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Biomedical subjects

T Kitagawa

Publications and source records attributed to T Kitagawa.

At least 739 records · Page 41Linked to original sources
PubMed1978

Enzyme immunoassay of viomycin. New cross-linking reagent for enzyme labelling and a preparation method for antiserum to viomycin.

A new cross-linking reagent of the hetero-bisfunctional type, a N-(maleimidobenzoyloxy)-succinimide (MBS) was prepared and used for enzyme labelling of viomycin under mild aqueous conditions by a two-step process. In the first step a maleimide residue was selectively introduced onto the N1-amino group of viomycin with a limited amount of MBS. The second step consisted of thioether formation between the maleimide residue and free thiol groups of beta-D-galactosidase. An antiserum to viomycin was raised in rabbit by immunization with a viomycin-BSA conjugate. The conjugate was prepared by protecting N6-amino group of viomycin with an acetyl group and succinylating the N1-amino group, activating the carboxyl group by a mixed anhydride method and coupling it with the amino groups of bovine serum albumin (BSA). The specificity of the antiserum was proved by an enzyme immunoassay based on the competition between viomycin and its enzyme conjugate toward diluted solutions of the antiserum. By use of the viomycin-enzyme conjugate and the antiserum to viomycin, enzyme immunoassay of viomycin was successfully performed by the competitive binding procedure with the double-antibody method, and 0.1 to 4 ng of the antibiotic could be detected.

Galactosidases↗
PubMed1978

A new method for preparation of an antiserum to penicillin and its application for novel enzyme immunoassay of penicillin.

A new method for the preparation of ampicillin-BSA conjugate by a three step procedure was developed. The first step is the introduction of a maleimide residue to ampicillin by a cross-linking reagent, MBS. The second step is reductive cleavage of disulfide bonds in BSA. The third step is thioether formation between the introduced maleimide residues and the reduced thiol groups. The obtained ampicillin-BSA conjugated raised an anti-ampicillin serum in rabbits. A new reagent, MPGS, was used for enzyme labelling of ampicillin to avoid immunological cross reaction. Using the enzyme labelled ampicillin and anti-ampicillin serum, enzyme immunoassay of ampicillin was successful in detecting 4 ng to 1 mug. Cross reactivities of anti-ampicillin to ampicillin analogs were studied by the enzyme immunoassay to find that the antiserum is specific to penicillin especially to ampicillin but hardly reacts with cephalosporins or the penicilloic acid derivative of ampicillin.

Ampicillin↗
PubMed1978

Resonance Raman spectra of riboflavin and its derivatives in the bound state with egg riboflavin binding proteins.

The resonance Raman spectra of riboflavin (RF) and its derivatives, including 3-deuterated (3-D RF), 3-methyl (3-CH3 RF), 3-carboxymethyl (3-CH2COOH RF), and 7,8-dichlororiboflavins (7,8-Cl RF), in H2O and D2O were observed in the 700-1700 cm-1 region. The fluorescence problem of riboflavin was overcome by complex formation of riboflavin with riboflavin binding proteins. The observed frequencies of Raman lines of RF are in good agreement with those of glucose oxidase obtained by Spiro et al. by the resonance CARS method, although the present spectral range is extended to much lower frequency with a higher signal-to-noise ratio than that for glucose oxidase. The observed Raman lines were assigned to the individual ring modes of isoalloxazine on the basis of the Raman spectra of appropriate model compounds such as uracil, pyrazine, and o-xylene. The 1253 cm-1 line of RF was shifted to ca. 1300 cm-1 for 3-D RF, 3-CH3 RF, and 3-CH2COOH RF, and accordingly can be assigned to the CN stretching mode of Ring III. The 1632 cm-1 line of RF was shifted for 7,8-Cl RF and was assigned to a Ring I mode. No Raman line mainly due to C = O stretching mode was observed in the present resonance Raman spectra.

Carrier Proteins↗
PubMed1978

Enzyme immunoassay of human chorionic gonadotropin employing beta-galactosidase as label.

An enzyme-linked immunoassay was applied to the determination of hCG, a glycoprotein hormone usually assayed by RIA. For this purpose, an enzyme hormone conjugate was prepared by reacting hCG with beta-D-galactosidase (beta-Gal.) of E. coli in the presence of N-(m-maleimidobenzoyloxy) succinimide (MBS) as coupling reagent. The conjugate, after purification by affinity and gel chromatographies, was shown to exhibit sufficient enzyme activity and immunoactivity. The immunoassay of hCG was performed by the double antibody method and, using this assay, 0.4-250 mIU/ml hCG were detectable. This was about 10 times as sensitive as the RIA. Difficulty was experienced when this method was utilized for the determination of hCG in plasma samples from patients. Since the presence of the plasma may have affected this assay method, the following improvements were made: 1) the same volume of hormone-free plasma was added to the standard solutions of hCG, and 2) the volume of plasma sample was 10 microliter. The performance and validity of this assay were comparable to the RIA using [125I]hCG as tracer. The dose-response curves of both assay have the same slope and there was no significant difference between the values (correlation coefficient, Y = 0.96X + 1.53).

Acylation↗
PubMed1978

DNA repair synthesis in rat retinal ganglion cells treated with chemical carcinogens or ultraviolet light in vitro, with special reference to aging and repair level.

A system in which the retinal tissues of noninbred Wistar rats were used in combination with autoradiography was developed for measurement of DNA repair synthesis in ganglion cells of the central nervous system. Retinal tissues in short-term organ culture were treated with various carcinogens plus tritiated thymidine ([methyl-3H]dThd) or were irradiated with UV light and then treated with [methyl-3H]dThd. Preliminary study with retinal tissues from rats at various ages revealed no age-associated changes in the levels of unscheduled DNA synthesis in ganglion cells.

4-Nitroquinoline-1-oxide↗
PubMed1978

Resonance Raman studies on the ligand-iron interactions in hemoproteins and metallo-porphyrins.

The resonance Raman spectra (RRS) were measured for various hemoproteins, as well as for free hemes, and analysed in terms of the interactions between the porphyrin ring and the heme iron and between the axial ligand and the heme iron. To establish vibrational assignments, the RRS of metallo-octae-thylporphyrin (M(OEP)), its 15N substituted and meso-deuterated derivatives were measured. It was found that the Raman line corresponding to the oxidation state marker of hemoproteins involves appreciable displacement of the pyrrolic nitrogen atoms toward metal ion but not that of methine bridges and that the frequencies of methine-bridge CC stretching vibrations are altered by the conjugation interaction between the porphyrin ring and the metal ion present. The prominent Raman lines of Ni(OEP) were assigned on the basis of the normal coordinate analysis. The Raman spectra of heme a bis-imidazole complex (Fe(A)(Im)2) in the presence of cyanide changed when pH was reduced to neutral. It implied formation of cyanhydrin at the peripheral formyl group of heme a. The Fe(A)(Im)2 formed an addition compound with NaHSO3 in a neutral solution but the added NaHSO3 was eliminated when the heme iron was reduced. The Raman spectra of hemoproteins were classified into four groups on the basis of the relative intensities and frequencies of the four key bands. It demonstrated the existence of two kinds of ferrous low-spin states. The difference between the two species was attributed to the difference in the nature of chemical bond between the heme iron and its sixth ligand (L). The Fe-L bonds of ferrous low-spin molecules in Groups A and C are mainly associated with the dz2(Fe)-lone pair and dz(Fe)-II(L) interactions, respectively. This interpretation of the Raman spectra appears consistent with the experimental facts that the ferrous low-spin molecules in Group C photodissociate upon illumination of light at the Soret band and also that the internal stretching frequencies of the sixth ligand are shifted to lower frequency upon coordination to the heme iron. The frequency of the oxidation state marker of reduced cytochrome P-450 (P-450cam) was unusually low in comparison with those of other hemoproteins. It was ascribed to delocalization of electrons from the thiolate anion to the porphyrin II*(ring) orbital through the pi-type molecular orbital (Eq.3). The RRS of reduced P-450cam.metyrapone adduct was quite close to that of reduced cytochrome b5. In conclusion, the Raman frequencies of hemoproteins may depend primarily upon the amount of electrons delocalized to the II* orbital of the porphyrin ring. The coordination of lone-pair electrons of the axial ligand to the iron dz2 orbital yields bond energy but scarcely affects the Raman frequencies. If the pi orbital of the axial ligand interacts with the iron dpi orbital, it perturbs the delocalization of the dpi(Fe) electrons to the II*(ring) orbital of porphyrin ring and thus affects the Raman frequencies.

Chemical Phenomena↗
PubMed1978

[Hypertyrosinemia].

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Amino Acid Metabolism, Inborn Errors↗
PubMed1978

In utero diagnosis of Gaucher disease.

Beta-Glucosidase activity measured by synthetic substrate at pH 4.6 was low in the cultured amniotic cells from two pregnant women at risk for juvenile and adult type Gaucher disease. The diagnosis was confirmed by showing a low activity of beta-glucosidase in the skin fibroblasts with a synthetic substrate or in the spleen with a natural substrate, and by ascertaining the presence of Gaucher cells in the fetal tissues. However, considerable activity of beta-glucosidase measured with synthetic substrate was found in the liver of both affected fetuses and in the spleen of one. It is advisable that the determination of beta-glucosidase to confirm prenatal diagnosis of Gaucher disease be done either in the cultured skin fibroblasts or in the spleen, and if in the spleen, with a natural substrate rather than a synthetic one.

Amniocentesis↗
PubMed1977

Resonance Raman study of the pH-dependent and detergent-induced structural alterations in the heme moiety of Rhodospirillum rubrum cytochrome c'.

The resonance Raman spectra and the structures of the heme moiety of Rhodospirillum rubrum cytochrome c' were investigated for its five states characterized by absorption spectra; Types-a and -n of the reduced form and Types-I, -II, and -III of the oxidized form. The frequency of the ligand-sensitive Raman line suggested the coordination of lysine (Nepsilon) at the sixth position of the heme iron of Type-n. The sixth ligand of Type-III was deduced to be either lysine or histidine but would not be methionine. Type-a and Type-II gave the Raman spectra of rather normal high spin type but Type-I was unusual in the sense that the frequencies of the Raman lines associated primarily with methine-bridge CC-stretching vibrations were relatively high in comparison with those of other high spin hemoproteins. Type-I was converted directly to Type-III upon the addition of SDS or 2-propanol but the conversion occurred via Type-II when pH was increased. Structural difference between the high spin hemes of Type-I and Type-II was discussed in detail.

Cytochrome c Group↗