnaK384-638 at 56.2 degrees C is mainly attributed to the unfolding of the beta-domain. The calorimetric scan of DnaK384-638D526N showed that the unfolding of the beta-domain was composed of two transitions. The polypeptide bound DnaK384-638 exhibited a symmetrical DSC peak at 58.6 degrees C, indicating that the substrate binding shifts the beta-domain toward a single cooperative unit. A low concentration of GdnHCl (<1.0 M) induced a conformational change in the beta-domain of DnaK384-638 without changes in the secondary structure. While the thermal unfolding of the beta-domain of DnaK384-638 was composed of two transitions in the presence of GdnHCl, the beta-domain of the substrate bound DnaK384-638 exhibited a single symmetrical DSC peak in the same condition. All together, our results indicate that complex between DnaK384-638 and substrate forms a rigid conformation in the beta-domain.

Escherichia coli Proteins↗
PubMed2005

Self-expandable metallic stent placement for palliation in gastric outlet obstructions caused by gastric cancer: a comparison with surgical gastrojejunostomy.

BACKGROUND: In patients with gastric outlet obstruction (GOO), palliative enteral stenting is a less invasive procedure compared with gastroenterostomy. Most diseases analyzed in previous studies of such stenting were pancreaticobiliary malignancies. METHODS: We reviewed the medical records of patients with GOO secondary to gastric cancer who were admitted to our institution between September 1994 and September 2004. The outcome of stent placement for GOO was compared with the outcome in patients who underwent palliative open gastrojejunostomy during the same period. Enrolled patients from both groups displayed symptomatic GOO. Patients with recurrent gastric cancer were excluded from this study. RESULTS: Twenty-two patients underwent palliative enteral stenting, and 22 patients were subjected to surgical gastrojejunostomy (bypass). There were no significant differences between the two groups regarding patient baseline characteristics. Technical success and clinical success were obtained in 100% and 77.3%, respectively, of both groups. The operating time was shorter in the stent group (30 vs 118 min; P<0.0001). The time from the procedure to the resumption of food intake was shorter in the stent group than in the bypass group (2 days vs 8 days; P<0.0001). An improvement in performance score after the procedure was observed in both groups (stent group; P=0.0264; bypass group; P=0.0235). No significant differences were observed regarding the possibility of discharge. In patients discharged, the median postoperative hospital stays were 19 days and 28 days (P=0.0558). The median survival periods were 65 days and 90 days. Minor complications were observed in 1 patient in the stent group and in 4 in the bypass group. No mortality or severe complications were observed for either group. CONCLUSIONS: Self-expandable metallic stent placement is a safe and efficacious procedure for palliation, with shorter operating time and more prompt restoration of oral intake, compared to surgical alternatives in patients with GOO caused by gastric cancer.

Aged↗
PubMed2005

Efficacy of an overtube for reducing the risk of peristomal infection after PEG placement: a prospective, randomized comparison study.

BACKGROUND: With the conventional pull method of PEG placement, there is a significant risk of wound infection from contamination of the gastrostomy catheter as it passes through the oral cavity. This study compared the occurrence of peristomal wound infection associated with PEG placement with and without use of an overtube. METHODS: Consecutive patients with dysphagia were randomized to undergo PEG placement with (Group I) or without (Group II) an overtube. For each patient, the peristomal area was evaluated daily for 1 week after PEG placement. The presence of erythema and of exudate were scored on a scale of 0 to 4; induration was scored on a scale of 0 to 3. Criteria for infection were a maximum combined score of 8 or higher, or the presence of microscopic and microbiologic evidence of suppurating exudate. In each group, cefazolin was administered prophylactically (2 g/d intravenously) for 3 days. For patients who had received an antibiotic(s) before PEG placement, the same antibiotic(s) was used. All procedures in both groups were performed by one of two investigators who used the pull method. RESULTS: A total of 76 patients were randomized; 3 were excluded from analysis, because death occurred within 1 week after the procedure. Two of 3 deaths were procedure-related (aspiration pneumonia in Group I, peritonitis in Group II). Data for 37 patients in Group I and 36 in Group II were analyzed. There was no significant difference between the groups with respect to baseline characteristics. The occurrence of peristomal infection within 1 week of PEG was significantly lower in Group I compared with Group II (2 vs. 12; p = 0.0029). The mean daily combined scores in Group I also were significantly lower than those in Group II ( p < 0.0001), and the median maximum parameter scores in Group I were significantly lower than those in Group II (erythema, p = 0.0062; induration, p = 0.0390; exudate, p < 0.0001), although the nominal significance for induration was removed by correction for the multiple testing of data. One patient excluded from Group II died from sepsis because of procedure-induced peritonitis. Among the 73 enrolled patients, there was no procedure-related mortality or clinically important wound infections that required surgical intervention in either group. CONCLUSIONS: Use of an overtube during PEG placement reduces the risk of peristomal wound infection.

Aged↗
PubMed2004

Interaction of the N-terminal domain of Escherichia coli heat-shock protein ClpB and protein aggregates during chaperone activity.

The Escherichia coli heat-shock protein ClpB reactivates protein aggregates in cooperation with the DnaK chaperone system. The ClpB N-terminal domain plays an important role in the chaperone activity, but its mechanism remains unknown. In this study, we investigated the effect of the ClpB N-terminal domain on malate dehydrogenase (MDH) refolding. ClpB reduced the yield of MDH refolding by a strong interaction with the intermediate. However, the refolding kinetics was not affected by deletion of the ClpB N-terminal domain (ClpBDeltaN), indicating that MDH refolding was affected by interaction with the N-terminal domain. In addition, the MDH refolding yield increased 50% in the presence of the ClpB N-terminal fragment (ClpBN). Fluorescence polarization analysis showed that this chaperone-like activity is explained best by a weak interaction between ClpBN and the reversible aggregate of MDH. The dissociation constant of ClpBN and the reversible aggregate was estimated as 45 muM from the calculation of the refolding kinetics. Amino acid substitutions at Leu 97 and Leu 110 on the ClpBN surface reduced the chaperone-like activity and the affinity to the substrate. In addition, these residues are involved in stimulation of ATPase activity in ClpB. Thus, Leu 97 and Leu 110 are responsible for the substrate recognition and the regulation of ATP-induced ClpB conformational change.

Animals↗
PubMed2004

Molecular cloning in yeast by in vivo homologous recombination of the yeast putative alpha1 subunit of the voltage-gated calcium channel.

Saccharomyces cerevisiae has only one gene encoding a putative voltage-gated Ca2+ channel pore-forming subunit, CCH1, which is not possible to be cloned by conventional molecular cloning techniques using Escherichia coli. Here, we report the successful cloning of CCH1 in yeast by in vivo homologous recombination without using E. coli. Overexpression of the cloned CCH1 or MID1 alone, which encodes a putative stretch-activated Ca2+ channel component, does not increase Ca2+ uptake activity, but co-overexpression results in a 2- to 3-fold increase. Overexpression of CCH1 does not substantially complement the lethality and low Ca2+ uptake activity of a mid1 mutant and vice versa. These results indicate that co-overproduction of Cch1 and Mid1 is sufficient to increase Ca2+ uptake activity.

Base Sequence↗
PubMed2004

Subcellular localization and oligomeric structure of the yeast putative stretch-activated Ca2+ channel component Mid1.

The yeast Mid1 protein with an apparent molecular mass of 100 kDa is required for Ca2+ influx stimulated by the mating pheromone and by a capacitative calcium entrylike mechanism acting in response to Ca2+ depletion from the endoplasmic reticulum (ER) and functions as a stretch-activated Ca2+ -permeable channel when expressed in mammalian cells. Our previous work with protease protection experiments has indicated that Mid1 is present in the plasma membrane. In this study, we examined a possible intracellular localization of this protein by indirect fluorescence microscopy and found that Mid1 is present in the ER membrane as well as the plasma membrane. Intracellular fluorescence images for Mid1 were the same as those for the ER marker protein Sec71 but quite different from those of the Golgi protein Ypt1. The results were confirmed by membrane fractionation using Angiografin density gradient analysis. We also investigated the oligomeric structures and protein levels of Mid1 and found that Mid1 forms a 200-kDa oligomer by disulfide bonding. The protein level and modification of Mid1 in the plasma membrane and the ER membrane were unchanged by the mating pheromone. These findings provide new insight into the function of Mid1 in relation to localization, modification, and activation mechanisms.

Calcium Channels↗
PubMed2004

Phe356 in the yeast Ca2+ channel component Mid1 is a key residue for viability after exposure to alpha-factor.

The yeast Mid1 protein is an integral membrane protein required for the viability of differentiated cells and Ca2+ influx induced by mating pheromone. Our previous study has identified a loss-of-function mutation, F356S. The F356S mutant is completely unable to maintain viability, but still has Ca2+ accumulation activity near the wild-type level. Here we further examined in detail the F356S mutation to unravel the function of Phe356. After exposure to the pheromone, the F356S mutant was not fully rescued by high extracellular Ca2+, like the mid1 null mutant, suggesting that Phe356 and Mid1 itself are also required for viability maintenance mechanism that does not involve Ca2+ signalling. Substitutions of hydrophilic amino acids for Phe356 caused lethality and low Ca2+ accumulation, but those of hydrophobic amino acids did not. Substitutions of small amino acids for Phe356 caused a significantly reduced viability, but did not affect Ca2+ accumulation. We suggest that the hydrophobicity of the Phe356 residue is important for both viability maintenance and Ca2+ uptake, and that its size for viability maintenance.

Amino Acid Sequence↗
PubMed2004

Self-expandable metallic stent placement as palliative treatment of obstructed colorectal carcinoma.

BACKGROUND: Stent placement in palliation of unresectable colon cancer is an alternative to surgical treatment. The through-the-scope stent for the exclusive treatment of colorectal cancer is not available in Japan. This report describes the use of an esophageal stent and the technical modifications required for its success in the treatment of colorectal strictures. We describe various technical strategies for colorectal stent placement and report on the outcomes. METHODS: Medical records of patients who underwent palliative colonic stenting between June 1997 and March 2003 were reviewed retrospectively, and the clinical outcome was evaluated. RESULTS: Insertion of a metallic esophageal stent was attempted in 12 patients (mean age, 73.0 years; 5 male, 7 female). Location of the stricture was in the rectum in 4 patients and in the sigmoid, descending, or transverse segments of the colon in 5, 1, and 2 patients, respectively. Two patients had recurrent colon cancer after surgery. The remaining 10 patients did not undergo surgery. Stent placement was technically successful in 11 patients, giving a technical success rate of 92%. Following successful stent placement, all but 1 patient obtained clinical success, generating a clinical success rate of 83%. Late complications occurred in 4 patients and included 2 migrations, 2 bleeds, and 1 obstruction. The complication rate of the procedure was 33.3%. There was no mortality or severe complications. The median survival period was 120 days. CONCLUSIONS: Stent placement can be considered safe and effective palliation for unresectable colorectal cancer. With technical modification of an esophageal stent, this procedure is now feasible. Stent placement in palliation of unresectable colon cancer is an alternative to surgical treatment. The through-the-scope stent for the exclusive treatment of colorectal cancer is not available in Japan. This report describes the use of an esophageal stent and the technical modifications required for its success in the treatment of colorectal strictures. We describe various technical strategies for colorectal stent placement and report on the outcomes.

Adult↗
PubMed2003

Molecular dissection of the hydrophobic segments H3 and H4 of the yeast Ca2+ channel component Mid1.

The Saccharomyces cerevisiae MID1 gene product, Mid1, is composed of 548 amino acid residues, has four relatively hydrophobic segments named H1-H4, and functions as a Ca(2+)-permeable, stretch-activated channel when expressed in mammalian cells. In some conditions Mid1 cooperates with Cch1, a yeast homolog of the alpha1 subunit of mammalian voltage-gated channels. To identify the important regions or amino acid residues necessary for Mid1 function, we employed in vitro site-directed mutagenesis on H3 and H4 of Mid1 and expressed the resulting mutant genes in a mid1 null mutant to examine whether the mutant gene products are functional or not in vivo. Mutant Mid1 proteins lacking the whole H3 or H4 segment, H3De or H4De, did not complement the lethality and low Ca(2+) accumulation activity of the mid1 mutant, although their localization and contents appeared to be normal, indicating that H3 and H4 are required for Mid1 function itself. Single amino acid exchange experiments on individual amino acid residues of H3 and H4 showed that 10 of 20 residues in H3 and 14 of 23 residues in H4 were important for the normal function of Mid1. In particular, we found four severe loss-of-function mutations, D341E, F356S, C373D, and C373R, and two interesting mutations leading to a high level of Ca(2+) accumulation with a slightly low complementing activity, G342A and Y355A. The importance of these amino acid residues will be discussed.

Amino Acids↗
PubMed2003

PEG with introducer or pull method: a prospective randomized comparison.

BACKGROUND: PEG by the conventional pull method has the potential drawback of being associated with a higher frequency of wound infection, presumably caused by contamination of the gastrostomy catheter as it passes through the oral cavity. This study investigated the occurrence of peristomal wound infection after PEG placement by using the pull and introducer techniques. METHODS: Between September 1999 and May 2002, consecutive patients with dysphagia for whom PEG was recommended were enrolled in the study and randomly assigned to two groups: PEG with the introducer method (Group I) or PEG with the pull method (Group II). The peristomal area of each patient was evaluated on a daily basis for one week after PEG. Erythema and exudate were scored on a scale from 0 to 4 and induration on a scale of 0 to 3. Criteria for infection were a maximum combined score of 8 or higher, or the presence of microscopic and microbiologic evidence of suppurating exudate. In each group, the endoscope was passed once during the procedure, and an antibiotic (piperacillin) was given prophylactically. All procedures were performed by one investigator with the assistance of another physician. RESULTS: Of the 60 patients enrolled, 30 were assigned to each group. PEG was successful in all patients. One patient was excluded from each group because of death (Group I, stroke; Group II, myocardial infarction) within one week of the procedure. Therefore, 58 patients, 29 in each group, were evaluated. There was no significant difference between the groups in terms of clinical parameters (age, gender, disease, performance score, mode of previous feeding, and recent antibiotic exposure). The occurrence of peristomal infection within one week of PEG was lower in Group I (introducer method) (0 vs. 9; p = 0.00094). The mean daily combined scores in Group I were significantly lower than those in Group II. Median of maximum parameter scores in Group I were significantly lower than those in Group II. There were no procedure-related mortalities or clinically significant wound infections that required surgical intervention. CONCLUSIONS: The risk of peristomal wound infection after PEG is lower with the introducer method compared with the pull method.

Aged↗
PubMed2002

Solid cystic tumor of the pancreas: report of six cases and a review of the Japanese literature.

Although many cases of solid cystic tumor of the pancreas (SCT) have been reported, its nature and histogenesis remain controversial. We herein report six cases of SCT, including three cases of noncystic type. A review of 22 cases of noncystic type SCT, including our 3 cases, was carried out to compare their features with those of 173 cases of classic SCT reported in Japan. Noncystic type SCTs tend to occur in male patients and are smaller in size and less frequently symptomatic, although they show histological characteristics similar to those of classic SCTs. The developmental process might be a cause of cyst formation. The accumulation and analysis of many, at present, "atypical" cases for clarification of its nature, will, it is hoped, lead to a new nomenclature for this condition that adequately describes its biological origin.

Adolescent↗
PubMed2002

Anorexia in space and possible etiologies: an overview.

Space travelers experience a flight duration-dependent loss in weight and body mass while in a microgravity environment, despite the absence of increased energy expenditure. Anorexia in space can lead to in-flight caloric deficits of 1330 kcal per 70 kg astronaut per day in the presence of abundant food and has a critical effect on endurance and performance. Microgravity, alterations in the light-and-dark cycle, and exposure to radiation energy are the environmental stresses believed to influence appetite, food intake, and gastrointestinal function during space flight. Review of data and recent studies in rodents during microgravity showed a release of stress hormones and complex neuroendocrine and physiologic changes involving the modulation of hypothalamic activity, food intake-related hormones, and cytokines. The shift of dietary preference to carbohydrates, which occurs in astronauts, denotes a stress physiologic response and augments free-plasma tryptophan concentration in the brain, the precursor of the potent anorexic agent, serotonin (5-HT). Alterations of other neuroendocrine mediators, including corticotropin-releasing factor (CRF), coordinate the stress response, leading to a decrease in appetite and gastrointestinal function. Our laboratories used the antiorthostatic tail-suspension technique to successfully mimic some of these anorexia-related stress responses and to directly demonstrate the role of 5-HT in microgravity-related decreased food intake and delayed gastric emptying. Further rodent studies from our laboratories demonstrated the adverse effect of altered dark-and-light cycles on food intake and body weight. Radiation energy, through its documented effects on appetite, probably contributes to the decreased caloric intake by astronauts. Modulation of hypothalamic activity, 5-HT, and CRF play a critical role in anorexia related to microgravity and circadian rhythm alterations. Specific gene knockout mice (e.g., 5-HT or CRF and their respective receptors) may prove fruitful in defining the pathways by which anorexia in space occurs. An understanding of these pathophysiologic problems as they relate to appetite, food intake, gastric emptying and gastrointestinal function, sufficiently to derive successful practical solutions, may lead to a quantitative enhancement of physiologic well-being and performance status, serving as a productive countermeasure in space.

Adaptation, Physiological↗
PubMed2002

Central mechanisms involved with catabolism.

PURPOSE OF REVIEW: Catabolism conjures up an end-metabolic process in which muscle and fat tissue are broken down into their constituent parts to provide nutrients for the body, secondary to a noxious stimulus that prevents the organism from adequately nourishing itself. However, catabolism is a primary event, initiated in the brain in response to perceived or real stresses or noxious stimuli, which has a secondary effect of inhibiting food intake and consequently the break down of skeletal muscle and adipose tissues to provide nutrients for the body to survive. RECENT FINDINGS: This is achieved via a cascade of neurohormonal monoaminergic and peptidergic mediators in the central nervous system, invoking the cortex, the limbic system and the hypothalamus. Among the most detailed mediators studied are corticotropin-releasing factor and serotonin which, via the hypothalamic-pituitary-adrenal axis and the sympathetic and parasympathetic nervous system, stimulate catecholamines and cortisol and inhibit anabolic hormones, insulin, leptin, ghrelin, including neuropeptide Y and other neuropeptides, among them the paracrine-acting cytokines. Simultaneously, there occurs stimulation of the counter-regulatory hormones cortisol, glucagon and the melanocortin family of neuropeptides. SUMMARY: The net effect is anorexia, with the inhibition of food intake, body weight loss, delayed gastric emptying and functions, the stimulation of gluconeogenesis, glycogenolysis and ketogenesis as sources of metabolic fuel, which if unabated leads ultimately to cachexia. The use of antagonists and the removal of stress or noxious stimuli experimentally test different pathways of this dynamic metabolic picture. Several studies have demonstrated important progress towards our understanding of the central mechanisms involved in anorexia and weight loss, which we summarize in this review.

Anorexia↗
PubMedDate not supplied

Mechanism for the phase transition of a genetically engineered elastin model peptide (VPGIG)40 in aqueous solution.

The concentration dependence of the pressure- and temperature-induced cloud point transition (Pc and Tc, respectively) of aqueous solutions of an elastin-like polypeptide with a repeating pentapeptide Val-Pro-Gly-Ile-Gly sequence (MGLDGSMG(VPGIG)40VPLE) was investigated by using apparent light scattering, differential scanning calorimetry, and circular dichroism methods. In addition, the effects of salts and surfactants on these properties were investigated. The Pc and Tc of the present peptide in aqueous solution were strongly concentration dependent. The calorimetric measurements showed that the enthalpy of transitions was 300-400 kJ/mol, i.e., 7-10 kJ/mol per VPGIG pentamer. The Tc of the (VPGIG)40 solution was highly affected by the addition of inert salts or SDS. The effects of salts were consistent with those observed in the lyotropic series or Hoffmeister series. The CD spectrum at low peptide concentrations indicated that the present peptide forms type II beta-turn-like structure(s) at higher temperatures, but the temperature dependence of random coil diminishment (195 nm) and beta-turn formation (210 nm) were not exactly coincident. A hypothetical mechanism of the (VPGIG)40 phase transition that could account for these observations was postulated. Observations suggest that the temperature-responsive properties of the elastin model peptides occur via a mechanism involving conformational change-association-aggregation and that the first two are strongly interactive.

Amino Acid Sequence↗