Bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO) was used to examine the peroxyoxalate chemiluminescence (CL) reaction for the detection of fluorescent compounds. Some fluorescent compounds (perylene, eosine, rhodamine, Rose Bengal, fluorescein and umbelliferone) gave higher CL intensities as the proportion of water in the reaction medium increased to ca. 40%, whereas dansylalanine, 8-anilinonaphthalene-1-sulphonic acid, 7-nitrobenzo-2-oxa-1,3-diazol-4-ylproline and dihydronicotinamide adenine dinucleotide gave opposite results. The effects of temperature and time on the post-column reaction in reversed-phase high-performance liquid chromatography (HPLC) were investigated. Under the optimal conditions, the detection limit for dansylamino acids was at the sub-femtomole level. The advantage of using TDPO in HPLC is its stability in the presence of hydrogen peroxide [ca. 10% loss of activity per 8 h vs. 60% per 8 h for bis(2,4,6-trichlorophenyl) oxalate].

Amino Acids↗

PubMed1987

[VAB-3 chemotherapy of endodermal sinus tumor of the ovary].

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Adult↗

PubMed1987

HPLC electrochemical fluorometric detection of amino acids including tryptophan using 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole.

It has been shown that by electrochemical oxidation 7-nitrobenzo-2-oxa-1,3-diazole-tryptophan (NBD-T) is converted to fluorophores having the same emission and excitation spectra as those for other NBD-amino acids. NBD-dioxindolylalanine was tentatively assumed to be a main electrochemical oxidation product of NBD-tryptophan. A coulochemical cell placed between an analytical column and a fluorometer showed no detrimental effect on the separation of NBD-amino acids by reversed phase HPLC. Highly sensitive fluorescence detection was achieved for amino and imino acids at 10-100 fmol levels. The detection limit for tryptophan was 50 fmol.

4-Chloro-7-nitrobenzofurazan↗

PubMed1987

Micro-scale sequence analysis from the N-terminus of peptides using the fluorogenic Edman reagent 4-N,N-dimethylamino-1-naphthyl isothiocyanate.

The fluorogenic Edman reagent 4-N,N-dimethylamino-1-naphthyl isothiocyanate (DNTC) was reacted with amino acids and peptides, cyclized by acid and the liberated 4-N,N-dimethylamino-1-naphthyl thiohydantoin (DNTH) amino acids were then separated and detected by HPLC. The fluorescence intensities of DNTH-amino acids except DNTH-proline and -serine were dramatically increased in the alkaline solution and organic solvent. Thus, the postcolumn reaction with alkaline acetonitrile solution was adopted in HPLC. The polar and aromatic amino acids afforded two DNTH-amino acids on derivatization with DNTC and cyclization with acids. These were suggested to be stereoisomers of DNTH-amino acids. The sequence analysis of 0.5 nmol Leu-enkephalin was achieved by the double coupling method with DNTC and phenyl isothiocyanate followed by the proposed HPLC system.

1-Naphthylisothiocyanate↗

PubMed1987

The calcium-buffering phase of bone mineral: some clues to its form and formation.

Devitalized, neonatal, rat calvariae incubated in a buffered solution release Ca2+ and Pi during the first day into the medium until levels reach 1.2 mM and 2.6 mM, respectively. Thereafter levels gradually decrease to stabilize by the fifth day at 0.5 mM and 2 mM. Comparison of these solubility changes with those of known calcium phosphate salts suggests that calvarial solubility is controlled by a mixture of impure, soluble, apatite precursors, which at cell death transform to an impure apatite. Increasing concentrations of Mg2+ increased the rate of Ca2+ release and, at 3 mM, stabilized Ca2+ by the fifth day at 0.9 mM. Decreasing the pH from 7.4 to 7.2 produced similar solubility changes as did the addition of citrate at 0.3 mM, phosphocitrate at 0.3 mM, ATP at 0.1 mM, and the bisphosphonate, HEBP, at 0.1 mM. Osteocalcin did not increase calvarial solubility but was able to slow the rate of calcium phosphate phase transition if present at the time of in vitro crystal formation. Phosphocitrate and HEBP were also more effective when present during in vitro crystal formation. HEBP was most effective when present during biological crystal formation, as shown by the increased solubility of devitalized calvariae removed after in vivo administration of HEBP. In vivo manipulations of osteoclast activity produced changes in plasma calcium which correlated with the solubility of the corresponding calvariae after removal and devitalization. Low milk intake increased calvarial solubility. Increasing doses of 1,25(OH)2D3 increased plasma calcium and calvarial solubility, both of which were reversed by injection of acetazolamide. It was concluded from this survey of devitalized bone solubility that calcium exchange between bone and body fluids can buffer calcium homeostasis in the young rat. The exchange is passive. The active components appear to be osteoblastic formation of soluble apatite precursors and their stabilizers and, in reverse, osteoclastic transformation of apatite to precursors by H2CO3 secretion.

Acetazolamide↗

PubMed1987

Immunohistochemical demonstration of metallothionein in the rat prostate.

Metallothionein in the rat prostate was demonstrated by using an indirect immunoperoxidase technique. Strong staining was found mainly in the supranuclear area of the cytoplasm of the glandular epithelium in the dorsal and the lateral glands. Diffuse cytoplasmic staining and nuclear staining were found with less frequency. Secretory granules were found in the lumen with various intensities of staining. Most of the epithelial cells in the ventral lobes and the coagulating glands were negatively stained. Immunoreactive cells were found sporadically. Two types of immunoreactive cells, round cells and spindle-shaped cells, were scattered in the stroma. Vascular endothelial cells, red blood cells, and smooth muscle cells were negatively stained. These results suggest the synthesis and secretion of metallothionein in the rat prostate.

Animals↗

PubMed1987

Structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of Newcastle disease virus.

The nucleotide sequence of the mRNA encoding the fusion (F0) protein of a virulent strain of Newcastle disease virus was determined. A single open reading frame in the sequence encodes a protein of 553 amino acids with a calculated molecular weight of 59058. The amino acid sequence predicted several structural features involving the fusion-inducing hydrophobic stretch (residues 117-142) and the cleavage-activation site (residues 112-116) to generate the disulfide-linked F1 and F2 subunits. The cleavage-activation site as well as a part of the fusion-inducing sequence were compared among a series of virulent and avirulent strains by the chain-termination method using a synthetic oligonucleotide primer. It was found that without exception, the cleavage-activation site of virulent strains consisted of two dibasic residues with an intervening glutamine, Arg-Arg-Gln-Arg-Arg, whereas the corresponding region of avirulent strains was made of a sequence with single basic residues scattered among uncharged residues, Gly-LysArg-Gln-GlySer-Arg. On the basis of these observations and the previous results showing a strict correlation between the pathogenicity and the cleavability of the fusion protein of NDV (Y. Nagai, H-D. Klenk, and R. Rott, Virology, 72, 494-508, 1976), we propose the importance of the dibasic residues for efficient proteolytic activation of the fusion protein and for the pantropic property of NDV. Some strains were found to have Leu-Ile-Gly as the N-terminus of F1, whereas others contained Phe-Ile-Gly, indicating that Phe-X-Gly is not always conserved at F1 N-terminus of paramyxovirus.

Amino Acid Sequence↗

PubMed1987

Fluorometric assay of thiols with fluorobenzoxadiazoles.

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Amino Acids↗

PubMed1987

Studies of the slow releasing of testosterone from radiation-polymerized testicular prostheses implanted subcutaneously in the back of castrated rabbits.

A controlled release testicular prosthesis containing testosterone, which was previously dissolved in 2-hydroxyethyl methacrylate (HEMA) at a temperature of 80 degrees C, was prepared by radiation-induced polymerization in the supercooled state at a low temperature. The daily dose of testosterone released in vitro from the poly(HEMA) testicular prosthesis was kept constant at a rate of 5.5 +/- 1.5 mg/d throughout an experimental period of 900 d. In the in vivo experiments, the poly(HEMA) testicular prosthesis was implanted subcutaneously in the back of castrated rabbits over a maximum period of 11 mnth. The cumulative amounts of testosterone released in vitro and in vivo from the poly(HEMA) testicular prosthesis for a period of 11 mnth were found to be 2.1 g (30.0 wt% of initial drug) and 0.9 g (12.8 wt% of initial drug), respectively. The serum testosterone level in castrated rabbits with a poly(HEMA) testicular prosthesis rapidly decreased for periods up to 2 mnth (after increasing during the first 2 wk), then showed a moderate decrease for a few months, and finally held constant at a level of 10 ng/ml throughout the experimental period. It was concluded that a slight amount of testosterone is continuously released in vivo from the radiation-polymerized poly(HEMA) testicular prosthesis over a long period analogous with that in vitro.

Animals↗

PubMed1987

High frequency of loss of human kappa light chain expression in mouse-human heterohybridomas.

The expression of human immunoglobulin (Ig) chains in human-mouse heterohybridomas was investigated on the days after cell fusion. Heterohybridomas were made by fusion of mediastinal lymphocytes from lung carcinoma patients and murine (BALB/c) myeloma P3U1. Most (more than 76%) of heterohybridomas easily lost the light chain rather than the heavy chain expression in the early stages after cell fusion. Frequency of loss of kappa-chain expression was predominant, whereas the expression of heavy chain and lambda-chain was rather stable in comparison with that of kappa-chain. These were also confirmed at the clonal level, demonstrating that only lambda-chain but not kappa-chain producers were successfully enriched. In fact some heterohybridomas producing human Ig of lambda-chain type established were found to be quite stable for more than 2 years without any recloning procedures.

Animals↗

PubMed1987

The binding of hemoglobin to red cell membrane lowers its oxygen affinity.

The mode of interaction of human hemoglobin (Hb) with the red cell membrane was investigated with special reference to the effect on oxygen binding properties and Hb-membrane binding constants. Compared to free native Hb, the membrane-bound native Hb showed a strikingly lowered oxygen affinity and smaller response to organic phosphates such as 2,3-diphosphoglycerate and inositol hexaphosphate. Similar effects of membrane binding were also observed for intermediately cooperative Hbs such as N-ethylmaleimide-treated Hb (NES-Hb) and iodoacetamide-treated Hb (AA-Hb), but very small effects were observed for non-cooperative Hb, i.e., carboxypeptidase A-treated Hb (des-His-Tyr Hb). The magnitude of the affinity lowering was in the order: NES-Hb greater than native Hb greater than AA-Hb much greater than des-His-Tyr Hb. In the presence of inositol hexaphosphate, the three chemically modified Hbs showed an increased oxygen affinity when bound to the red cell membrane, probably due to partial replacement of bound inositol hexaphosphate by membrane. The binding to membrane caused a slight decrease in cooperativity for native Hb, but no distinct change in cooperativity was observed for the three modified Hbs. These results imply: a) the red cell membrane binds to deoxyHb more strongly than to oxyHb; b) the difference in membrane binding affinity between oxyHb and deoxyHb is closely related to the quaternary structure change in the Hb molecule occurring upon oxygenation. The higher affinity of the membrane for deoxyHb than for oxyHb apparently disagrees with the conclusion drawn by earlier investigators. However, the present binding experiments by means of ultrafiltration proved that the red cell membrane actually binds to deoxyHb much more strongly than to oxyHb, validating the present conclusion based on oxygenation experiments. Our results are consistent with those obtained recently by other investigators using a synthetic peptide or the cytoplasmic fragment of red cell membrane band 3.

Carboxypeptidases↗

PubMed1987

Transverse polarization transfer DNN(0 degrees) measurements for the (p,n) reaction on 58Ni and 90Zr at Ep

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Journal Article↗

PubMed1987

Application and limitations of differential hybridization in the isolation of T cell-specific cDNA clones.

We investigated the limitations and effectiveness of differential hybridization in the cloning of T cell-specific cDNA (complementary DNA) molecular clones. By using the technique with T cell and B cell cDNA probes, together with Northern blot analysis, we successfully isolated cDNA clones exclusively expressed in T cells from 1 X 10(4) plaque-forming units of a T cell hybridoma. These clones represent 0.068% of the mass of the cytoplasmic mRNA. Our result shows that differential hybridization is an effective procedure when used in combination with Northern blot analysis for screening of genes selectively expressed in T cells.

Animals↗

PubMed1987

Structure and function of a new hemoglobin variant, Hb meilahti (alpha 2 beta 2 36(C2)Pro----Thr), characterized by mass spectrometry.

An abnormal hemoglobin was found by isoelectric focusing in the blood of a Finnish woman with erythrocytosis. Oxygen equilibrium curves of the patient's hemolysate indicated the presence of a variant with a very high oxygen affinity. Structural analysis was carried out by mass spectrometry. In the spectrum of the tryptic digest, an abnormal peptide was found at m/z1278 which corresponded to the mass number of the protonated molecular ion of beta T4 with 36Pro----Thr substitution. The structure was confirmed by the mass spectrum of the chymotryptic digest.

Genetic Variation↗