Conformational change of the triple-helical structure. IV. Kinetics of the helix-folding of (Pro-Pro-Gly)n (n = 10, 12, and 15)

The kinetic curves of the helix-refolding of (PPG)_n (n = 10, 12, and 15) were analyzed with an all-or-none model. The Arrhenius plot of the overall rate constant of the helixfolding k_F showed a negative activation energy at high temperature. With the aid of a sequential model, it was concluded that the reason for the anomaly was the instability of short helices (shorter than seven helical units in a trimeric molecule), and/or the more rapid rates of helix-folding and helix-opening for shorter helices. The rate constant of the formation of one helical unit composed of three tripeptides at an end of a long helix was calculated to be 10^2–4 sec^?1. It was much smaller than that for other kinds of helices, such as an α helix (10¹⁰ sec^?1) or a double helix of nucleic acids (10^7–9sec^?1).     

Karyotypes and serum transferrin patterns of hybrids between Asian and Oceanian black rats,Rattus rattus

Karyotypes and serum transferrin patterns were examined in Asian and Oceanian black rats (R. rattus). Japanese R. r. tanezumi and Malayan R. r. diardii had 2n=42, but Australian and New Guinea R. r. rattus showed 2n=38 chromosomes. F₁ hybrids between Japanese and Australian rats and Malayan and New Guinea rats had 2n=40 chromosomes which consists of the two genomes of both parents. Although various matings between the F₁ hybrids were made, only one F₂ male rat with 2n=39 chromosomes was obtained. The F₁ hybrids seem to be semisterile. Parental transferrin phenotypes were TfR in Japanese rats and TfCD in Oceanian rats. F₁ hybrids examined showed TfRD in both male and female and one F₂ hybrid had TfR type transferrin. Based on the above investigations, it is suggested that Asian and Oceanian black rats are geographically isolated and evolved different chromosomal and serum transferrin characteristics, but the sexual isolation of the two groups is incomplete at the present time.Contribution No. 826 from the National Institute of Genetics, Japan. Supported by a grant-in-aid from the Ministry of Education of Japan (Scientific Expedition in 1968, No. 8801 in 1969 and No. 9001 in 1970).     

Comparative examination of terrestrial plant leaves in terms of light-induced absorption changes due to chloroplast rearrangements

Light-induced chloroplast rearrangements were studied for leavesof 17 species of terrestrial plants (8 species of monocotyledonaeand 9 of dicotyledonae) by measuring their absorbance changeswith a dual-wavelength positional scanner. Almost all of theseleaves examined underwent two types of absorbance change; absorbanceincrease on illumination with weak blue light and absorbancedecrease with strong light. The maximal increase and decreaseand the light intensities for these maximal responses as wellas the intensity for mutual compensation of these opposite responseswere determined and compared for these species. (Received February 25, 1974; )     

Cytochrome P450 associated with free hepatic polyribosomes

On phenobarbital administration to rabbits, the concentration of hepatic cytochrome P450, an unstable constitutive microsomal enzyme, increased sharply in the heavy fraction of the free polyribosomes. The fraction had following properties: (1) its cytochrome P450 content was unusually high; the content was much lower in the lighter polyribosomes, the cytochrome P450 could not be extracted from post-mitochondrial supernatant solutions or microsomes with polyribosomes. (2) The fraction was membrane-free. (3) The fraction had RNA-to-protein ratios characteristic of polyribosomes; (4) it had characteristically low phospholipid content; (5) its sucrose density-gradient centrifugation profiles were characteristic of heavy polyribosomes, not microsomes. (6) The heavy polyribosomal fraction failed to catalyze mixed-function oxidations dependent on cytochrome P450, and the system was not activated by mixed mono- and dilaurylphosphatidylcholine. (7) Cytochrome P450 was released from the fraction by ribonuclease, and (8) cytochrome P450 was partially released from the fraction by puromycin.     

Aminooxy‐naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l‐tryptophan aminotransferase: structure–activity relationships

We previously reported l ‐α‐aminooxy‐phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis, but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from AOPP to study the structure–activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These active compounds reduced the endogenous indole‐3‐acetic acid (IAA) content upon application to Arabidopsis seedlings. Among the compounds, we selected 2‐(aminooxy)‐3‐(naphthalen‐2‐yl)propanoic acid (KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169 showed typical auxin‐deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine ammonia‐lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly developed compounds constitute a class of inhibitors of TAA1. We designated them ‘pyruvamine’.     

Segregative cell division and the cytoskeleton in two species of the genus Struvea (Cladophorales,Ulvophyceae, Chlorophyta)

The detailed segregative cell division (SCD) processes and changes in the arrangement of cortical microtubules and actin filaments were examined in two species of Struvea. SCD was initiated by the appearance of annular constrictions along the lateral side of a mother cell. The constrictions decreased in diameter, became thin, tubular in shape, and pinched the protoplasm of the mother cell into several protoplasmic sections. The protoplasmic sections expanded and developed into daughter cells, which appressed each other, and were arranged in a single row. Lateral branches protruded from the upper parts of the daughter cells. The protoplasm of the lateral branches was divided by secondary SCDs and was distributed amongst the new daughter cells. SCD and lateral branch formation were essential for morphogenesis in Struvea. Cortical microtubules were arranged parallel and longitudinally to the cell axis before SCD. When SCD was initiated, there was considerable undulation of the cortical microtubules and several transverse bundles appeared in the cytoplasmic zone where annular constrictions occurred. A microtubule‐disrupting drug (amiprophos methyl) inhibited SCD. Actin filaments maintained reticulate patterns before and during SCD. These results demonstrated that SCD in Struvea species was quite distinct from that in Dictyosphaeria cavernosa reported previously.     

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

In the budding yeast Saccharomyces cerevisiae, osmostress activates the Hog1 mitogen-activated protein kinase (MAPK), which regulates diverse osmoadaptive responses. Hkr1 is a large, highly glycosylated, single-path transmembrane protein that is a putative osmosensor in one of the Hog1 upstream pathways termed the HKR1 subbranch. The extracellular region of Hkr1 contains both a positive and a negative regulatory domain. However, the function of the cytoplasmic domain of Hkr1 (Hkr1-cyto) is unknown. Here, using a mass spectrometric method, we identified a protein, termed Ahk1 (Associated with Hkr1), that binds to Hkr1-cyto. Deletion of the AHK1 gene (in the absence of other Hog1 upstream branches) only partially inhibited osmostress-induced Hog1 activation. In contrast, Hog1 could not be activated by constitutively active mutants of the Hog1 pathway signaling molecules Opy2 or Ste50 in ahk1Δ cells, whereas robust Hog1 activation occurred in AHK1⁺ cells. In addition to Hkr1-cyto binding, Ahk1 also bound to other signaling molecules in the HKR1 subbranch, including Sho1, Ste11, and Pbs2. Although osmotic stimulation of Hkr1 does not activate the Kss1 MAPK, deletion of AHK1 allowed Hkr1 to activate Kss1 by cross talk. Thus, Ahk1 is a scaffold protein in the HKR1 subbranch and prevents incorrect signal flow from Hkr1 to Kss1.     

Vitamin B6 suppresses apoptosis of NM-1 bovine endothelial cells induced by homocysteine and copper

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B(6)in vivo. In the present study, we examined effects of vitamin B(6) on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.