Two Amino-acid Carriers in Pea Chloroplasts

CHLOROPLASTS are more permeable to amino-acids^1,2 than are cell membranes³ and also discriminate between their optical isomers². This has led to the suggestion that the passage of amino-acids through the limiting membranes of chloroplasts is facilitated by carriers². Using the additional criteria of saturation and competition, we support this suggestion and show that at least two carriers are probably involved.     

Chromophore: apoprotein interactions in phytochrome A*

Phytochromes are molecular light switches by virtue of their photochromic red/far-red reversibility. The His-324 residue next to the chromophore-linked Cys-323 plays a critical role in conferring photochromism to the tetrapyrrole chromophore in native phytochrome A. The chromophore appears to be enclosed between the amphiphilic α-helical chains in a hydrophobic pocket. The absorbance maxima of both the Pr and the Pfr forms of pea phytochrome A are blue-shifted by 10 and 20 nm, respectively, upon C-terminal truncation. We speculate that the quaternary structure of the phytochrome A molecule involves some interactions of the C-terminal half with the chromophore domain. The Pfr conformation of phytochrome includes an amphiphilic α-helix of the amino terminal chain, which occurs in 113 ms after picosecond photoisomerization of the Pr form. Compared to α-helical folding, unfolding of the α-helix occurs faster in about 310 μs upon phototransformation of the Pfr form of phytochrome A. The photochromic transformation of phytochrome A modulates protein kinase-catalysed phosphorylation sites in vivo and in vitro, but only a subtle local change in conformation is detectable in the phosphorylated phytochromes. This suggests that the post-translational modification serves as a surface label, rather than a transducer-activating trigger, for the recognition of a putative phytochrome receptor.     

Ceramide Inhibits Cell Proliferation through Akt/PKB Inactivation and Decreases Melanin Synthesis in Mel‐Ab Cells

Ceramide is a bioactive sphingolipid that mediates a variety of cell functions. However, the effects of ceramide on cell growth and the melanogenesis of melanocytes are not known. In the present study, we investigated the actions of cell‐permeable ceramide and its possible role in the signaling pathway of a spontaneously immortalized mouse melanocyte cell line, Mel‐Ab. Our results show that C₂‐ceramide inhibits DNA synthesis in Mel‐Ab cells and G361 human melanoma cells in a dose‐dependent manner. Cell cycle analysis confirmed the inhibition of DNA synthesis by a reduction in the S phase. To investigate the ceramide signaling pathway, we studied whether C₂‐ceramide is able to influence extracellular signal‐regulated kinase (ERK) and/or Akt/protein kinase B (PKB) activation. We demonstrated that phosphorylated Akt/PKB is decreased by C₂‐ceramide, whereas phosphorylated ERK was only slightly affected. Therefore, the C₂‐ceramide‐induced inactivation of Akt/PKB may be closely related to the reduced cell proliferation of Mel‐Ab cells. Furthermore, we assessed the effects of C₂‐ceramide on the pigmentation of Mel‐Ab cells. The results obtained showed that the melanin content of cells was significantly reduced by C₂‐ceramide at concentrations in the range of 1–10 μM, and that the pigmentation‐inhibiting effect of C₂‐ceramide is much greater than that of kojic acid at 1–100 μM. In addition, we found that the activity of tyrosinase is reduced by C₂‐ceramide treatment. Our results demonstrate that C₂‐ceramide reduces the pigmentation of Mel‐Ab cells by inhibiting tyrosinase activity.     

Production of Recombinant Hinnavin II/MSH Hybrid in Insect Cells

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. For the purpose of this study, a cDNA encoding hinnavin II‐α‐melanocyte stimulating hormone (hin/MSH) hybrid was chemically synthesized, annealed, and then cloned into transfer vector pBacPAK 9 for expression in Sf21 insect cells. Recombinant hin/MSH (rhin/MSH) hybrid was efficiently produced in baculovirus expression vector system (BEVS) as a hybrid peptide. The antibacterial activity of the rhin/MSH hybrid was compared to that of the recombinant hinnavin II (rhin), using inhibition zone and overlay assay. This new recombinant hybrid peptide may serve as an attractive candidate for powerful novel class of antimicrobial pharmaceuticals.