Gas chromatographic method for the determination of urinary acetylpolyamines

A gas chromatographic method was developed for the determination of monoacetylputrescine, monoacetylcadaverine, N¹-acetylspermidine and N⁵-acetylspermidine in human urine. The amines were isolated from urine by silica gel column chromatography. 1, 10-Diaminodecane was used as internal standard. The amines were reacted with ethyl chloroformate in aqueous medium to four ethyloxycarbonyl derivatives prior to application to gas chromatography using a flame ionization detector. Separation and determination of the derivatives were carried out on a Uniport HP column (1.0 m) impregnated with 0.5% SP-1000 under temperature-programmed conditions. The monoacetylpolyamines could be measured accurately at the nanomole level. The method was used for the determination of the monoacetylpolyamines in urine of healthy volunteers. The values obtained were in the range of the published data.     

A fluorescence probe study of the phosphorylation reaction of the calcium ATPase of sarcoplasmic reticulum

The fluorescent thiol reagent N-(1-anilinonaphthyl-4)maleimide (ANM) reacts covalently with the Ca2+ ATPase moiety of fragmented sarcoplasmic reticulum in two phases as determined by the increase of fluorescence intensity and optical density at 350 nm. In the rapid phase, 5.5 nmol of ANM reacts with 1 mg of fragmented sarcoplasmic reticulum protein. Assuming that 55% of the total membrane protein is the Ca2+ ATPase, this is equivalent to 1 mol of SH/10(5) g of ATPase, designated as SH1-ANM. ANM reacts with the second SH (SH2-ANM) at a much slower rate. Reaction of ANM with both SH1-ANM and SH2-ANM produces no inhibition of phosphoenzyme (EP) formation. Upon addition of Mg . ATP in the micromolar range, at [Ca2+] = 1 microM there is an increase in the fluorescence intensity of ANM attached to SH2-ANM, while the ANM attached to SH1-ANM does not respond to Mg . ATP. Under conditions in which there is no EP formation, there is no fluorescence change. Furthermore, the enhancement of ANM fluorescence produced by Mg . ATP is reversed by ADP as it reacts with EP to form ATP. Thus, it appears that the Mg . ATP-induced fluorescence increase reflects changes of enzyme conformation produced by EP formation.     

The Amino Acid Sequence of the Tryptophan-Containing Subunit (α-Subunit) of Bovine Brain S-100 Protein

Abstract: The tryptophan-containing subunit (α-subunit) of bovine brain S-100 protein was purified from a S -aminoethyl derivative of S-100a protein, and its amino acid sequence was determined. The α-subunit contained 93 residues, including one tryptophan, and had a molecular weight of 10,400. The sequence shows an extensive homology (58% identity) to the sequence of another "tryptophan-free" subunit (β-subunit) found in both S-100a and S-100b protein, and has a calcium binding site characteristic of the "E-F hand" proteins, such as calmodulin or troponin C. The tryptophan residue is located at position 90 which is presumably adjacent to the C-terminal end of the α-helix following the calcium binding loop, and thus appears likely to serve as a specific probe in structure-function studies of S-100a protein.     

Angeloylcumambrin-B,an antimicrobial sesquiterpene lactone from Chrysanthemum ornatum var. Spontaneum

Angeloylcumambrin-B, a new antimicrobial guaianolide sesquiterpene lactone, was isolated from Chrysanthemum ornatum and the structure was determined by a combination of chemical and physical methods.     

Lignans of Larix leptolepis

Five lignans have been isolated from wood of Larix leptolepis. They are identified as 1-(4-hydroxy-3-methoxyphenyl)-2-4-[2-formyl-(E)-vinyl]-2-methoxyphenoxy-propane- 1,3-diol, 1-(4-hydroxy-3-methoxyphenyl)-2-2-methoxy-4-[1-(E)-propen-3-ol]-phenoxy- propane-1,3-diol, 1-(4-hydroxy-3-methoxyphenyl)-2-(4-formyl-2-methoxyphenoxy)-propane-1,3-diol, 1,2-bis-(4-hydroxy-3-methoxyphenyl)-propane-1,3-diol and a trilignol, leptolepisol C.     

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.     

Gene copy number effects in the mer operon of plasmid NR1.

The level of resistance to Hg2+ determined by the inducible mer operon of plasmid NR1 was essentially the same for three gene copy number variants in Escherichia coli, less in Proteus mirabilis, and intermediate in P. mirabilis "transitioned" to a high r-determinant gene copy number. Cell-free volatilization rates of radioactive mercury indicated increasing levels of intracellular mercuric reductase enzyme from low- to high-gene copy number forms in P. mirabilis and from low- to high-copy number forms in E. coli, but the additional enzyme in E. coli was effectively cryptic.     

Cloning and physical mapping of the dnaA region of the Escherichia coli chromosome.

The dnaA gene of Escherichia coli K-12, supposedly present in the deoxyribonucleic acid (DNA) of specialized transducing phase lambda i21 dnaA-2, was cloned onto plasmid pBR322. The new plasmid was named pMCR501. Physical analyses of DNAs of lambda i21 dnaA-2 and pMCR501 revealed the following. The lambda i21 dnaA-2 DNA retained the delta sr I lambda 1-2 and ninR5 deletions and imm21 substitution which were originally present in the parental phage. The size reduction was compensated for by the insertion-substitution segment (tna-dnaA region) in lambda i21 dnaA-2 DNA. The fractional size of this segment was approximately 7 megadaltons (Md), or 10 kilobases, which was found to be the sum of the tna insertion subsegment of ca. 3.5 Md and the dnaA substitution subsegment of ca. 3.5 Md. Phage P1-mediated transductional mapping between the dnaA46 and tna mutations gave a cotransduction frequency of 84%, corresponding to approximately 5 kilobases. Thus, it is strongly suggested that the dnaA gene resides in the lambda i21 dnaA-2 DNA. Cleavage mapping with the restriction endonuclease of pMCR501 DNA confirmed that it was constructed by excising a BamHI fragment of 4.29 Md, containing the 3.5-Md dnaA substitution segment, from the lambda i21 dnaA-2 DNA, inserting it into the sole BamHI cleavage site on pBR322.     

A synthesis of 3′,4-́dideoxykanamycin B

3′,4′-Dideoxykanamycin B, the kanamycin B derivative that is active against resistant bacteria, was prepared from kanamycin B viaN-tosylation, 3′,4′-O-sulphonylation, 3′,4′-unsaturation, and hydrogenation. The unsaturated intermediate was obtained from the 3′,4′-di-O-sulphonyl derivatives by the action of sodium iodide in N,N-dimethylformamide; if zinc dust was added in this reaction, aziridine derivatives were formed, Removal of the tosyl group was successfully performed by using sodium in ammonia-ethylamine.     

Distribution of ferritin-conjugated lectins on sialidase-treated membranes of human erythrocytes

The labeling of sialidase-treated, human erythrocyte membranes with ferritin-conjugates of four plant lectinss, concanavalin A, Ricinus communis hemagglutinin, Bauhinia purpurea hemagglutinin and Arachis hypogoea hemagglutinin, is reported. Among these ferritin-conjugated lectins, ferritin-conjugated concanavalin A and ferritin-conjugated R. communis hemagglutinin were found in clusters on the sialidase-treated membranes, whereas ferritin-conjugated B. pupurea hemagglutinin and ferritin-conjugated A. hypogoea hemagglutinin were found in a random distribution on the membranes. Furthermore, when the membranes were labeled with a mixture of concanavalin A and ferritin-conjugated B. purpurea hemagglutinin, ferritin particles were found in clusters, indicating that the membrane receptors for B. purpurea hemagglutinin were forced to move together with those for concanavalin A. A method for thentitative estimation of the clustering of ferritin particles on the membranes was also devised and applied to the labeling of sialidase-treated, human erythrocyte membranes with the above four ferritin-conjugated lectins.