Biomedical subjects

Makoto Takehana

Publications and source records attributed to Makoto Takehana.

7 recordsLinked to original sources

PubMed2006

NADH binding properties of rabbit lens lambda-crystallin.

PURPOSE: The present investigation aims to evaluate the NADH binding ability of lambda-crystallin, a taxon-specific enzyme-crystallin, in the rabbit lens. METHODS: A lambda/betaL1-crystallin fraction was separated from the rabbit lens soluble fraction by gel filtration and the enzyme-crystallin was partially purified by subsequent affinity column chromatography. Analysis of NADH bound to the lambda-crystallin preparation was performed using spectrophotometric and enzymological methods. Binding of added NADH to the enzyme-crystallin preparation was also analyzed using a simple ultrafiltration method, which was theoretically equivalent to equilibrium dialysis, to study additional NADH binding to the protein. RESULTS: The prepared lambda-crystallin samples clearly exhibited an absorption maximum at 340 nm, even though they were thoroughly dialyzed. This was due to the presence of nondialyzable NADH bound tightly to the protein. The bound NADH was removed by charcoal treatment, and extracted by 0.1% SDS or 70 degrees C heat treatment. A dissociation constant (Kd) of less than 5 nM indicated tight binding of NADH. The quantity of bound NADH in the 88% purified 33 kDa enzyme-crystallin was estimated to be 20.5 nmol/mg protein, suggesting a stoichiometry of 0.7 mol of the nucleotide/mol of the 33 kDa protein. Additional looser binding of added NADH to lambda-crystallin was observed in both the lambda/betaL1-crystallin fraction (including the full-length 33 kDa protein: 34%; 25-30 kDa proteins, most of which might be generated by cleavage of the 33 kDa protein: 64%) and the partially purified enzyme-crystallin. It was assumed from the analysis of binding titration that some (about 30%) of the 33 kDa protein and most of the lower molecular weight proteins still possessed the ability to loosely bind NADH. Kd values of their lower affinity binding were determined to be 2 or 6 microM. CONCLUSIONS: From the present study, we conclude that lambda-crystallin plays a sufficiently important role as a NADH binding protein to maintain high levels of this nucleotide in the rabbit lens.

Animals↗

PubMed2004

Isolation of ascorbate free radical reductase from rabbit lens soluble fraction.

Ascorbate free radical (AFR) reductase with diaphorase activity was isolated from the rabbit lens soluble fraction to characterise some molecular properties of the enzyme. The isolation was accomplished using gel filtration (Sephadex G-75 superfine or Sephacryl S-200 HR), affinity chromatography (Affi-Gel Blue), native isoelectric focusing and two-dimensional gel electrophoresis. A major soluble AFR reductase was found at an isoelectric point of 8.4 and a molecular weight of 31 kDa, and a few minor enzymes were also detected in the range of pI 7.0-8.6. An unknown N-terminal partial amino acid sequence was determined in one peptide fragment prepared from the major enzyme fraction. From the sequence analysis, it is discussed that the lens soluble AFR reductase may differ from NADH-cytochrome b5 reductase reported to be involved in the membrane-bound AFR reductase activity of mitochondria, microsomes and plasma membrane.

Amino Acid Sequence↗

PubMed2004

Effects of dorzolamide hydrochloride on ocular tissues.

We studied the intraocular pharmacokinetics of dorzolamide hydrochloride eye drops and the effect of dorzolamide on carbonic anhydrase activity and localization in ocular tissues. Carbonic anhydrase activity was detected in normal ocular tissues. The activity was inhibited in corneal endothelial cells, the ciliary body, lens epithelial cells, or the retina 1 to 8 hours after instillation of dorzolamide eye drops. In lens epithelial cells and the retina, the enzyme activity had not recovered even 10 hours after instillation of the drug. Immunostaining did not reveal any differences between the group administered dorzolamide eye drops and the control group administered a physiologically balanced solution. Time-related changes in dorzolamide concentrations in ocular tissues were measured by high-performance liquid chromatography (HPLC). In the cornea, anterior aqueous, iris, ciliary body and retina, drug concentrations increased 15 minutes after the instillation and peaked within 1 hour. These results suggest that dorzolamide immediately suppresses carbonic anhydrase activity in ocular tissues, and is rapidly distributed among the tissues of the eye when administered as eye drops.

Animals↗

PubMed2002

Type XVI collagen is expressed in factor XIIIa+ monocyte-derived dermal dendrocytes and constitutes a potential substrate for factor XIIIa.

We have previously reported that connective tissue cells in the superficial dermis preferentially express alpha1(XVI) collagen rather than those in the lower dermis. Double immunofluorescence labeling using the antibodies for alpha1(XVI) collagen and factor XIIIa (plasma transglutaminase), which is a marker of dermal dendrocytes, demonstrated that both antibodies reacted with the same cells in the superficial dermis of normal skin as well as the lesional skins of dermal dendrocyte-related disorders, dermatofibroma, and psoriasis. Dermal dendrocytes are considered to be established by a culture of peripheral blood monocytes in the presence of granulocyte macrophage-colony stimulating factor and interleukin-4. Reverse transcription--polymerase chain reaction, metabolic labeling, and immunofluorescence studies demonstrated that treatment of CD14+ peripheral blood monocytes with granulocyte macrophage-colony stimulating factor/interleukin-4 over a period of 8 d resulted in the induction of alpha1(XVI) collagen as well as factor XIIIa. The physiologic significance of colocalization of alpha1(XVI) collagen and factor XIIIa in the tissue and their coordinate induction in CD14+ monocyte-derived dendritic cells in vitro was studied. Considerable incorporation of [3H]putrescine by factor XIIIa into recombinant noncollagenous domain (NC) 11 but not into collagenous domain (COL) 1.NC1 domain of the alpha1(XVI) polypeptide was found. Incubation of recombinant NC11 of alpha1(XVI) polypeptide with factor XIIIa in vitro produced a covalent cross-linking complex on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The results indicate that alpha1(XVI) collagen is constitutively expressed by most dermal dendrocytes in the skin and dendritic cells differentiated from peripheral blood monocytes in vitro. Type XVI collagen is expressed in factor XIIIa+ dermal dendrocytes and may form an intermolecular cross-linking through NC11 domain by the reaction catalyzed by factor XIIIa contributing to the structural integrity of factor XIIIa+ dendritic cell-rich tissues.

Adult↗

PubMed2002

Comparison of Lp82- and m-calpain-mediated proteolysis during cataractogenesis in Shumiya cataract rat (SCR).

PURPOSE: It is well known that m-calpain, a ubiquitous calpain, is involved in cataract formation in rodent lens. Involvement of Lp82, a lens-specific calpain, in the cataract formation is also suggested. However, the exact relationship between Lp82-mediated proteolysis and lens opacification has not yet been established. We therefore compared Lp82- and m-calpain-mediated proteolyses of alphaA-crystallin during cataractogenesis to clarify whether Lp82 is involved in cataract formation. METHODS: In order to analyze the Lp82- and m-calpain-mediated proteolyses, we developed antibodies exclusively specific to the proteolytic products of alphaA-crystallin produced by Lp82 and m-calpain actions, respectively. The proteolytic profiles of alphaA-crystallin by Lp82 and m-calpain during cataractogenesis in SCR lenses were analyzed by Western blotting and immunohistochemical staining. RESULTS: While m-calpain-mediated proteolysis was detected predominantly in cataractous lenses, Lp82-mediated proteolysis was detected not only in cataractous but in normal lenses. The m-calpain-mediated proteolysis was observed in restricted areas developing and destined to develop opacification, i.e., the nuclear and perinuclear regions of lens. On the other hand, Lp82-mediated proteolysis was observed not only in the same regions but also in the cortical region where opacity does not develop. Unlike m-calpain-mediated proteolysis, Lp82-mediated proteolysis was not inhibited by the oral administration of aminoguanidine (AG), which acts to prevent lens opacification. CONCLUSIONS: From these results, it is shown that there is no direct contribution of Lp82-mediated proteolysis to cataract formation in SCR. Rather, Lp82 may function in fiber cell development and/or fiber cell remodeling during lens maturation under physiological conditions, since Lp82-mediated proteolysis occurs in the cortical region of normal lens.

Aging↗

PubMed2002

Heterogeneity of ascorbate free radical reductase in the human lens.

The soluble ascorbate free radical (AFR) reductases in the human lens were separated into many isoforms in the range of pI 5-7 by native isoelectric focusing. In the two-dimensional gel electrophoresis, however, two main proteins with molecular weights of 20-25 kD were commonly identified to each isoform. The observed heterogeneity of the human lens AFR reductase is very similar to those reported for beta- and gamma-crystallins in aged and cataractous human lenses. From these results, it is suggested that some of the isoforms of the lens AFR reductase, especially the more acidic isoforms, may be formed by posttranslational modifications.

Cataract↗

PubMedDate not supplied

lambda-crystallin related to dehydroascorbate reductase in the rabbit lens.

PURPOSE: To evaluate the relationship of lambda-crystallin to reduced nicotinamide adenine dinucleotide (NADH)-dependent dehydroascorbate (DHA) reductase found specifically in the rabbit lens. METHODS: DHA reductase Fractions I-IV were separated from the lambda/betaL1-crystallin fraction of rabbit lens soluble protein by diethylaminoethyl (DEAE)-cellulose ion-exchange column chromatography, and then the enzyme was partially purified from Fraction II by rechromatography on the same ion-exchange column. The isolated DHA reductase fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, native isoelectric focusing and two-dimensional gel electrophoresis. RESULTS: Using Western blot and a probe of antiserum to recombinant lambda-crystallin, the main 33-kDa protein band was strongly stained in all the rabbit lens DHA reductase fractions, and most of the additional protein bands of approximately 25-30 kDa were also detectable. In the partially purified enzyme, the 33-kDa subunit alone was identified as a distinct protein band by SDS-PAGE, and a main basic protein was found at pI 7.6 by native isoelectric focusing. In addition, many bands of more acidic proteins were separated from other enzyme fractions, and protein spots corresponding to the 33 and/or approximately 25-30-kDa subunits were detected in each of the more acidic proteins by two-dimensional gel electrophoresis. CONCLUSION: These results suggest that lambda-crystallin is closely related to the DHA reductase in the rabbit lens. The above heterogeneity of the enzyme-crystallin may arise from posttranslational modifications.

Amino Acid Sequence↗