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Biomedical subjects

Jun Hasegawa

Publications and source records attributed to Jun Hasegawa.

34 records · Page 2Linked to original sources

Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro.

Recent studies have suggested that Spt6 participates in the regulation of transcription by RNA polymerase II (RNAPII). However, its underlying mechanism remains largely unknown. One possibility, which is supported by genetic and biochemical studies of Saccharomyces cerevisiae, is that Spt6 affects chromatin structure. Alternatively, Spt6 directly controls transcription by binding to the transcription machinery. In this study, we establish that human Spt6 (hSpt6) is a classic transcription elongation factor that enhances the rate of RNAPII elongation. hSpt6 is capable of stimulating transcription elongation both individually and in concert with DRB sensitivity-inducing factor (DSIF), comprising human Spt5 and human Spt4. We also provide evidence showing that hSpt6 interacts with RNAPII and DSIF in human cells. Thus, in vivo, hSpt6 may regulate multiple steps of mRNA synthesis through its interaction with histones, elongating RNAPII, and possibly other components of the transcription machinery.

Animals↗

Spectral analysis of colonic intraluminal pressure in patients who received a colonic replacement following radical esophagectomy.

The aim of this study was to evaluate the motor activity of the interposed colonic segment in patients who had received a colonic replacement following radical esophagectomy using spectral analysis and a 24 hr activity graph. The 24-hr ambulatory pressure waves were recorded in the replaced colon after esophagectomy (n=8) using a solid-state manometric catheter (MicroDigitrapper, Synetics). Motility and spectral analyses of the intraluminal pressure waves were performed by Multigram and Gastrosoft (Synetics). It was revealed that after a meal the 3 cpm (cycles per minute) component of the motility index increased but the 12-15 cpm component decreased. The diurnal rhythm showed that colonic motility was high in the daytime and low during sleep. In contrast, duodenal motility was relatively high even during sleep. The motility index increased as the postoperative period increased. The motility of the replaced colon was higher during the daytime and after meals. The higher motility after meals was characterized by an increase in the 3 cpm component. These motor characteristics may help the function of the replaced colon as a substitute for the esophagus.

Aged↗

Electrogastrographic activity in patients who received proximal gastrectomy plus jejunal interposition or total gastrectomy plus jejunal interposition.

Electrogastrograms (EGGs) were recorded in patients both before and after receiving proximal gastrectomy plus jejunal interposition (PGJI) or just after receiving total gastrectomy plus jejunal interposition (TGJI). Intraluminal pressure was also recorded in some postoperative patients. The EGG 3 cpm component (2.5-4.9 cpm) remained after PGJI, but subsequently decreased with a significant reduction in the preoperative to postoperative ratio of the 3 cpm components (P<0.05). The mean frequency of the 3 cpm components increased significantly after PGJI (P<0.05) and its instability factor increased. The EGG 10 cpm components became relatively dominant compared to other frequency components in 2 out of 8 of patients having PGJI but the mean amplitude of 10 cpm decreased. In TGJI patients, only the 10 cpm component was conspicuous in EGG as in the case of total gastrectomy and Roux en Y anastomosis procedures. The spectral frequencies of intraluminal pressure in the interposed jejunum were similar to the EGG of 10 cpm components both in the case of PGJI and TGJI patients. In conclusion, surface EGG could record the electrical activities of the interposed jejunum more easily in patients having had TGJI than in PGJI.

Anastomosis, Roux-en-Y↗

Relationship between redox function and protein stability of cytochromes c.

Electrochemical, 1H NMR, and optical studies on mesophile Pseudomonas aeruginosa cytochrome c551, its single (F34Y) and quintuple (F7A/V13M/F34Y/E43Y/V78I) mutants, and thermophile Hydrogenobacter thermophilus cytochrome c552 at wide temperature range demonstrated that the stable protein exhibits the low redox potential predominantly due to the enthalpic contribution to the redox reaction. The overall stability of the oxidized form was shown to determine the stability of the Fe-methionine coordination bond, which then directly regulates the redox function.

Cytochromes c↗

Topographical electrogastrograms after radical esophagectomy with colonic replacement.

PURPOSE: To characterize the functional substitution of colon for the esophagus, we compared the electrogastrogram (EGG) maps and spectral frequencies and power of preoperative controls with patients who had undergone colonic replacement. METHODS: Monopolar EGGs were recorded and spectrally analyzed at 27 locations on the thoracoabdominal surface. The spectral powers of five frequency groups were converted into EGG maps. RESULTS: In contrast to the epigastric concentrations of maximal power foci in a preoperative 3-cpm (cycles per minute) group, those of the colon replacement subjects seemed not to be concentrated in the epigastric region. Power in the 6-cpm colon replacement group were significantly greater and those in the 3-cpm colon replacement group were significantly less than those in the preoperative controls. Spectral frequencies in the 1- and 3-cpm colon replacement groups were significantly higher than those in the preoperative controls. CONCLUSIONS: The colonic and gastric EGG activities had 3-cpm, and probably 6-cpm in common. However, the colonic EGG activities were significantly different from the gastric EGG activities in frequency in the 3-cpm group, and in amplitude in both the 3- and 6-cpm groups. Thus, the replaced colon seems to preserve the original colonic EGG activity.

Aged↗

Foreign body in the maxillary sinus--possible plastic tube: a case report.

We experienced the case of a patient who had a foreign body in the maxillary sinus. The patient complained of recurrent unilateral badly smelling rhinorrhea. The computed tomography and the magnetic resonance images revealed a foreign body in the maxillary sinus. The foreign body was removed via the Caldwell-Luc approach. The foreign body was a plastic tube, which was supposed to be designed for the drainage of the maxillary sinus. However, the patient did not remember having been subjected to a treatment using such kind of tube. After the surgical removal, the patient became free from the nasal symptoms.

Adult↗

A rapid purification method for human RNA polymerase II by two-step affinity chromatography.

The molecular dissection of transcription mechanisms is greatly facilitated by constructing and manipulating defined transcription systems in vitro. This approach requires highly purified transcription factors. A major enzyme participating in the transcription reaction is RNA polymerase II (RNAPII), which is composed of at least 12 subunits (RPB1-12). Due to its complex structure, it is difficult to prepare highly pure RNAPII by the conventional purification procedure. We transfected HeLa cells with a plasmid expressing RPB3 with a double FLAG-histidine tag on its amino-terminus. A high yielding clone was isolated and its extracts were subjected to immunoaffinity purification and then Co(2+) affinity chromatography. This resulted in a preparation of RNAPII complexes that consisted of all the core subunits, including the double-tagged RPB3 protein. Transcription reactions with oligo (dC)-tailed templates and transcription assays involving general transcription factors revealed that the double-tagged RNAPII complexes are active and functional in basal and activated transcription. Our method is superior to the conventionally used purification procedure in that the final preparation is markedly more pure (92% versus 40%), and the procedures are much less time-consuming. Thus, this two-step affinity purification method is an uncomplicated and effective method by which active and functional RNAPII can be prepared.

Cell Line↗

Human transcription elongation factor NELF: identification of novel subunits and reconstitution of the functionally active complex.

The multisubunit transcription elongation factor NELF (for negative elongation factor) acts together with DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF)/human Spt4-Spt5 to cause transcriptional pausing of RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, A to E. NELF-A has sequence similarity to hepatitis delta antigen (HDAg), the viral protein that binds to and activates RNAPII, whereas NELF-E is an RNA-binding protein whose RNA-binding activity is critical for NELF function. To understand the interactions of DSIF, NELF, and RNAPII at a molecular level, we identified the B, C, and D proteins of human NELF. NELF-B is identical to COBRA1, recently reported to associate with the product of breast cancer susceptibility gene BRCA1. NELF-C and NELF-D are highly related or identical to the protein called TH1, of unknown function. NELF-B and NELF-C or NELF-D are integral subunits that bring NELF-A and NELF-E together, and coexpression of these four proteins in insect cells resulted in the reconstitution of functionally active NELF. Detailed analyses using mutated recombinant complexes indicated that the small region of NELF-A with similarity to HDAg is critical for RNAPII binding and for transcriptional pausing. This study defines several important protein-protein interactions and opens the way for understanding the mechanism of DSIF- and NELF-induced transcriptional pausing.

Amino Acid Sequence↗

Bone resorption and inflammatory inhibition efficacy of intermittent cyclical etidronate therapy in rheumatoid arthritis.

OBJECTIVE: Osteoclast activation or cartilage and bone destruction are developed in patients with rheumatoid arthritis (RA). The efficacy of etidronate with respect to osteoporosis, inhibition of bone resorption and destruction, and antiinflammation in RA was examined for 72 weeks. METHODS: Sixty-three patients with RA (56 women, 7 men) were divided into a group that received intermittent cyclical etidronate therapy (ICET) (31 patients) and a non-ICET group (32 patients). Over a 72 week followup period, the urinary deoxypyridinoline (DPD), serum bone alkaline phosphatase (BAP), bone mineral density (BMD), Larsen damage score, Lansbury activity index, and concentrations of serum C-reactive protein (CRP) and serum interleukin 6 (IL-6) of the 2 groups were compared. RESULTS: In the non-ICET group, a significant decrease in BMD and a significant increase in the Larsen damage score were observed. In the ICET group, the level of DPD started to decrease 12 weeks after etidronate administration and progression of the Larsen damage score was significantly inhibited. IL-6 concentration was significantly decreased 72 weeks after etidronate administration. Concentrations of BAP and CRP and the Lansbury activity index were not significantly different between the ICET and the non-ICET groups. A significant correlation between the IL-6 and DPD concentrations was observed. CONCLUSION: Etidronate was effective at inhibiting bone resorption and destruction in study patients with RA, while not increasing BAP concentrations; and a correlation was observed between the concentration of DPD and IL-6, indicating the antiinflammatory effect of etidronate.

Aged↗

Utilization of PCIA (patient-controlled intravenous analgesia) for postoperative analgesia of spine fusion.

We examined the effect of patient-controlled intravenous analgesia (PCIA) using metoclopramide hydrochloride and morphine hydrochloride in 30 patients (15 males, 15 females) who underwent spine fusion surgery under general anesthesia. The intravenous administration of 2 mg morphine hydrochloride was done prior to the completion of surgery. Subsequently, morphine hydrochloride was administed via venous injection at a dose of 1 ml/hr. Additionally, a 1 ml bolus dosage was administered at a 60-minute lockout interval employing a pump which contained 36 mg of morphine hydrochloride, 30 mg of metoclopramide hydrochloride in 62 ml of physiologic saline. At approximately 72 hours, we observed the resting VAS (visual analog scale) and the side effects of morphine hydrochloride and thereafter the button pressing frequency (1 button have 0.5 mg morphine hydrochloride) was investigated. The resting VAS was stable through 72 hours in 13 males and 15 females of the 30 cases exhibiting from 2 to 3. The frequency of button pressing was investigated regarding time and gender. One male and two female patients exhibited metastatic tumors of the spine but demonstrated no button manipulation. For the remaining 27 subjects the button pressing frequency increased in most instances furthermore, from 24-48 hours after surgery, the frequency for male patients clearly increased. From 48 hours after surgery, no gender differences were evident regarding the button frequency. However, differences were observed regarding the disease, gender and postoperative time course of analgesia. As a result, no alteration in the background dose appeared to provide the best result, as did a short duration lockout interval.

Aged↗

Blind endobronchial insertion of a movable bronchial blocker attached to an endotracheal tube, Univent tube.

The endotracheal tube with a movable bronchial blocker, Univent tube, used to effect one-lung ventilation is easy to use in endotracheal intubation. However, problems are often encountered when inserting the blocker into the bronchus. We herein describe a method which enables the easy blind insertion of the blocker into the left or right bronchus. The techniques of inserting the blocker into the left main stem bronchus will be described. With the patient in a supine position, the head of the patient is moved to the left. The operator then places his right hand fingers near the cricoid and presses to displace the larynx of the patient toward the right. While performing this procedure, the operator advances the blocker using the left hand. Finally, using a bronchoscope, the placing of the blocker is to be ascertained in an appropriate position inside the left bronchus. When strong resistance is left, the blocker should be retracted, the force of laryngeal displacement is altered and, then, the blocker is inserted again in a resistance-free manner. We have so far experienced no complications such as severe tracheobronchial hemorrhage, tracheobronchial perforation of laryngeal dislocation. We herein describe a useful and simple method for inserting the bronchial blocker of the Univent tube into the bronchus.

Bronchoscopy↗

Influence of amino acid side chain packing on Fe-methionine coordination in thermostable cytochrome C.

Paramagnetic NMR and optical studies of the oxidized forms of mesophile Pseudomonas aeruginosa cytochrome c(551) and its quintuple mutant (F7A/V13M/F34Y/E43Y/V78I), and thermophile Hydrogenobacter thermophilus cytochrome c(552) demonstrated that the amino acid side chain packings in the protein interior influence the coordination bond between the heme iron and the axial methionine in the proteins. The strength of heme axial coordinations was found to correlate with the overall protein thermostability.

Bacterial Proteins↗

Solution structure of ascidian trypsin inhibitor determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of ascidian trypsin inhibitor (ATI), a 55 amino acid residue protein with four disulfide bridges, was determined by means of two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy. The resulting structure of ATI was characterized by an alpha-helical conformation in residues 35-42 and a three-stranded antiparallel beta-sheet in residues 22-26, 29-32, and 48-50. The presence of an alpha-helical conformation was predicted from the consensus sequences of the cystine-stabilized alpha-helical (CSH) motif, which is characterized by an alpha-helix structure in the Cys-X(1)-X(2)-X(3)-Cys portion (corresponding to residues 37-41), linking to the Cys-X-Cys portion (corresponding to residues 12-14) folded in an extended structure. The secondary structure and the overall folding of the main chain of ATI were very similar to those of the Kazal-type inhibitors, such as Japanese quail ovomucoid third domain (OMJPQ3) and leech-derived tryptase inhibitor form C (LDTI-C), although ATI does not show extensive sequence homology to these inhibitors except for a few amino acid residues and six of eight half-cystines. On the basis of these findings, we realign the amino acid sequences of representative Kazal-type inhibitors including ATI and discuss the unique structure of ATI with four disulfide bridges.

Amino Acid Sequence↗

Migration of CX3CR1-positive T cells producing type 1 cytokines and cytotoxic molecules into the synovium of patients with rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by chronic inflammation of multiple joints. Large numbers of T cells, which produce type 1 cytokines, infiltrate into RA synovium. Chemokines and chemokine receptors are considered to contribute to the T cell infiltration. In this study, we examined the role of CX3CL1/fractalkine and its receptor CX3C chemokine receptor 1 (CX3CR1) in the T cell migration into RA synovium. METHODS: Using flow cytometry, immunohistochemistry, and reverse transcription-polymerase chain reaction, we analyzed CX3CR1 expression by peripheral blood and synovial T cells, and CX3CL1 expression in synovium from patients with RA. Cytokine and cytotoxic molecule expression by CX3CR1-positive T cells was analyzed by flow cytometry. RESULTS: CX3CR1 expression by peripheral CD4+ and CD8+ T cells was up-regulated in RA patients. The peripheral CD4+ and CD8+ T cells expressing CX3CR1 predominantly produced interferon-gamma and tumor necrosis factor alpha, and expressed cytotoxic molecules such as granzyme A and perforin. Furthermore, CX3CR1+,CD3+ T cells infiltrated into RA synovium. CX3CL1, the unique ligand of CX3CR1, was expressed by endothelial cells and synoviocytes in RA synovium, but not in osteoarthritis synovium. CONCLUSION: Our findings suggest that the interactions of CX3CL1 and CX3CR1 might contribute to the accumulation of CX3CR1+ T cells expressing type 1 cytokines and possessing cytotoxic granules in RA synovium.

Arthritis, Rheumatoid↗

Cytochrome c from a thermophilic bacterium has provided insights into the mechanisms of protein maturation, folding, and stability.

Cytochrome c is widely distributed in bacterial species, from mesophiles to thermophiles, and is one of the best-characterized redox proteins in terms of biogenesis, folding, structure, function, and evolution. Experimental molecular biology techniques (gene cloning and expression) have become applicable to cytochrome c, enabling its engineering and manipulation. Heterologous expression systems for cytochromes c in bacteria, for use in mutagenesis studies, have been established by extensive investigation of the biological process by which the functional structure is formed. Mutagenesis and structure analyses based on comparative studies using a thermophile Hydrogenobacter thermophilus cytochrome c-552 and its mesophilic counterpart have provided substantial clues to the mechanism underlying protein stability at the amino-acid level. The molecular mechanisms underlying protein maturation, folding, and stability in bacterial cytochromes c are beginning to be understood.

Amino Acid Sequence↗

Thermodynamic characterization of variants of mesophilic cytochrome c and its thermophilic counterpart.

Thermal stability was measured for variants of cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and a thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c-552 (HT c-552), by differential scanning calorimetry (DSC) at pH 3.6. The mutated residues in PA c-551, selected with reference to the corresponding residues in HT c-552, were located in three spatially separated regions: region I, Phe7 to Ala/Val13 to Met; region II, Glu34 to Tyr/Phe43 to Tyr; and region III, Val78 to Ile. The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpic but also entropic contributions, which reflected improved packing of the side chains. Meanwhile, the mutated region II made enthalpic contributions to the stability through electrostatic interactions. The obtained differences in the Gibbs free energy changes of unfolding [Delta(DeltaG)] showed that the three regions contributed to the overall stability in an additive manner. HT c-552 had the smallest heat capacity change (DeltaC(P)), resulting in higher DeltaG values over a wide temperature range (0-100 degrees C), compared to the PA c-551 variants; this contributed to the highest stability of HT c-552. Our DSC measurement results, in conjunction with mutagenesis and structural studies on the homologous mesophilic and thermophilic cytochromes c, provided an extended thermodynamic view of protein stabilization.

Bacteria↗