Isolation and identification of 25-hydroxy-24-oxocholecalciferol: A metabolite of 25-hydroxycholecalciferol

A metabolite of 25-hydroxycholecalciferol has been isolated in pure form from chicken kidney homogenates. It has been identified as 25-hydroxy-24-oxocholecalciferol by means of ultraviolet absorption spectrophotometry, mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and specific chemical reactions.     

Phlegmacins and anhydrophlegmacinquinones: Dimeric hydroanthracenes from seedlings of Cassia torosa

From seedlings of Cassia torosa four dimeric hydroanthracenes have been isolated. Two, a pair of atropisomeric dimers consisting of two molecules o     

Myosin and actin from Escherichia coli K12 C600.

Myosin-like protein and actin-like protein from E. coli formed filaments very similar in structure to those of myosin and actin from skeletal muscle. At 0.2 M KCl, a large number of "thick filaments" of uniform size (about 0.6-0.7 micron long and about 20 nm wide) was present. These thick filaments aggregated as the KCl concentration decreased to less than 0.2 M. Filaments of actin-like protein were decorated with muscle heavy meromyosin, showing "arrowheads". The arrowhead structure disappeared in the presence of ATP. A mixture of E. coli myosin-like protein and rabbit skeletal actin exhibited a gelation phenomenon on the additon of ATP. The phenomenon was reversible and showed ATP specificity. However, the gelation phenomenon was not observed with the mixture of E. coli actin-like protein and E. coli myosin-like protein. These results provide compelling evidence that the E. coli myosin-like protein and actin-like protein we isolated are essentially identical to myosin and actin, respectively.     

Purification of membrane-bound polyvinyl alcohol oxidase in Pseudomonas sp. VM15C

Abstract Polyvinyl alcohol (PVA) was utilized by a symbiotic mixed culture which was composed of Pseudomonas putida VM15A and Pseudomonas sp. VM14C. The PVA oxidase was found in the culture fluid, membrane, and cytosol fractions of VM15C. The membrane-bound PVA oxidase was purified by several steps of chromatography. The enzyme (p I = 9.6) exhibited the maximum activity at pH 8.0 to 8.4 and 45°C, and utilized secondary alcohol as well as PVA. The enzyme showed the PVA dehydrogenating activity linking with phenazine ethosulfate, indicating the possibility that PVA oxidation is coupled with an electron transport chain on the bacterial membrane.     

Biphasic uptake of iron-transferrin complex by L1210 murine leukemia cells and rat reticulocytes

The kinetics of the cellular uptake of iron-transferrin complex was studied in L1210 murine leukemia cells and rat reticulocytes using ¹²⁵I-transferrin. Saturation of transferrin with iron was necessary for optimal uptake. Following the incubation of cells with the radiolabeled complex a biphasic pattern of uptake was observed. The initial phase was rapid and relatively temperature-independent and was not altered by ethylamine, an inhibitor of transglutaminase activity which is necessary for receptor-mediated endocytosis. This phase was considered to result from receptor-ligand interaction which could be reversed to a great degree by replacement with unlabeled transferrin. A plateau was then reached, indicating a saturation of receptors. After 30 min a second phase of uptake was indicated by the second rise in the curve. This phase was slow, relatively temperature-dependent and could be abolished by ethylamine. It was interpreted as evidence of internalization of the ligand. Analysis of the data from competition studies with unlabeled transferrin indicated that the first phase might itself comprise a reversible and an irreversible step with a ratio of 5 to 1.4 for bound transferrin. Thus, the cellular uptake of iron-transferrin complex may consist of a reversible ligand-receptor interaction. Conformational changes may render this interaction irreversible and the internalization of the ligand may then follow.     

Correspondence of homologies in amino acid sequence and tertiary structure of protein molecules

According to the method developed previously (Kubota, Y., Takahashi, S., Nishikawa, K. and Ooi, T. (1981) J. Theor, Biol. 91, 347-361), homology among proteins may be estimated quantitatively. We extended the method to investigate the relationship of an amino acid sequence to its teritary structure and identify homologous segments which have homologous native conformations in proteins. First, we selected proper indices for the computation of correlation coefficients from 32 properties inherent to amino acids, such as hydrophobicity. The arithmetic average of correlation coefficients using six indices gave rise to a good correlation for the CD- and EF-hand regions (Ca2+ binding sites) in carp parvalbumin, but poor ones for other segments. We then applied the method to homologous proteins, the three-dimensional structures of which are known: horse hemoglobin alpha-chain and beta-chain; cytochrome c and c2; serine proteases, chymotrypsinogen and elastase; alpha-lytic protease and protease A from prokaryotic organisms. The results show that the sequence homology estimated by the present method has a good correspondence to the homology in three-dimensional structures and therefore the method is promising for the identification of important sites in sequences which have similar native conformations. For an example of the application of the method, two sequences of human interferon, one from fibroblast and the other from leukocyte, are compared, suggesting functional sites in the molecule.     

Monoclonal antibodies recognizing amino-terminal and carboxy-terminal regions of human aldolase A: probes to detect conformational changes of the enzyme.

Three monoclonal antibodies (MAbs1A2, 3C5, and 4C2) for human aldolase A [EC 4.1.2.13] were established. MAbs1A2, 3C5, and 4C2 were shown to belong to subclasses IgM, IgG1, and IgG2a, respectively. None of the MAbs inhibits aldolase A activity. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs to chimeric proteins between aldolases A and B [Kitajima et al. (1990) J. Biol. Chem. 265, 17493-17498] in ELISA and to the CNBr-cleaved fragments of aldolase A in immuno-blotting. MAbs1A2 and 3C5 reacted with sites located within amino acid residues 306-363 at the C-terminal region of the enzyme. MAb4C2 recognized an epitope of the enzyme present within amino acid residues 34-108 at the N-terminal region. In a competitive binding assay, MAbs1A2 and 3C5 competed with each other for binding to the antigen and also interfered with the binding of MAb4C2, whereas MAb4C2 failed to inhibit the binding of MAbs1A2 and 3C5 to the antigen. MAb3C5 showed a species-specificity in the reaction with the antigen; it reacted with human and rabbit aldolase A with similar reactivity but not at all with the rat and mouse enzymes, which differ from the human and rabbit enzymes in two amino acid residues at positions 328 and 348. Reactivities of MAbs to aldolase A were further examined with engineered enzymes containing an amino acid substitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ejaculations induced by p-chloroamphetamine (PCA) in rats, hamsters and mice.

The ejaculatory response induced by p-chloroamphetamine (PCA) in male rats, hamsters and mice was observed during 2 hours after the injection. The animals were treated intraperitoneally with PCA at doses ranging from 0.78125 to 160 mg/kg. The ED50 (effective dose in 50% of animals) values of PCA for the initiation of ejaculation in rats and hamsters were 1.3397 (1.0732-1.6725) and 0.1105 (0.0802-0.1522) mg/kg, respectively. On the other hand, no ejaculation was observed in any mice at any doses examined. So we concluded that there are species differences in the ejaculatory response, induced by PCA, among rats, hamsters and mice.     

Pre- and post-implantation losses of embryos in aging female IVCS mice

Mated female mice of the IVCS strain, aged 90 (control group), 180, 210, 240, 270, 300, 330, 360 and 420 days old, were studied for pre- and post-implantation loss of embryos. The percentage of pre-implantation loss in mice aged 90 to 210 days was 1.7/11.8 (14.4%) to 2.7/11.7 (23.1%). In mice aged 240 to 300 days it increased significantly as compared to the controls (46.5-90.2% versus 14.4%). It reached 100% in 300-days-old mice. The post-implantation loss of embryos and/or fetuses in mice aged 90 to 240 days was 1.0/10.2 (9.8%) to 2.5/9.0 (27.8%). In mice aged 270 to 300 days it increased significantly compared to the controls (100% versus 9.8%). The decrease in reproductive activity appeared first in a decrease in litter size, followed by a decrease in the number of blastocyst and implantation sites, and finally by anovulation during the process of aging in IVCS mice.