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Biomedical subjects

N Fujii

Publications and source records attributed to N Fujii.

At least 289 records · Page 16Linked to original sources
PubMed1994

Simultaneous racemization and isomerization at specific aspartic acid residues in alpha B-crystallin from the aged human lens.

We provide evidence that the racemization and isomerization of aspartyl(Asp) residues occur simultaneously in the alpha B-crystallin in the lens of aged (mean age: 80 years) and young (age: 11 months) humans. We purified alpha B-crystallin and subjected it to tryptic digestion. The resulting peptides were separated by reverse-phase high-performance chromatography (RP-HPLC) and were characterized by amino-acid composition, sequence analysis and mass spectrometry. Two specific sites, Asp-36 (D/L of Asp: 0.92) and Asp-62(D/L of Asp: 0.57), among 13 Asp/asparginyl (Asn) residues in aged alpha B-crystallin, were found to be highly racemized and isomerized to form beta-Asp residues. The beta-Asp-containing peptides were clearly distinguished from normal Asp-containing (alpha-Asp) peptides by RP-HPLC. The racemization and isomerization of Asp residues in aged alpha B-crystallin may occur via a succinimide intermediate. In young alpha B-crystallin, we observed neither racemization nor isomerization. We also found that Met-68 was oxidized to form Met sulfoxide to a greater extent in aged alpha B-crystallin than in young alpha B-crystallin. We concluded that racemization, isomerization, and oxidation of alpha B-crystallin occur spontaneously in the aging process.

Aged↗
PubMed1994

Krabbe disease: isolation and characterization of a full-length cDNA for human galactocerebrosidase.

Human galactocerebrosidase, the enzyme deficient in Krabbe disease, was purified, through several hydrophobic column steps and gel filtration, 22,650-fold from human lymphocytes. Using information on its N-terminal and internal amino acid sequences, and the polymerase chain reaction method, we cloned a full-length cDNA for the enzyme. The deduced amino acid sequence matched all amino acid sequences determined. The 3780 nucleotide sequence included 2007 nucleotides which encoded a single chain peptide of 669 amino acid residues with a 26 amino acid N-terminal signal peptide and six potential asparagine-linked glycosylation sites. The galactocerebrosidase cDNA detected an about 4 kb mRNA band material in human cultured skin fibroblasts. A nonsense mutation was found at codon 369 (GAA-->TAA) in the coding sequence of cDNA amplified from cultured skin fibroblast mRNA from a patient with typical Krabbe disease.

Amino Acid Sequence↗
PubMed1994

New disulfide bond-forming reactions for peptide and protein synthesis.

1. In order to solve the difficult problems for the synthesis of peptides or proteins containing several disulfide bonds, three new disulfide bond-forming reactions were developed. 2. Thallium(III) trifluoroacetate oxidation. This reagent, a mild oxidant with a soft acid character, cleaved various S-protecting groups of cysteine in trifluoroacetic acid, with spontaneous formation of cystine. Thus, some model peptides containing a disulfide bond were prepared by direct oxidative conversion of the respective S-substituted cysteine peptides. 3. Sulfoxide-directed disulfide bond-forming reaction. The disulfide bond was formed at the sulfur atom of Cys(R)(O) (sulfoxide) intermolecularly as well as intramolecularly following liberation of the SH group from the coreactant, Cys(R'), by a suitable acid. Thus, formation of the disulfide bond was found to be feasible at the position of Cys(R)(O). 4. Silylchloride-sulfoxide procedure. In the presence of diphenylsulfoxide, methyltrichlorosilane in trifluoroacetic acid cleaved various S-protecting groups to form cystine within 10 to 30 min at 4 degrees C. By combination of this new disulfide bond-forming reaction and others, some peptides containing two or three disulfide bonds were synthesized successfully.

Amino Acid Sequence↗
PubMed1994

Cervical spinal cord injury associated with ossification of the posterior longitudinal ligament.

Among 231 patients with cervical injuries treated over 12 years, 15 had cervical spinal cord injury associated with ossification of the posterior longitudinal ligament. All of them were male and most had injuries due to a relatively weak external force. Four of them underwent surgery. There was little difference in improvement of paralysis between the conservatively and the surgically treated group.

Aged↗
PubMed1994

Mechanisms of potentiation in sweating induced by long-term physical training.

To evaluate the mechanism of potentiation of sweating after long-term physical training, we compared sweating function in trained and untrained subjects using the frequency of sweat expulsion (fsw) as an indicator of central sudomotor activity. Nine trained male subjects (trained group) and eight untrained male subjects (untrained group) performed 30-min cycle exercise at 35% maximal oxygen uptake at 25 degrees C ambient temperature and 35% relative humidity. Oesophageal temperature (T(oes)), mean body temperature (Tb), chest sweating rate (msw,chest), forearm sweating rate (msw,forearm), and fsw were measured. The slopes of the msw,chest versus body temperature (T(oes) and Tb) and versus fsw relationships in the trained group were significantly greater than those in the untrained group (both, P < 0.05), while there was no difference between the groups in the slopes of the msw,forearm versus body temperature or versus fsw relationships. Neither the body temperature threshold for initiation of chest or forearm sweating nor the slope of the fsw-Tb relationship differed between groups. We concluded that, during light exercise at moderate ambient temperature, the msw,chest in the subjects who had undergone long-term physical training was greater than that in the untrained subjects while the msw,forearm was not changed. The greater msw,chest in the trained subjects was concluded to be due to an increase of sensitivity of peripheral mechanisms.

Adult↗
PubMed1994

Hypernatremic myopathy.

We describe a 21-year-old man presenting with proximal muscle weakness associated with hypernatremia. His manifestations other than muscle weakness included dry skin, loss of axillary and pubic hair, decreased libido and loss of thirst sensation. His serum sodium level was elevated to 169-171 mEq./l but all other electrolytes were normal. In addition, serum CK was elevated and an EMG study showed myogenic changes. Endocrinological studies revealed hypothalamic hypopituitarism, while MRI revealed a suprasellar mass. A partial correction of hypernatremia led to an immediate recovery of the muscle weakness as well as a normalization of both the serum CK level and EMG findings, suggesting a direct association between the muscle weakness and hypernatremia. The phosphocreatine/inorganic phosphorus (PCr/Pi) ratios in the resting calf muscle, obtained using 31P magnetic resonance spectroscopy (MRS), were very low during the state of muscle weakness, while they returned to nearly normal values after clinical improvement, suggesting that the muscle weakness in hypernatremic state was caused by a depletion of the intramuscular energy stores, probably due to an overworking Na-K pump to correct the intracellular electrolyte imbalance.

Adult↗
PubMed1994

Simultaneous stereoinversion and isomerization at specific aspartic acid residues in alpha A-crystallin from human lens.

We characterized the primary structure of alpha A-crystallin from the lens of the human eye by detailed analyses of the amino acid sequence, mass, and stereoisomers, and found the biologically uncommon D- and beta-aspartic acid (Asp) residues in the protein. The stereoconfiguration of the Asp151 and Asp58 residues in alpha A-crystallin from old subjects (mean age: 80 years) was inverted to the D-isomer, and the residues were simultaneously isomerized to beta-aspartyl residues, which may occur via a succinimide intermediate. This is thought to be the first observation of stereoinversion of amino acids in protein in vivo. It is noteworthy that similar stereoinversion was observed in the same residues of alpha A-crystallin from young subjects (age: 11 months), with simultaneous isomerization, although the extent of isomerization was low compared with that in the aged. The conversion may take place in the early life of the lens and the resulting isomers may accumulate with aging. We also found that terminal serine173 was cleaved in aged alpha A-crystallin. Since the cleavage was not observed in young alpha A-crystallin, it may result from the in vivo aging process. The present findings also make it necessary to revise the amino acid sequence of human alpha A-crystallin presented previously: human alpha A-crystallin is composed of 173 (formerly reported as 172) amino acid residues, and the sequence from residues 153 to 155 is THA (formerly reported as -HT).

Aging↗
PubMed1994

Analysis of the rolC promoter region involved in somatic embryogenesis-related activation in carrot cell cultures.

In cell cultures of carrot (Daucus carota L.), somatic embryogenesis can be induced by transferring cells from a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) to one devoid of 2,4-D. Previous analysis of transgenic carrot cells containing the 5' non-coding sequence of the Ri plasmid rolC and a structural gene for bacterial beta-glucuronidase (uidA) has shown that the chimeric gene is actively expressed after induction of somatic embryogenesis. In this study, we demonstrate that activation of the rolC promoter is dependent on the process of embryo development but not on the duration of the cell culture in 2,4-D-free medium. We also analyzed the cis region of the rolC promoter that is responsible for somatic embryogenesis-related activation (SERA), namely relatively low beta-glucuronidase (GUS) activity in calli and proembryogenic masses (PEM) and high GUS activity in heart- and torpedo-stage embryos. When the -255-bp region of the rolC gene was used, SERA was retained. Internal deletions within this -255-bp region did not alter SERA by the rolC promoter. Furthermore, when a rolC promoter fragment (-848 to -94 bp) was fused to the cauliflower mosaic virus (CaMV) 35S core region (-90 to +6 bp), it conferred relatively low GUS activity in calli and PEM but high GUS activity in heart and torpedo embryos. When -848 to -255-bp or -255- to -94-bp fragments of the rolC promoter were fused to the same CaMV 35S core region, GUS activity patterns were not related to somatic embryogenesis. These results suggest that the combination of several regulatory regions in the rolC promoter may be required for SERA in carrot cell cultures.

Base Sequence↗
PubMed1994

Helicobacter pylori induces inflammation in mouse urinary bladder and pelvis.

Helicobacter pylori was transurethrally inoculated into the mouse urinary tract. The organism established infection and induced inflammation in the urinary bladder and pelvis. During the infection, urinary pH was elevated, probably due to the production of NH3 by bacterial urease. H. pylori was recovered from the urinary bladder, kidney and urine of the infected mice. Histopathologically, severe neutrophil infiltration was observed in the mucosal layer of both organs. H. pylori was detected on the surface of the epithelial cells. These results indicate that low pH and bacterial flora were not essential factors in establishing the mucosal infection with H. pylori. This experimental system is useful to investigate the pathogenicity of H. pylori in mucosal organs.

Animals↗
PubMed1994

Heat shock protein produced by Helicobacter pylori.

The cells of Helicobacter pylori were suspended in the medium containing 35S-methionine. After a heat shock of the cells at 42 C for 5, 10, and 30 min, the production of proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Out of many proteins produced by the cells, only 66 kDa protein production was dramatically increased by heat treatment. The N-terminal amino acid sequence of 66 kDa protein was quite similar to that of 62 kDa and 54 kDa proteins previously suggested as heat shock protein (HSP) of H. pylori based on the reaction with polyclonal and monoclonal antibodies against HSP 60 family proteins produced by other bacteria. Therefore, it was concluded that H. pylori produces the 66 kDa protein as its major heat shock protein which belongs to HSP 60 family.

Amino Acid Sequence↗
PubMed1994

Complement-mediated killing of Borrelia garinii--bactericidal activity of wild deer serum.

The susceptibility of Borrelia garinii to fresh wild deer sera was determined by incubating strain SIKA2 at 10% serum concentration for 1 hr at 37 C in an in vitro bactericidal assay. Each serum showed bactericidal effects at various levels. The effect was dependent on the concentration of antibody to the spirochetes. Complement was essential in the bactericidal assay because the inactivated deer serum showed greatly decreased activity. Our results suggest that B. garinii is sensitive to deer serum, in the presence of antibody and the bactericidal effect is important for preventing Lyme disease in wild sika deer.

Animals↗
PubMed1994

Fimbria-specific immune response in various inbred mice inoculated with Porphyromonas gingivalis 381.

We studied the genetic control of Porphyromonas gingivalis fimbriae response. Inbred mice with different H-2 haplotypes and/or different genetic backgrounds were inoculated with viable P. gingivalis 381 cells and tested for fimbria-specific T cell responses in vivo (delayed-type hypersensitivity). H-2d mice showed a strong footpad response, whereas H-2b mice showed a weak response to fimbriae from P. gingivalis. Similar evidence of genetic control was obtained with an enzyme-linked immunosorbent assay for the detection of IgG antibody in inbred mice (BALB/c, C3H/HeN and C57BL/6). Several immunoglobulin G (IgG) subclass responses were associated with H-2 in these B10 congenic mice. However, quantification of IgG antibody to fimbriae was not controlled by H-2 in B10 congenic mice. The results indicate that, in mice, the responsiveness to fimbriae of P. gingivalis can be controlled by several genes, including the H-2 complex. C3H/HeN mice were inoculated with the fimbriae intravenously, and the expression of surface antigens on spleen T cells was measured in a fluorescent antibody cell sorter. Stimulation by fimbriae resulted in a changed expression of surface antigens on T cells. Thus, the fimbriae can induce T cell activation.

Animals↗
PubMed1994

Experimental Borrelia garinii infection of Japanese quail.

Japanese quail inoculated subcutaneously with Borrelia garinii responded to infection. B. garinii was reisolated mainly from skin and randomly from several organs between 7 and 56 days postinoculation. Skin lesions were occasionally observed in association with spirochete recovery. All birds were positive for antibody at 53 to 56 days postinoculation. These results suggest that the Japanese quail is a model for experimental infection with B. garinii and that birds are reservoirs for Lyme disease spirochetes.

Animals↗
PubMed1994

Fine structure of a virus-encoded helper T-cell epitope expressed on FBL-3 tumor cells.

Antigen peptide fn20 representing Friend murine leukemia virus env122-141 (DEPLTSLTPRCNTAWNRLKL) is recognized by two independent Friend virus-induced, FBL-3 tumor-specific helper T-cell (Th) clones. We isolated more Th clones from mice immunized with fn20 peptide. We examined the fine structure of the peptide required to activate a large group of fn20-specific Th clones. A systematic analysis of peptides of decreasing lengths eliciting Th proliferation defined the minimum core length as 13 amino acids (LTSLTPRCNTAWN). Functional proliferation and competition assays with variant peptides with alanine substitutions permitted the assignment of five peptide residues in two major histocompatibility complex-interacting and three T-cell-receptor-interacting sites. Th clones were different in their reactivities toward peptides of various lengths and the variant peptides.

Amino Acid Sequence↗
PubMed1994

Oligo-2',5'-adenylate synthetase in pulmonary sarcoidosis and idiopathic pulmonary fibrosis.

Oligo-2',5'-adenylate synthetase (2,5AS) is an enzyme induced by all types of interferon (IFN). We measured the levels of 2,5AS activity in peripheral blood mononuclear leukocytes (PBML) and bronchoalveolar lavage fluid (BALF) cells of patients with pulmonary sarcoidosis (SAR), idiopathic pulmonary fibrosis (IPF), and normal controls (NC). In NC, the levels of BALF cell 2,5AS activity were approximately seven times as high as the levels of PBML 2,5AS activity. The measurement 2,5AS activity from isolated cells showed that the levels of 2,5AS activity are independent of cell differential from PBML and BALF cells. The levels of PBML and BALF cell 2,5AS activity in SAR were both significantly high in comparison with those in NC. In patients with IPF, the levels of PBML 2,5AS activity were significantly increased as compared with those in NC, whereas there was no significant difference regarding the levels of BALF cell 2,5AS activity between patients with IPF and NC. These results suggest the following: (1) in patients with SAR, IFN production is enhanced both in the alveolar space and peripheral circulation; (2) in patients with IPF, IFN production is greatly enhanced in the circulation, whereas IFN production is not enhanced in the alveolar space; and (3) IFN may contribute to the pathogenesis of SAR and IPF.

Adult↗
PubMed1994

Rupture of inferior phrenic artery aneurysm. An unusual complication of mesenteric arteritis due to postcoarctectomy syndrome.

Postcoarctectomy syndrome was developed in a boy with Williams syndrome. Angiogram revealed widespread changes of visceral arteries and extravasation of the contrast material from the right inferior phrenic artery. Emergency laparotomy was successful. This unusual complication makes clear that the mesenteric arteritis after coarctectomy can cause aneurysm with a risk of rupture.

Abnormalities, Multiple↗
PubMed1994

Subgingival temperature in rats with natural gingivitis.

Elevated temperature, normally a characteristic of inflammation, is a potential indicator of periodontal disease. The present investigation examined the relationship of subgingival temperature and various indicators of gingivitis in rats with spontaneous gingivitis (SUS rat) and in control rats (RES rat). In 24 SUS rats with gingivitis, the subgingival temperature rose, but not in 6 SUS and 20 RES rats with healthy gingiva. Subjects with high mean subgingival temperatures had significantly higher mean percentages of Prevotella intermedia and Fusobacterium nucleatum than age-matched RES rats. The results show that dental plaque, including specific bacteria P. intermedia and F. nucleatum, can play some role in causing the elevated temperatures.

Age Factors↗