Molecular phylogeny based on the κ-casein and cytochrome b sequences in the mammalian suborder Ruminantia

Nucleotide sequences for the -casein precursor proteins have been determined from the genomic DNAs or hair roots of the Ruminantia. The coding regions, exons 2, 3, and 4, were amplified separately via the three kinds of PCRs and then directly sequenced. The primers were designed from the sequence of bovine -casein gene; they were applicable for the amplification of the -casein genes from the 13 species in the Ruminantia except exon 2 of the lesser mouse deer. These results permitted an easy phylogenetic analysis based on the sequences of an autosomal gene. A phylogenetic tree was constructed from the mature K-casein sequences and compared with the tree of the cytochrome b genes which were sequenced from the same individuals. The Cervidae (sika deer, Cervus nippon) were separated from the branch of the Bovidae on the tree of -casein genes with a relatively high confidence level of the bootstrap analysis, but included in the branch of the Bovidae on the tree of cytochrome b genes. The -casein tree indicated a monophyly of the subfamily Caprinae, although the internal branches were uncertain in the Caprinae. The tree based on the nucleotide sequences of cytochrome b genes clearly showed the relationships of the closely related species in the genus Capricornis consisting of serow (C. smatorensis), Japanese serow (C. crispus), and Formosan serow (C. swinhoei). These results would be explained by the difference of resolving power between the -casein and the cytochrome b sequences. Correspondence to: K. Chikuni     

Immunostimulatory activities of monoglycosylated α-d-pyranosylceramides

We compared the immunostimulatory effects of chemically synthesized α-galactosylceramides (α-GalCers), α-glucosylceramides (α-GluCers), 6″-monoglycosylated α-GalCer and 6″- or 4″-monoglycosylated α-GluCer and made the following observations: (1) the length of the fatty acid side chain in the ceramide portions greatly affects the immunostimulatory effects of α-GalCers and α-GluCers; (2) the configuration of the 4″-hydroxyl group of the inner pyranose moiety plays an important role in the immunostimulatory effects of monoglycosylated α- -pyranosylceramides; (3) the free 4″-hydroxyl group of the inner pyranose of monoglycosylated α- -pyranosylceramides plays a more important role in their immunostimulatory effects than the free 6″-hydroxyl group.     

Establishment and characterization of a new,spontaneously immortalized,pancreatic ductal cell line from the Syrian golden hamster

Spontaneously immortal pancreatic cell lines are not available. By use of a defined culture medium, such a line (TAKA-1) was established from the Syrian golden hamster. Cytological, cytogenetic, molecular biological, enzymatic and receptor patterns as well as antigenicity were studied and were compared with those of the normal hamster pancreatic ductal cells in vivo. TAKA-1 cells grew exponentially in a monolayer on collagen gel in a defined medium but did not proliferate in soft agar. Ultrastructurally, the cells closely resembled the normal hamster pancreatic ductal cells. Similarities and dissimilarities were found between the normal ductal cells and TAKA-1 cells. Similarities included the presence of cytokeratin, carbonic anhydrase and some tumor-associated antigens. However, unlike the normal ductal cells, TAKA-1 cells expressed blood group A angigen and anti-vimentin, showed affinity to selected lectins, and an abnormality of chromosome 3, which is suggested to be associated with immortality. Moreover, unlike the hamster pancreatic ductal cancer cells but like the normal hamster pancreatic ductal cells, TAKA-1 cells did not have a c-Ki-ras mutation. EGF, TGF- and secretin, but not CCK or GRP, bound to the TAKA-1 cells. TAKA-1 cells produced TGF-, and their growth was stimulated by exogenous EGF in serum-free medium. This cell line presents a suitable model for biologic and pathologic study of the hamster pancreatic ductal cells in vitro.     

(+)-Arctigenin,a lignan from Wikstroemia indica

A new lignan, (+)-aretigenin has been isolated from the roots of Wikstroemia indica (Nan-Ling-Jao-Hua) and identified as 8(R) 8′(S)-4′-hydroxy-3, 4,3′-trimethoxylignan-olid (9, 9′) on the basis of spectral evidence as well as a direct comparison with its enantiomer, (?)-arctigenin.     

Phosphatidylcholine liposomes containing cholesterol analogues with side chains of various lengths

The effect of the length of the side chain of sterols on their interaction with phosphatidylcholine was studied by measuring the permeability properties of liposomes constituted with sterol analogues with side chains of various lengths. The sensitivities of liposomes constituted with these sterol analogues toward digitonin and polyene antibiotics were also examined.The effects of sterols on phase transition of phosphatidylcholine were examined by measuring their effects on permeability increase due to perturbation of phase equilibrium and by differential scanning calorimetry. An analogue with a short side chain, isopropyl (C-22), had a very similar effect to cholesterol in suppressing the permeability increase, suggesting that the full length of the side chain is not necessary for this effect.The permeability of egg yolk phosphatidylcholine at 42°C was suppressed as much by the analogue C-22 as by cholesterol. Androstene-3-β-ol, an analogue without a side chain, however, had little suppressive effect. Thus it is concluded that the condensing effect of sterol requires a side chain, but not the full length of side chain.Liposomes constituted with analogues having a side chain with more than 5 carbon atoms showed maximum reactivity with a polyene antibiotic, amphotericin B, whereas those constituted with analogues having a side chain with less than 4 carbon atoms showed weaker reactivity. These findings indicate that a side chain with more than 5 carbon atoms is essential for the maximum interaction of liposomes with amphotericin B. Unlike amphotericin B, filipin reacted almost equally well with liposomes containing C-22 and with those containing cholesterol. Thus the chain length of the side chain of sterol is less important for interaction of liposomes with filipin than for their interaction with amphotericin B.Liposomes containing analogues having a side chain with more than 6 carbon atoms showed maximum reactivity with digitonin. Thus for the maximum interaction of liposomes with digitonin, the side chain of sterol should be longer than 6 carbon atoms.     

Surface charge of protoplasts and their significance in cell-cell interaction

-potential of mesophyll protoplasts of tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida Hort.), turnip (Brassica rapa L.) and cowpea (Vigna unguiculata L. Walpers) was determined by use of a cell electrophoresis apparatus. All protoplasts examined showed a constant negative value of-10 to-35 mV. The addition of CaCl₂ nullified the -potential of tobacco protoplasts. This phenomenon is explained by DLVO theory of colloid science, which has been successfully applied to animal cells. Furthermore, positively charged polymers reversed the -potential to positive values. Treatment of the protoplast surface with several enzymes was carried out to characterize the chemical nature of suface charges. The removal of surface charges was most conspicuous by the treatment of acid phosphatase (EC 3.1.3.2), but did not occur upon treatment with -neuraminidase (EC 3.2.1.18) or Streptomyces griseus pronase. Thus a major part of the surface charge originates from the phosphate groups at the cell membrane. The significance of these studies for the properties of the protoplast surface in cell adhesion is discussed.     

Automated determination of orotic acid, uracil and pseudouridine in urine by high-performance liquid chromatography with column switching

A column-switching high-performance liquid chromatographic method, requiring no sample preparation apart from filtration, is described for quantification of urinary orotic acid, uracil and pseudouridine. The analyses were carried out using a reversed-phase octadecylsilane-bonded column for sample clean-up and a cation-exchange column for separation; 5–20 ]sml samples of urine were directly analysed, and more than 100 samples could be analysed consecutively. Each sample required only 30 min. Detection limits of these compounds were 5 pmol. Creatinine-related urinary uracil excretion was lowest in the newborn period (17.3 ± 14.4 μmol/g of creatinine). A patient with partial ornithine transcarbamylase deficiency and his mother usually excreted a high level of uracil during the period of normal orotic acid excretion and normal serum ammonia level.     

Identification of a novel amino acid, o-bromo-L-phenylalanine, in egg-associated peptides that activate spermatozoa

Eight sperm-activating peptides containing a novel amino acid were isolated from the egg jelly of the sea urchin Tripneustes gratilla. Accurate mass measurement of the peptide in FAB mass spectrometry showed that the mass of the novel amino acid residue was 224.978. On the basis of the isotopic ion distribution and the degree of unsaturation, the mass value indicated that the elemental composition of the amino acid residue was C9H8O1N1Br1, suggesting that the novel amino acid was bromophenylalanine. Proton NMR spectroscopy, amino acid analysis, and RP-HPLC with three synthetic isomers of bromophenylalanine demonstrated that o-bromophenylalanine was the novel amino acid. Derivatization of the amino acid with Marfey's reagent, (1-fluoro-2,4-dinitrophen-5-yl)-L-alanine amide (FDAA), further indicated that the amino acid was the L-isomer. In other sperm-activating peptides isolated from the egg jelly of the sea urchin, both m- and p-bromophenylalanines were discovered. The presence of m-bromophenylalanine has not been previously reported in natural products, while p-bromophenylalanine is found in theonellamide F, an antifungal bicyclic peptide from a marine sponge.