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Biomedical subjects

Noriko Fujii

Publications and source records attributed to Noriko Fujii.

At least 19 recordsLinked to original sources
PubMed2006

Differential analysis of D-beta-Asp-containing proteins found in normal and infrared irradiated rabbit lens.

Although proteins are generally composed of l-alpha-amino acids, d-beta-aspartic acid (Asp)-containing proteins have been reported in various elderly tissues. Our previous study detected several d-beta-Asp-containing proteins in a rabbit lens derived from epithelial cell line by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for d-beta-Asp-containing proteins. The identity of each spot was subsequently determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Ms-Fit online database searching algorithm. In this study, we discovered novel d-beta-Asp-containing proteins from rabbit lens. The results indicate that beta-crystallin A3, beta-crystallin A4, beta-crystallin B1, beta-crystallin B2, beta-crystallin B3, gamma-crystallin C, gamma-crystallin D, and lambda-crystallin in rabbit lens contain d-beta-Asp residues. Furthermore, the occurrence of d-beta-Asp residues increases with infrared ray (IR) irradiation. Additionally, some d-beta-Asp-containing proteins only appear after IR irradiation. One such protein is the alpha-enolase, which shows homology to tau-crystallin.

Animals↗
PubMed2006

Kinetic study of racemization of aspartyl residues in recombinant human alphaA-crystallin.

Asp58 and Asp151 in human lens alphaA-crystallin invert and isomerize to d-beta-aspartyl residues with age. Here, we report that the racemization rate constants (k) of Asp58 and Asp151 residues in human recombinant alphaA-crystallin at 37 degrees C are 3.72 +/- 0.8 x 10(-4) and 10.7 +/- 0.7 x 10(-4)/day, respectively. The activation energy of racemization of Asp58 and Asp151 in the protein was 27.0 +/- 0.5 kcal/mol and 21.0 +/- 0.5 kcal/mol, respectively. The time required for the D/L ratio of Asp58 and Asp151 to approximate to 1.0 (D/L ratio of Asp = 0.99) at 37 degrees C was estimated as 20.9 +/- 3.7 and 6.80 +/- 0.4 years, respectively. Thus, Asp151 is more susceptible to racemization than Asp58, consistent with data from short model peptides. However, the racemization rates of both Asp58 and Asp151 residues in the protein were twice as rapid as in model peptides. These results indicate that the racemization of Asp residues in alphaA-crystallin may be influenced not only by the primary structure but also by the higher order structure around Asp residues in the protein.

Amino Acid Sequence↗
PubMed2006

Astrobiology and biological chirality.

The emerging discipline of astrobiology could gain valuable support from research dealing with the problems of biological chirality. The most profitable fields of common interest are: (a) living organisms under extraterrestrial conditions, (b) extraterrestrial signatures of life and (c) origin(s) of biological chirality. These areas of complementary and overlapping fields are analysed on the basis of selected references.

Astronomy↗
PubMed2005

Characterization of new D-beta-aspartate-containing proteins in a lens-derived cell line.

Although proteins are generally composed of l-alpha-amino acids, biologically uncommon D-beta-aspartic acid (Asp)-containing proteins have been reported in various tissues from elderly individuals. Our previous study indicated that the N/N1003A cell line, derived from rabbit lens, includes D-beta-Asp-containing proteins of approximately 50 kDa by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for D-beta-Asp-containing proteins. In this study, we identified the D-beta-Asp-containing proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm. The results indicate that one of these 50 kDa proteins is an enolase showing homology with tau-crystallin. Other D-beta-Asp-containing proteins, which we have recently discovered include lamin A/C, cytoplasmic NADP+-dependent isocitrate dehydrogenase, fructose-bisphosphate aldolase A, aldose reductase, L-lactate dehydrogenase A or calponin H2, phosphoglycerate mutase 1, phosphatidylethanolamine-binding protein, alpha-B-crystallin, and peptidyl-prolyl cis-trans isomerase A (PPlase).

Animals↗
PubMed2005

D-amino acid in elderly tissues.

All living organisms on earth are composed of L-amino acids and D-sugars. Therefore the presence and function of D-amino acids in living organisms have not been studied. Recently, however, D-aspartic acid (D-Asp) which is a D-amino acid, has been detected in various proteins from human tissue samples such as eye lens, brain, skin, bone, teeth, aorta from elderly individuals. It has been explained that the presence of D-Asp is the result of racemization of Asp residues in the protein during the life span, inasmuch as the proteins in these tissues are metabolically inert. The Asp-151 and Asp-58 residues in alpha A-crystallin from elderly human lens are especially stereochemically labile and the D/L ratios of these residues were greater than 1.0. A D/L ratio greater than 1.0 is not defined as racemization, but as the inversion of configuration. This was the first observation that inversion occurred in the configuration of amino acids in vivo during the natural aging process. In this review, we summarize the D-Asp in the various tissues reported by many researchers and describe the mechanism of D-Asp formation in protein. We suggest that a chiral reaction field exists in the native higher order structure of protein which induces the inversion of L-Asp to D-Asp residue.

Aged↗
PubMed2005

[A woman complicated with immune thrombocytopenic purpura, subclinical Graves disease and peripheral neuropathy 5 years after allogeneic bone marrow transplantation].

A 21-year-old woman developed immune thrombocytopenia (ITP), subclinical Graves disease and peripheral neuropathy without typical chronic graft-versus-host disease (GVHD) 5 years following an allogeneic bone marrow transplantation from an HLA-identical sibling. She received high-dose intravenous immunoglobulin (IVIG) and prednisolone (PSL), which resulted in transient recovery of platelet numbers and muscle weakness. A combination of cyclosporine and PSL induced a durable response against not only the thrombocytopenia but also her high levels of thyroid stimulating antibody (TSAb), muscle weakness and sensory abnormality. The level of thyroglobulin in the donor, who had not developed Graves disease, was also elevated, indicating that late onset-subclinical Graves disease was caused by donor lymphocytes that were autoreactive to the thyroid glands.

Adult↗
PubMed2004

The damaging effect of UV-C irradiation on lens alpha-crystallin.

PURPOSE: To evaluate the effect of UV-C irradiation on the structural properties of alpha-crystallin and its chaperone activity. METHODS: alpha- and betaL-crystallins were isolated from bovine lenses using gel chromatography. The purified alpha-crystallin was subjected to UV-C irradiation (254 nm; 1, 2, 5, 10, 20, 50 J/cm2). We measured the tryptophan fluorescence, circular dichroism (CD) spectroscopy in the far UV, and the chaperone activity of both irradiated and non-irradiated alpha-crystallin. RESULTS: The tryptophan fluorescence of alpha-crystallin decreased, whereas the N-formylkynurenine fluorescence increased markedly with increasing doses of UV-C irradiation. Both the oxidation of Met1 and the racemization of Asp151 of alphaA-crystallin increased at a dose of 1-2 J/cm2 and then gradually decreased. The CD spectrum showed that the secondary structure of alpha-crystallin altered with increasing radiation dose, and almost all of the beta-sheet structure was lost at doses above 50 J/cm2. The chaperone activity of alpha-crystallin irradiated with doses under 5 J/cm2 remained intact. However, it was reduced to only 40% after irradiation at 10 J/cm2. CONCLUSIONS: Our study suggests that photo-oxidation of tryptophan residues in alpha-crystallin may be one of the events that affects the three-dimensional packing array and chaperone activity of this lens protein.

Animals↗
PubMed2004

Homochirality and life.

Before the emergence of life, left-handed amino acids (L-enantiomers) were selected and right-handed amino acids (D-enantiomers) were eliminated on the primal earth. Nevertheless, with the progress of analytical methods, D-amino acids have recently been found in higher order living organisms in the form of free amino acids, peptides, and proteins. Free D-amino acids have numerous physiological functions. D-amino acids containing animal peptides are well known as opioid peptides. D-amino acids in protein are related to aging. In this review, we describe the D-amino acids that are present and function as D-amino acid biosystems in our bodies.

Aging↗
PubMed2004

Immunohistochemical study of chronological and photo-induced aging skins using the antibody raised against D-aspartyl residue-containing peptide.

BACKGROUND: Biologically uncommon D-aspartyl residues have been reported in the elderly tissues such as tooth, eye lens, aorta, and brain. We have previously prepared the antibody against D-aspartyl residue-containing peptide and found that it reacted with elastotic material of actinic elastosis. METHODS: Immunoreactivity of the normal skins obtained from sun-exposed and sun-protected skins of varied ages with this antibody was studied. RESULTS: In the sun-exposed skins, the antibody showed negative reaction with the skin specimens of young donors, whereas it reacted with elastotic materials of actinic elastosis of the elderly. In the sun-protected skins, the antibody recognized elastic fiber-like structures and inner layer of vessels found from the mid to lower dermis of old donors but showed no positive reaction to skin specimens of young donors. CONCLUSIONS: The results suggest that the antibody is a potent marker for chronological and ultraviolet (UV)-induced skin aging. Unusual eosinophilic bodies seen in the superficial dermis in the sun-exposed area of the elderly skins were also immunoreactive with the antibody, suggesting that the eosinophilic bodies resulted from UV-induced skin damage.

Adolescent↗
PubMed2004

Non-T-cell-depleted HLA-haploidentical hematopoietic stem cell transplantation from a family donor based on fetomaternal microchimerism in pediatric hematologic malignancies.

Based on recent fetomaternal microchimerism/tolerance theory, two children with acute lymphoblastic leukemia underwent non-T-cell-depleted hematopoietic stem cell transplants (SCT) from haploidentical HLA 2 loci-mismatched family donors (one from a sibling, one from the mother). Engraftment was achieved in two patients. In two recipients, acute graft-versus-host disease was limited to grade II or less, and no chronic graft-versus-host disease developed. Both of these patients have maintained complete remission for more than 8 months post-SCT. Non-T-cell-depleted SCT from haploidentical HLA 2 loci-mismatched family donors seems feasible if microchimerism is detectable.

Child↗
PubMed2003

Characterization of alphaA-crystallin from high molecular weight aggregates in the normal human lens.

PURPOSE: Lens alpha-crystallins, composed of two subunits of alphaA- and alphaB-crystallin, form low molecular weight (LMW) water soluble aggregates with an average molecular mass of approximately 800 kDa. In the intact lens, some of the alpha-crystallins are associated with even larger high-molecular-weight (HMW) aggregates which are thought to be precursors of components found in the water insoluble fraction. Although the mechanism of HMW aggregation and insolubilization are not known, the process is considered to be related to cataract formation. The purpose of the present study is to compare alphaA-crystallins from HMW and LMW forms in order to help understand the mechanism of insolubilization. METHODS: HMW and LMW alphaA-crystallins were isolated from lenses of 50 year old and 2 year old human donors and compared with respect to chaperone activity and fluorescence. We also analyzed isomerization and racemization of Asp-58 and Asp-151 residues in alphaA-crystallin from HMW and LMW fractions. RESULTS: The chaperone activity of HMW alphaA-crystallin was lower than that of LMW alphaA-crystallin. Fluorescence spectra indicated that HMW alphaA-crystallin was more hydrophobic than that of LMW. Isomerization of normal alpha-linkage to abnormal beta-linkage at both Asp-58 and Asp-151 residues markedly increased in HMW alphaA-crystallin compared with that of LMW alphaA-crystallin. The D/L ratio of beta-Asp-58 of either HMW or LMW forms were higher than 1.0, showing that inversion occurred in this site. In addition, the D/L ratio of the Asp-151 residue from HMW alphaA-crystallin was significantly lower than that of the LMW form. CONCLUSIONS: These results were qualitatively the same as those previously found in alphaA-crystallin from total proteins of cataractous versus normal lenses, suggesting that changes in the native structure of alphaA-crystallin associated with the HMW fraction of normal lenses reflect the same changes that occur to a greater degree in total proteins of human cataractous lens.

Amino Acids↗
PubMed2003

Immunological detection of D-beta-aspartate-containing protein in lens-derived cell lines.

PURPOSE: Although the presence of biologically uncommon D-beta-aspartate (D-beta-Asp) in lens protein is thought to be related to aging, we recently found this isomer in lens alphaA-crystallin from human newborns. The objective of this study was to examine whether D-beta-Asp occurs in protein from lens-derived cell lines. METHODS: We examined the expression of D-beta-Asp-containing protein in the lens-derived cell lines alphaTN4-1 and N/N1003A, by western blot and immunoprecipitation analysis using a polyclonal antibody against Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr (peptide 3R), which corresponds to three repeats of positions 149-153 in human alphaA-crystallin. The anti-peptide 3R antibody, prepared in a previous study, is a useful tool for investigating D-beta-Asp-containing peptides. RESULTS: Western immunoblot and immunoprecipitation analysis showed that a 50 kDa protein in N/N1003A cells was strongly immunoreactive with the anti-peptide 3R antibody. Antibodies against alphaA- and alphaB-crystallin also stained this protein. On the other hand, the alphaTN4-1 cell line only expressed proteins of about 20 kDa, which also reacted to antibodies against alphaA-crystallin and alphaB-crystallin. CONCLUSIONS: The results indicate that the N/N1003A cell line expressed a 50 kDa D-beta-Asp-containing protein, which may share a common amino acid sequence with alphaA- and alphaB-crystallin.

Animals↗
PubMed2003

Quantification of the isomerization of Asp residue in recombinant human alpha A-crystallin by reversed-phase HPLC.

A method for determining the isomerization of Asp residues in proteins is described and demonstrated by quantifying the isomerization of Asp(151) in recombinant human alphaA-crystallin. First, four types of dodecapeptide fragment ((146)IQTGLD(151)ATHAER(157)) in which the Asp residue was either L-Asp, D-Asp, L-isoAsp or D-isoAsp were synthesized, and RP-HPLC conditions were established for their separation. Next, the Asp(151)-containing peptide fragments isolated from the tryptic hydrolysate of recombinant alphaA-crystallin were analyzed under these conditions. New peaks, the retention times of which were the same as those of peptides containing D-Asp, L-isoAsp and D-isoAsp, were generated when alphaA-crystallin was incubated for 140 days at 37 degrees C. An amino acid composition, amino acid sequence, and enantiomeric analysis revealed that two peaks with retention times identical to those of peptides containing L-isoAsp and D-isoAsp represented dodecapeptide fragments containing L-isoAsp(151) and D-isoAsp(151), respectively. RP-HPLC analysis under other condition suggested that the peak with retention time identical to that of peptide containing D-Asp represented dodecapeptide fragments containing D-Asp(151). The present method does not require acid hydrolysis, which generates further isomerization products as artifacts, and thus make possible the sensitive quantification of each type of Asp isomer individually at a specific site in a protein. In our analysis of the Asp(151) residue in human alphaA-crystallin, the degree of isomerization from L-Asp to D-Asp can be determined to a level as low as 0.3%.

Aspartic Acid↗
PubMed2002

The presence of D-beta-aspartic acid-containing peptides in elastic fibers of sun-damaged skin: a potent marker for ultraviolet-induced skin aging.

Biologically uncommon d-aspartyl residues have been reported in proteins of various elderly tissues. We prepared a polyclonal antibody against d-beta-Asp-containing peptide and examined its immunoreactivity in the skin. The antibody recognized integrated or disintegrated elastic fibers in the sun-exposed skin but not in the sun-protected skin of the elderly donors. Western blot analysis of the proteins isolated from sun-damaged skin demonstrated that the 50 kDa protein was immunoreactive with both antibodies for d-beta-Asp-containing peptide and elastin. Ultraviolet (UV) irradiation on normal skin caused the appearance of d-beta-Asp-containing peptide-immunoreactive fibers in the dermis. These results suggest that UV irradiation is closely related to the formation of d-beta-Asp in the elastic fibers of skin. We propose that the antibody could be a useful indicator for sun damage of the skin.

Aged↗
PubMed2002

Isolation of cathepsin B inhibitory peptides, Cabin-A1 and -A2, from a tryptic and chymotryptic hydrolysate of human serum albumin.

Two novel peptides that inhibit cathepsin B were isolated from a tryptic and chymotryptic hydrolysate of human serum albumin, and designated as Cabin-A1 and -A2. Cabin-A1 and -A2 were purified by reversed-phase HPLC and identified as Ser-Leu-His-Thr-Leu-Phe and Phe-Gln-Asn-Ala-Leu, respectively. These peptides correspond to f(65-70) and f(403-407) of human serum albumin. Human albutensin A (Ala-Phe-Lys-Ala-Trp-Ala-Val-Ala-Arg), which corresponds to f(210-218), was also isolated as a potent cathepsin B inhibitor. Synthetic Cabin-A1, -A2, and human albutensin A showed dose-dependent inhibition of cathepsin B, with K(i) values of 2.4, 290, and 3.8 microM, respectively.

Cathepsin B↗
PubMed2002

D-amino acids in living higher organisms.

The homochirality of biological amino acids (L-amino acids) and of the RNA/DNA backbone (D-ribose) might have become established before the origin of life. It has been considered that D-amino acids and L-sugars were eliminated on the primitive Earth. Therefore, the presence and function of D-amino acids in living organisms have not been studied except for D-amino acids in the cell walls of microorganisms. However, D-amino acids were recently found in various living higher organisms in the form of free amino acids, peptides, and proteins. Free D-aspartate and D-serine are present and may have important physiological functions in mammals. D-amino acids in peptides are well known as opioid peptides and neuropeptides. In protein, D-aspartate residues increase during aging. This review deals with recent advances in the study of D-amino acids in higher organisms.

Amino Acid Sequence↗
PubMed2002

Successful transplantation of haploidentical CD34+ selected bone marrow cells for an infantile case of severe combined immunodeficiency with aspergillus pneumonia.

A 5-month-old boy with X-linked severe combined immunodeficiency (SCID) and aspergillus pneumonia was successfully transplanted. Before and during transplantation, the patient received O2 administration, systemic amphotericin B, and itraconazole. The transplant was performed with a conditioning regimen of busulfan/cyclophosphamide and 2.9 x 10(6)/kg of CD34+ selected bone marrow cells from his HLA haploidentical mother. Acute grade II graft-versus-host disease (GvHD) was well controlled. Neutrophil counts reached >0.5 x 10(9)/L by day 15 and platelet counts reached > 50 x 10(9)/L by day 48. The T-cell subset (counts) in peripheral blood increased to 42.2% (0.31 x 10(9)/L) by day 46. The pneumonia improved by day 54. The patient has been doing well with limited chronic GvHD of the gut with a follow-up of longer than 40 months after BMT. Conquest of aspergillus pneumonia in SCID infants could be achieved by CD34+ bone marrow cell transplantation together with appropriate anti-fungal treatment.

Antigens, CD34↗