Biomedical subjects

Hiroshi Nakayama

Publications and source records attributed to Hiroshi Nakayama.

At least 19 recordsLinked to original sources

PubMed2007

Site-specific functionalization of proteins by organopalladium reactions.

A new carbon-carbon bond has been regioselectively introduced into a target position (position 32 or 174) of the Ras protein by two types of organopalladium reactions (Mizoroki-Heck and Sonogashira reactions). Reaction conditions were screened by using a model peptide, and the stability of the Ras protein under the reaction conditions was examined by using the wild-type Ras protein. Finally, the iF-Ras proteins containing a 4-iodo-L-phenylalanine residue were subjected to organopalladium reactions with vinylated or propargylated biotin. Site-specific biotinylations of the Ras protein were confirmed by Western blot and LC-MS/MS.

Mass Spectrometry↗

PubMed2006

Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K.

Thiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, gammaCys131-SO(2)H. When the SCNase alpha, beta and gamma subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (alphabetagamma)(4), like native SCNase but exhibited no catalytic activity. Metal analysis indicated that SCNase was expressed as an apo-form irrespective of the presence of cobalt in the medium. On the contrary, SCNase co-expressed with P15K, encoded just downstream of SCNase genes, in cobalt-enriched medium under the optimized condition (SCNase((+P15K))) possessed 0.86 Co atom/alphabetagamma trimer and exhibited 78% of the activity of native SCNase. SCNase((+P15K)) showed a UV-Vis absorption peak characteristic of the SCNase cobalt center. About 70% of SCNase((+P15K)) had the gammaCys131-SO(2)H modification. These results indicate that SCNase((+P15K)) is the active holo-SCNase. P15K is likely to promote the functional expression of SCNase probably by assisting the incorporation of cobalt ion.

Amino Acid Sequence↗

PubMed2006

Familial tauopathy mimicking corticobasal degeneration an autopsy study on three siblings.

We describe a family with tauopathy involving three autopsied siblings. All of the three siblings, as well as their grandmother, exhibited parkinsonism responsive to levodopa in one of the three, later associated with a possible reduction in spontaneity, compatible with frontal lobe dysfunction. Autopsy findings shared by the three siblings included, marked degeneration in the basal ganglia, especially in the substantia nigra and mild to moderate spongiosis in the cerebral cortices (mainly frontal) with achromatic neurons and tau/Gallyas-positive spherical aggregates resembling astrocytic plaques. Widespread deposition of tau in neurons, thread-like structures and oligodendrocytes as coiled body was robust in these lesions, whereas alpha-synuclein-positive deposits were absent. These features are compatible with sporadic corticobasal degeneration (CBD). Hippocampal neurons were characterized by its non-fibrillary tau deposits in the cytoplasm, as similarly visualized with Gallyas silver stain (Gal), while the Campbell-Switzer method (CS) failed to exhibit argyrophilia. This staining profile of neuronal cytoplasm with non-fibrillary structure (tau+/Gal+/CS-) is characteristic for tau-positive cortical neurons in CBD, distinct from typical neurofibrillary tangles of Alzheimer type, positive for both Gal and CS. Moreover, degenerative change was more evident in CA2 than in CA1, as noted in some cases of tauopathy with accumulation of 4-repeat tau such as CBD. This familial tauopathy can be classified as an extremely rare example of CBD based on the distribution of characteristic lesions and staining profiles of tau-deposits.

Adult↗

PubMed2006

Thiocyanate hydrolase is a cobalt-containing metalloenzyme with a cysteine-sulfinic acid ligand.

Thiocyanate hydrolase (SCNase) purified from Thiobacillus thioparus THI115 hydrolyzes thiocyanate to carbonyl sulfide and ammonia. DNA sequences of the cloned genes revealed the close relation of SCNase to nitrile hydratase (NHase). The consensus sequences for coordination of the metal ion found in NHases were also conserved in the gamma subunit of SCNase. Here, we showed that the SCNase contained one cobalt atom per alphabetagamma heterotrimer. UV-vis absorption spectrum suggested that the cobalt exists as a non-corrin ion. Reduced SCNase showed an ESR signal characteristic of low-spin Co2+, which closely resembled that of the Co-type NHases. Mass spectrometry for the peptide fragment containing the metal-binding motif of the SCNase gamma subunit indicated that the cysteine residue at position 131 was post-translationally oxidized to a cysteine-sulfinic acid. From these results, we concluded that SCNases and NHases form a novel non-corrin and/or non-heme protein family having post-translationally modified cysteine ligands.

Amino Acid Sequence↗

PubMed2006

Regioselective carbon-carbon bond formation in proteins with palladium catalysis; new protein chemistry by organometallic chemistry.

Palladium-catalyzed reactions have contributed to the advancement of many areas of organic chemistry, in particular, the synthesis of organic compounds such as natural products and polymeric materials. In this study, we have used a Mizoroki-Heck reaction for site-specific carbon-carbon bond formation in the Ras protein. This was performed by the following two steps: 1) the His6-fused Ras protein containing 4-iodo-L-phenylalanine at position 32 (iF32-Ras-His) was prepared by genetic engineering and 2) the aryl iodide group on the iF32-Ras-His was coupled with vinylated biotin in the presence of a palladium catalyst. The biotinylation was confirmed by Western blotting and liquid chromatography-mass spectrometry (LC-MS). The regioselectivity of the Mizoroki-Heck reaction was furthermore confirmed by LC-MS/MS analysis. However, in addition to the biotinylated product (bF32-Ras-His), a dehalogenated product (F32-Ras-His) was detected by LC-MS/MS. This dehalogenation resulted from the undesired termination of the Mizoroki-Heck reaction due to steric and electrostatic hindrance around residue 32. The biotinylated Ras showed binding activity for the Ras-binding domain as its downstream target, Raf-1, with no sign of decomposition. This study is the first report of an application of organometallic chemistry in protein chemistry.

Carbon↗

PubMed2006

A new protein engineering approach combining chemistry and biology, part I; site-specific incorporation of 4-iodo-L-phenylalanine in vitro by using misacylated suppressor tRNAPhe.

An Escherichia coli suppressor tRNA(Phe) (tRNA(Phe) (CUA)) was misacylated with 4-iodo-L-phenylalanine by using the A294G phenylalanyl-tRNA synthetase mutant (G294-PheRS) from E. coli at a high magnesium-ion concentration. The preacylated tRNA was added to an E. coli cell-free system and a Ras protein that contained the 4-iodo-L-phenylalanine residue at a specific target position was synthesized. Site-specific incorporation of 4-iodo-L-phenylalanine was confirmed by using LC-MS/MS. Free tRNA(Phe) (CUA) was not aminoacylated by aminoacyl-tRNA synthetases (aaRSs) present in the E. coli cell-free system. Our approach will find wide application in protein engineering since an aryl iodide tag on proteins can be used for site-specific functionalization of proteins.

Acylation↗

PubMed2006

In vitro synergistic effects of double combinations of beta-lactams and azithromycin against clinical isolates of Neisseria gonorrhoeae.

In Japan, Neisseria gonorrhoeae, a sexually transmitted pathogen, has recently shown significant resistance to various antimicrobial agents. In this study, a checkerboard method was utilized to investigate the in vitro activities of cefixime (CFIX), cefteram (CFTM), or amoxicillin (AMPC) in combination with azithromycin (AZM) against 25 clinical isolates of N. gonorrhoeae. Synergy, defined as a fractional inhibitory concentration (FIC) index of less than or equal to 0.50, was observed in 32% of isolates with CFIX+AZM, 12% of isolates with CFTM+AZM, and 4% of isolates with AMPC+AZM. Moreover, partial synergy, defined as an FIC index of greater than 0.50 and less than 1, was observed in 44% of isolates with CFIX+AZM, 68% of isolates with CFTM+AZM, and 52% of isolates with AMPC+AZM. In particular, as a result of the combination of CFIX and AZM, for all isolates, significant reductions were observed in the median CFIX minimum inhibitory concentration (MIC; from 0.25 to 0.008 microg/ml; P < 0.0001) and the median AZM MIC (from 0.12 to 0.03 microg/ml; P < 0.0001). However, antagonism, defined as an FIC index of greater than 1, was observed in only 4% of the isolates with both CFIX+AZM and CFTM+AZM, while it was seen in 12% of the isolates with AMPC+AZM. To our knowledge, this is the first study to demonstrate that the in vitro activity of CFIX against N. gonorrhoeae can be significantly enhanced in combination with AZM.

Amoxicillin↗

PubMed2006

Complete coverage of in situ aortograft by total omental pedicle flap as the most reliable treatment of aortoesophageal fistula.

Aortoesophageal fistula secondary to a thoracic aortic aneurysm is usually a fatal disease with few survivors reported previously. We encountered 2 consecutive patients who were treated successfully with esophagectomy and in situ aorta reconstruction using cryopreserved homograft that was wrapped completely with omental pedicle flap. For the construction of omental flap, the right gastroepiploic artery was resected at the root and all the vessels entering the greater curvature and the transverse colon were resected at the adherent edges. Because the gastroepiploic arcade is totally preserved, large amounts of omental tissue could be obtained, with an excellent blood supply mainly from the left gastroepiploic artery. This type of omental flap is highly mobile, easily transferred to the left hemithorax, and has enough volume to cover the in situ aortic graft completely including anastomosis lines. Thus, our omental coverage appears to be the most reliable method to prevent postoperative graft infection.

Aged↗

PubMed2006

Mutational study on alphaGln90 of Fe-type nitrile hydratase from Rhodococcus sp. N771.

Nitrile hydratase (NHase) from Rhodococcus sp. N771 is a non-heme iron enzyme having post-translationally modified cysteine ligands, alphaCys112-SO2H and alphaCys114-SOH. We replaced alphaGln90, which is conserved in all known NHases and involved in the hydrogen-bond network around the catalytic center, with glutamic acid or asparagine. The kcat of alphaQ90E and alphaQ90N mutants decreased to 24% and 5% that of wild type respectively, but the effect of mutations on Km was not very significant. In both mutants, the alphaCys114-SOH modification appeared to be responsible for the catalysis as in native NHase. We crystallized the nitrosylated alphaQ90N mutant and determined its structure at a resolution of 1.43 A. The structure was basically identical to that of native nitrosylated NHase except for the mutated site and its vicinity. The structural difference between native and alphaQ90N mutant NHases suggested the importance of the hydrogen bond networks between alphaGln90 and the iron center for the catalytic activity.

Crystallography, X-Ray↗

PubMed2005

Analysis of mutations within multiple genes associated with resistance in a clinical isolate of Neisseria gonorrhoeae with reduced ceftriaxone susceptibility that shows a multidrug-resistant phenotype.

A Neisseria gonorrhoeae strain with a reduced susceptibility to ceftriaxone (minimum inhibitory concentration (MIC) = 0.5 microg/mL) was isolated among 398 clinical isolates obtained from 2000-2001 in Fukuoka City, Japan. The N. gonorrhoeae strain was negative for penicillinase production but it showed multidrug resistance against penicillin (MIC = 8 microg/mL), tetracycline (MIC = 4 microg/mL), azithromycin (MIC = 0.5 microg/mL) and ciprofloxacin (MIC = 16 microg/mL). The molecular mechanisms of the multidrug-resistant phenotype in this strain were analysed. Polymerase chain reaction and direct DNA sequencing were performed to identify mutations within the penA, ponA, mtrR, penB, gyrA and parC genes of the gonococcal strain, which thus explain the multidrug-resistant phenotype. The N. gonorrhoeae strain contained a significantly different sequence of the penA gene from that of the ceftriaxone-susceptible strains. Some regions of the transpeptidase domain within this penA gene were closely similar to those found in other Neisseria species such as Neisseria subflava, Neisseria flavescens or Neisseria perflava/sicca. This strain also included a ponA mutation that is associated with high-level resistance to penicillin, mtrR mutations that mediate overexpression of the MtrCDE efflux pump responsible for resistance to hydrophobic agents such as azithromycin, and penB mutations that reduce porin permeability to hydrophilic agents such as tetracycline. Moreover, this strain contained gyrA and parC mutations that confer high-level resistance to ciprofloxacin. These results indicate the emergence of a N. gonorrhoeae strain with reduced susceptibility to ceftriaxone, which also showed a multidrug-resistant phenotype that can be explained by the presence of multiple loci mutations associated with antibiotic resistance.

Amino Acid Sequence↗

PubMed2005

Externalization and recognition by macrophages of large subunit of eukaryotic translation initiation factor 3 in apoptotic cells.

We previously isolated a monoclonal antibody named PH2 that inhibits phosphatidylserine-mediated phagocytosis of apoptotic cells by macrophages. We report here the identification of the cognate antigen. A protein bound by PH2 in Western blotting was identified as the 170-kDa subunit of eukaryotic translation initiation factor 3 (eIF3 p170/eIF3a). When eIF3a was expressed in a culture cell line as a protein fused to green fluorescence protein, the fusion protein was detected at the cell surface only after the induction of apoptosis. The same phenomenon was seen when the localization of endogenous eIF3a was determined using anti-eIF3a antibody, and eIF3a seemed to be partially degraded during apoptosis. Furthermore, bacterially expressed N-terminal half of eIF3a fused to glutathione S-transferase bound to the surface of macrophages and inhibited phagocytosis of apoptotic cells by macrophages when it was added to phagocytosis reactions. These results collectively suggest that eIF3a translocates to the cell surface upon apoptosis, probably after partial degradation, and bridges apoptotic cells and macrophages to enhance phagocytosis.

Animals↗

PubMed2005

Distribution of cerebral cortical lesions in diffuse neurofibrillary tangles with calcification: a clinicopathological study of four autopsy cases showing prominent parietal lobe involvement.

We investigated clinicopathologically four Japanese autopsy cases of diffuse neurofibrillary tangles with calcification (DNTC), which has been believed to be characterized by temporal or temporofrontal circumscribed lobar atrophy, and examined the distribution of their cerebral cortical lesions using hemisphere specimens. The lesions were classified into three categories (slight, moderate, and severe). Severe lesions were present in the temporal lobes and insular gyri of all four cases, consistent with the studies reported to date. In contrast, severe lesions were encountered in the parietal lobe of case 1 and moderate lesions were found in the parietal lobes of cases 2-4. Furthermore, moderate lesions of the precentral gyrus were present in cases 2-4, and moderate lesions of the postcentral gyrus were encountered in all four cases. We postulate that the distribution of cerebral cortical lesions in DNTC is more widespread than previously assumed. Our data also indicate that the unusual clinical signs of DNTC reported by several Japanese researchers, including parietal signs such as apraxia and agnosia, are roughly consistent with the topographic distribution of cerebral cortical lesions in DNTC elucidated in this study.

Brain Diseases↗

PubMed2005

Prospective randomized trial comparing Billroth I and Roux-en-Y procedures after distal gastrectomy for gastric carcinoma.

To determine the clinical efficacy of Roux-en-Y reconstruction (RY) after distal gastrectomy, we compared postoperative outcomes of patients who underwent RY or conventional Billroth I reconstruction (B-I). A total of 50 patients were prospectively randomized to either B-I or RY reconstruction, and complications, postoperative course, and nutritional status were compared. Bile reflux and inflammation in the remnant stomach and lower esophagus were evaluated by postoperative follow-up endoscopy at 6 months. Operative time and blood loss as well as postoperative nutrition did not show significant differences between the two groups. As anticipated, 5 of 24 patients with RY reconstruction developed gastrojejunal stasis in the early postoperative period, which led to a longer postoperative hospital stay as compared with the B-I group (mean +/- S.D; B-I; 19.0 +/- 6.2, RY; 31.8 +/- 21.7 days) (P < 0.05). Endoscopic examination revealed that the frequency of bile reflux (P < 0.01) and degree of inflammation in the remnant stomach (P < 0.05) were less in the RY group than in the B-I group. However, inflammatory findings in the lower esophagus were observed in 7 (27%) of B-I, and 8 (35%) of the RY group, suggesting that late phase esophagitis was not improved in the RY group. Roux-en-Y reconstruction was effective in preventing duodenogastric reflux and resulting gastritis, but it did not prevent esophagitis. Because RY reconstruction induces the frequent complication of Roux-en-Y stasis, causing longer postoperative hospital stay, this method has limited advantages over B-I anastomosis after distal gastrectomy.

Anastomosis, Surgical↗

PubMed2004

Renal disease in Arima syndrome is nephronophthisis as in other Joubert-related Cerebello-oculo-renal syndromes.

Clinicopathological features of the renal disease in Arima syndrome were studied in five autopsy cases. All cases showed insidious development of end-stage renal disease during childhood, preceded by polyuria/polydipsia, anemia, and growth failure. Decreased urinary concentrating ability and excessive sodium loss were the characteristic laboratory findings. Gross examination showed that both kidneys were small and showed multiple cysts of various sizes. The histological examinations revealed chronic sclerosing tubulo-interstitial nephropathy with cystic tubuli predominantly located at cortico-medullary areas. These observations suggest that the renal disease in Arima syndrome is in accordance with nephronophthisis both clinically and pathologically. Contrary to the previous literature which described that Arima syndrome can be distinguished from other Joubert-related cerebello-oculo-renal syndromes by its unique renal disease, i.e., cystic dysplastic kidney (CDK), our study indicates that the phenotype of the renal disease is common among these syndromes as well as abnormalities in other organs, suggesting the underlying similar molecular mechanisms.

Adolescent↗

PubMed2004

Only a small subset of the horizontally transferred chromosomal genes in Escherichia coli are translated into proteins.

Horizontally transferred genes are believed to play a critical role in the divergence of bacterial strains from a common ancestor, but whether all of these genes express functional proteins in the cell remains unknown. Here, we used an integrated LC-based protein identification technology to analyze the proteome of Escherichia coli strain K12 (JM109) and identified 1,480 expressed proteins, which are equivalent to approximately 35% of the total open reading frames predicted in the genome. This subset contained proteins with cellular abundance of several dozens to hundreds of thousands of copies, and included nearly all types of proteins in terms of chemical characteristics, subcellular distribution, and function. Interestingly, the subset also contained 138 of 164 gene products that are currently known to be essential for bacterial viability (84% coverage). However, the subset contained only a very small population (10%) of protein products from genes mapped within K-loops, which are "hot spots" for the integration of foreign DNAs within the K12 genome. On the other hand, these genes in K-loops appeared to be transcribed to RNAs almost as efficiently as the native genes in the bacterial cell as monitored by DNA microarray analysis, raising the possibility that most of the recently acquired foreign genes are inadequate for the translational machinery for the native genes and do not generate functional proteins within the cell.

Bacterial Proteins↗

PubMed2004

Mitochondrial nucleoid and transcription factor A.

Nuclear DNA is tightly packed into nucleosomal structure. In contrast, human mitochondrial DNA (mtDNA) had long been believed to be rather naked because mitochondria lack histone. Mitochondrial transcription factor A (TFAM), a member of a high mobility group (HMG) protein family and a first-identified mitochondrial transcription factor, is essential for maintenance of mitochondrial DNA. Abf2, a yeast counterpart of human TFAM, is abundant enough to cover the whole region of mtDNA and to play a histone-like role in mitochondria. Human TFAM is indeed as abundant as Abf2, suggesting that TFAM also has a histone-like architectural role for maintenance of mtDNA. When human mitochondria are solubilized with non-ionic detergent Nonidet-P40 and then separated into soluble and particulate fractions, most TFAM is recovered from the particulate fraction together with mtDNA, suggesting that human mtDNA forms a nucleoid structure. TFAM is tightly associated with mtDNA as a main component of the nucleoid.

DNA, Mitochondrial↗

PubMed2004

Preoperative grading system for predicting operative conditions in laparoscopic cholecystectomy.

PURPOSE: In a previous retrospective study, we predicted the operative conditions for abdominal wall-lifting laparoscopic cholecystectomy (ALLC), using a new preoperative grading system. We conducted the present study to evaluate the validity of our grading system prospectively, and to improve the operative outcome. METHODS: Ninety-seven patients underwent cholecystectomy between January 2000 and March 2002, and were prospectively examined according to our preoperative grading system. Allotting 0-5 points for nine preoperative factors, the total combined score was defined as the predictive score. The postoperative score was defined by allotting 0-8 points to five operative factors. The ratio of the preoperative score / postoperative score was defined as the skill score. RESULTS: The mean postoperative score was significantly correlated with the predictive score (P < 0.01). The mean operation time and the mean postoperative score differed significantly among surgeons with skill scores higher or less than 1.25 (P < 0.05). They were significantly improved (P < 0.05) by choosing an operator according to the predictive score and skill score. CONCLUSION: Our preoperative grading system using the predictive score is a valid method of predicting the actual operative conditions of ALLC. An adequately skilled operator should be chosen according to the difficulty of each case, to ensure the best possible operative outcome.

Aged↗

PubMed2004

The anticancer natural product pironetin selectively targets Lys352 of alpha-tubulin.

Pironetin is a potent inhibitor of tubulin assembly and arrests cell cycle progression in M phase. Analyses of its structure-activity relationships suggested that pironetin covalently binds tubulin. To determine the binding site of pironetin, we synthesized biotinylated pironetin, which inhibited tubulin assembly both in vitro and in situ. The biotinylated pironetin selectively and covalently bound with tubulin. Partial digestion of biotinylated pironetin-treated tubulin by several proteases revealed that the binding site is the C-terminal portion of alpha-tubulin. By systematic alanine scanning, the pironetin binding site was determined to be Lys352 of alpha-tubulin. Lys352 is located at the entrance of a small pocket of alpha-tubulin, and this pocket faces the beta-tubulin of the next dimer. This is the first compound that covalently binds to the alpha subunit of tubulin and Lys352 of alpha-tubulin and inhibits the interaction of tubulin heterodimers.

Amino Acid Sequence↗