Trichoclin,a new furocoumarin from trichocline incana

The structure for trichoclin, (E)-8-(3-methyl-4-hydroxy-2-butenyloxy)-psoralen, a new furocoumarin isolated from Trichocline incana, has been established. Phellopterin and isopimpinellin were also obtained. The new side chain of trichoclin was confirmed by synthesis.     

On the selective elimination of Y-bearing sperm

The potential use of antibodies that selectively recognize either X-bearing or Y-bearing sperm is self-evident. Thus our attention was directed to the fact that under optimal conditions, H-Y antibody lyses 50% of mouse spermatozoa. Accordingly, we asked whether expression of H-Y antigen is haploid in spermatozoa from XY male mice heterozygous for the autosomal dominantSxr gene, for if H-Y expression were haploid, H-Y antibody would be expected to kill 75% of spermatozoa derived from these XY,Sxr/- males. However, maximal lysis remained at the 50% level, which indicates that haploid expression of H-Y antigen and the potential immunoselection of Y-(or X-) bearing spermatozoa are unlikely.     

Effect of the removal of the COOH-terminal region of bacteriorhodopsin on its light-induced H+ changes.

Removal of the COOH-terminal region of bacteriorhodopsin by digestion with trypsin or papain reduces the yield of light-induced H+ release by 50-70%. The rate of H+ release is not affected significantly, but the half time of H+ uptake increases almost twofold. However, there is no effect on the photocycle of bacteriorhodopsin as judged by the yield and decay kinetics of the M412 photointermediate. The H+:M ratio in enzyme-digested membranes is approximately 0.4-0.8, whereas untreated membranes have a H+:M ratio of approximately 2. Purple membrane sheets stored in distilled water at 4 degrees C for prolonged periods also have a low H+:M ratio, probably due to protease activity associated with bacterial contamination. Electrophoresis on sodium dodecylsulfate-polyacrylamide gels showed that both the enzyme-treated and the stored purple membrane samples have a higher electrophoretic mobility compared to the fresh preparation. The reduction in molecular weight can be accounted for by the loss of several residues from the COOH-terminal portion of the bacteriorhodopsin. We propose that the COOH-terminal region is partially responsible for the high yield of H+ release by the purple membrane.     

Interaction with phospholipid bilayers, ion channel formation, and antimicrobial activity of basic amphipathic alpha-helical model peptides of various chain lengths.

Basic amphipathic alpha-helical peptides Ac-(Leu-Ala-Arg-Leu)3 or 4-NHCH3 (4(3) or 4(4)) and H-(Leu-Ala-Arg-Leu)3-(Leu-Arg-Ala-Leu)2 or 3-OH (4(5) or 4(6)) were synthesized and studied in terms of their interactions with phospholipid membranes, biological activity, and ion channel-forming ability. CD study of the peptides showed that they form alpha-helical structures in the presence of phospholipid liposomes and thus they have amphipathic distribution of the side chains along the axis of the helix. A leakage study of carboxyfluorescein encapsulated in phospholipid vesicles indicated that the peptides possess a highly potent ability to perturb the membrane structure. Membrane current measurements using the planar lipid bilayer technique revealed that the peptide 4(6), which was long enough to span the lipid bilayer in the alpha-helical structure, formed cation-selective ion channels at a concentration of 0.5 microM in a planar diphytanoylphosphatidylcholine bilayer. In contrast, other shorter peptides failed to form discrete and stable channels though they occasionally induced an increase in the membrane current with erratic conductance levels. The probability of detecting a conductance increase was in the order of 4(6) greater than 4(5) greater than 4(4) greater than 4(3), which corresponds to the order of the peptide chain lengths. Furthermore, 4(6) but not 4(5) showed an antimicrobial activity against both Gram-positive and -negative bacteria. The structure of ion channels formed by 4(6) and the relationship between the peptide chain length and biological activity of the synthetic peptides are discussed.     

CD2 can mediate TCR/CD3-independent T cell activation.

T lymphocytes can be activated clonotypically through TCR/CD3 complex or polyclonally via the CD2 molecule. Whether CD2-mediated activation is dependent on TCR/CD3 expression or signaling is controversial. We have re-explored this issue by using a series of CD2-transfected, TCR/CD3 surface membrane-negative human and mouse T cells. Our results clearly show that such T cells can be triggered for IL-2 secretion and increases in intracellular Ca2+ through the CD2 molecule in the absence of surface expression of TCR/CD3 complexes. These responses are only observed when cells express high levels of CD2 and there is a critical threshold of CD2 expression necessary for such activation in the absence of CD3. Concomitant expression of TCR/CD3 complex markedly lowers the level of CD2 required for activation via the latter pathway. These results provide a clear resolution of the controversy concerning the requirement for surface CD3 expression in T cell activation through CD2 and further suggest a possible role for CD2 in activation of TCR/CD3-negative cells.     

Inter- and intra-population variations in diapause attribute of the two-spotted spider mite,Tetranychus urticae Koch,in Japan

Populations of the two-spotted spider mite, Tetranychus urticae Koch collected from various localities and from various host plants in Japan showed wide variations in diapause attribute. Diapause percentages at 18°C/9L15D varied from nearly 100% in the north to 0% in the south-west. At intermediate latitudes the mites showed wide inter-population variations. Populations on herbaceous hosts in vinyl- or glass-houses gave significantly lower incidence of diapause than those on roses and deciduous fruit trees. Presence of winter hosts and better host quality under protected environments seemed to favour non-diapausing mites. The temperature threshold for diapause expression also varied widely among local populations. Northern populations consistently had higher and less variable thresholds than populations at intermediate latitudes with thresholds between 15 and 18°C. Inbred lines derived from a population in Kyoto exhibited a wide variation in diapause percentage at 18°C. These results show that diapause in T. urticae is a quantitative threshold trait and that populations in central Japan consist of a variety of genotypes with different diapause traits. This might provide a genetic source for adaptation to local and temporal variations in environmental conditions.     

Structure and regulated expression of Kunitz chymotrypsin inhibitor genes in winged bean [Psophocarpus tetragonolobus (L.) DC.]

We analyzed the structure and the expression of Kunitz chymotrypsin inhibitor (WCI) genes in winged bean. WCI was encoded by a multigene family which comprised at least seven members. From their primary structures, four genes (WCI-2, WCI-3a, WCI-3b, and WCI-x) were expected to be functional ones and the other three (WCI-P1, WCI-P2, and WCI-P3) to be pseudogenes. The nucleotide sequences of the WCI-3a and WCI-3b genes were nearly identical, and they encoded the WCI-3 protein, the major chymotrypsin inhibitor in seeds. The WCI-2 gene also encoded the chymotrypsin inhibitor found in seeds and the WCI-x gene was expected to encode an unidentified chymotrypsin inhibitor. WCI messenger RNA and protein accumulated mainly in developing seeds and tuberous roots, small amounts of WCI mRNA being present in stems and pericarps. In seeds, transient accumulation of WCI mRNA was observed during the seed maturation period. These results suggest that the expression of WCI genes is regulated organ-specifically and developmentally in winged bean.     

Estrogen modulates Ca(2+)-independent lipid-stimulated kinase in the rabbit corpus luteum of pseudopregnancy. Identification of luteal estrogen-modulated lipid-stimulated kinase as protein kinase C delta.

Rabbit corpora lutea were tested for the presence of phosphorylative responses sensitive to estrogen. Luteal Ca(2+)-independent lipid-stimulated kinase activity was detected by phosphorylation of the endogenous substrate, p76. Estrogen treatment, by way of estradiol-17 beta implant, increased levels of the lipid-stimulated phosphoprotein 2-3-fold throughout pseudopregnancy. Midpseudopregnant rabbit luteal extracts were further evaluated to determine the identity of the lipid-stimulated kinase. Results of low pH-activated phosphorylation were consistent with the identification of p76 as an autophosphorylated member of the protein kinase C (PKC) family. Partial purification of the luteal lipid-stimulated kinase was performed using sequential DEAE-cellulose/hydroxylapatite chromatographies and using gel filtration. Western immunoblot with type-specific anti-PKC delta antiserum showed coelution of kinase p76 activity with immunoreactive PKC delta. Immunoblot analysis confirmed that luteal levels of PKC delta were increased by estrogen treatment.     

The effect of amino acid substitution at position 219 of Citrobacter freundii cephalosporinase on extension of its substrate spectrum.

The cephalosporinase of Citrobacter freundii GN346 is a class-C beta-lactamase comprising 361 amino acids. The substitution of the glutamic acid at position 219 in the enzyme by lysine was previously shown to broaden its substrate specificity to unfavorable substrates such as oxyimino cephalosporins [Tsukamoto, K., Ohno, R. & Sawai, T. (1990) J. Bacteriol. 172, 4348-4351]. To investigate the cause of this phenomenon, Glu219 was changed to glutamine, cysteine or tryptophan. All the resultant enzymes showed higher cefuroxime-hydrolytic activities than the wild type, the order of increasing cefuroxime-hydrolytic activity being as follows: Trp greater than Lys greater than Cys greater than Gln greater than Glu. The rate of hydrolysis of cefuroxime by the Trp219 enzyme was approximately 3 x 10(4) times that of the wild-type enzyme. The order of increasing cefuroxime hydrolysis was approximately proportional to the molecular volume of the amino acid substituted and independent of the ionic character of the amino acids. The cysteine residue at position 219 in the Cys219 enzyme allowed its complete reaction with an SH-blocking reagent, 4-chloromercuriphenylsulfonic acid. The modified enzyme with the bulkier residue showed a 45% higher cefuroxime-hydrolytic activity than the untreated enzyme. These results suggested that extension of the substrate spectrum may be attributed to alteration in the configuration of the enzyme around position 219.