Enhanced susceptibility of photosynthesis to low-temperature photoinhibition due to interruption of chill-induced increase of S-adenosylmethionine decarboxylase activity in leaves of spinach (Spinacia oleracea L.)

The possible involvement of polyamines in the chilling tolerance of spinach (Spinacia oleracea L.) was investigated focusing on photosynthesis. During chilling at 8/5C (day/night) for 6 d, S-adenosylmethionine decarboxylase (SAMDC) activity increased significantly in leaves in parallel with the increase in putrescine and spermidine (Spd) content in leaves and chloroplasts. Treatment of leaves with methylglyoxal-bis(guanylhydrazone) (MGBG), an SAMDC inhibitor, resulted in the deterioration of plant growth and photosynthesis under chilling conditions, which was reversed by the concomitant treatment with Spd through the roots. Plants treated with MGBG showed lower photochemical efficiency of PSII than either the control or plants treated with MGBG plus Spd during chilling and even after transfer to warm conditions, suggesting an increase of photoinhibition due to low Spd in chloroplasts. Indeed, MGBG-treated plants had much lower activities of thylakoid electron transport and enzymes in carbon metabolism as well as higher degrees of lipid peroxidation of thylakoid membranes compared to the control. These results indicate that the enhanced activity of SAMDC with a consequential rise of Spd in chloroplasts is crucial for the cold acclimation of the photosynthetic apparatus in spinach leaves.     

Establishment and characterization of a lymphoepithelial-like carcinoma cell line (HUUCLEC) derived from the human uterine cervix

A cell line designated HUUCLEC was established from a human uterine cervical lymphoepithelial carcinoma obtained from a 61-year-old Japanese woman. The cell line has grown slowly without interruption and serial passages were successively carried out 60 times within 3 years. The cultured cells were spindle or round in shape, showing anaplastic and pleomorphic features, a pavement cell arrangement and multilayering without contact inhibition. The population doubling time of the HUUCLEC line was 72 hours while the chromosomal number varied widely and showed aneuploidy. The modal chromosomal number was stable at the triploid range and marker chromosomes were present; the Ebstein-Barr virus was absent in the cultured cells.     

Structure and activity of insect cytokine GBP which stimulates the EGF receptor

Growth-blocking peptide (GBP) is an insect cytokine that possesses diverse biological activities such as larval growth regulation, cell proliferation, and stimulation of immune cells. GBP is a 25-amino acid peptide with one disulfide bond. It has been revealed that the tertiary structure of GBP consists of an N- and C-terminal disordered region and a well-structured core. Although there is only a slight similarity between the primary structures of GBP and EGF and the molecular weight of GBP is about half that of EGF, GBP directly binds and activates the EGF receptor of human keratinocyte cells. Furthermore, the tertiary structure of the well-defined region of GBP is similar to that of the C-terminal domain of EGF. This review will focus on the tertiary structure of GBP and its activities, as compared with those of EGF.     

Effect of Hb-encapsulation with vesicles on H2O2 reaction and lipid peroxidation

We encapsulated a purified and concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane to form Hb vesicles (particle diameter, ca. 250 nm) for the development of artificial oxygen carriers. Reaction of Hb inside the vesicle with hydrogen peroxide (H(2)O(2)) is one of the important safety issues to be clarified and compared with a free Hb solution. During the reaction of the Hb solution with H(2)O(2), metHb (Fe(III)) and ferrylHb (Fe(IV)=O) are produced, and H(2)O(2) is decomposed by the catalase-like reaction of Hb. The aggregation of discolored Hb products due to heme degradation is accompanied by the release of iron (ferric ion). On the other hand, the concentrated Hb within the Hb vesicle reacts with H(2)O(2) that permeated through the bilayer membrane, and the same products as the Hb solution are formed inside the vesicle. However, there is no turbidity change, no particle diameter change of the Hb vesicles, and no peroxidation of lipids comprising the vesicles after the reaction with H(2)O(2). Furthermore, no free iron is detected outside the vesicle, though ferric ion is released from the denatured Hb inside the vesicle, indicating the barrier effect of the bilayer membrane against the permeation of ferric ion. When vesicles composed of egg york lecithin (EYL) as unsaturated lipids are added to the mixture of Hb and H(2)O(2), the lipid peroxidation is caused by ferrylHb and hydroxyl radical generated from reaction of the ferric iron with H(2)O(2), whereas no lipid peroxidation is observed in the case of the Hb vesicle dispersion because the saturated lipid membrane of the Hb vesicle should prevent the interaction of the ferrylHb or ferric iron with the EYL.     

Solution structure of paralytic peptide of silkworm,Bombyx mori

Paralytic peptide of Bombyx mori (BmPP) is one of the multifunctional ENF-peptides; the name of “ENF” is the consensus N-terminal amino acid sequence of the family peptides. We revealed that BmPP significantly possesses growth-blocking activity and plasmatocyte-spreading activity and that its activity profiles are different from those of another ENF-family peptide, namely, the growth-blocking peptide of Pseudaletia separata (PsGBP). We also determined the NMR structures of BmPP and PsGBP under the same conditions, which revealed the structural differences of the first and second β-turn regions between the two peptides. On the basis of our results, it can be considered that the tertiary structural difference in these peptides may cause their different profiles of growth-blocking activity.     

Structural analysis of an insect lysozyme exhibiting catalytic efficiency at low temperatures

Bombyx mori lysozyme (BmLZ), from the silkworm, is an insect lysozyme. BmLZ has considerable activity at low temperatures and low activation energies compared with those of hen egg white lysozyme (HEWLZ), according to measurements of the temperature dependencies of relative activity (lytic and glycol chitin) and the estimation of activation energies using the Arrhenius equation. Being so active at low temperatures and low activation energies is characteristic of psychrophilic (cold-adapted) enzymes. The three-dimensional structure of BmLZ has been determined by X-ray crystallography at 2.5 A resolution. The core structure of BmLZ is similar to that of c-type lysozymes. However, BmLZ shows some distinct differences in the two exposed loops and the C-terminal region. A detailed comparison of BmLZ and HEWLZ suggests structural rationalizations for the differences in the catalytic efficiency, stability, and mode of activity between these two lysozymes.     

A new technique to co-localise membrane proteins with Homer/vesl

The minimal requirements were defined as necessary for cluster formation of the group 1 metabotropic glutamate receptor (mGluR), which is regulated by the Homer/vesl family of scaffolding proteins [Curr. Opin. Neurobiol. 10 (2000) 370]. Cluster formation of G-protein-coupled receptors (GPCRs) plays a fundamental role in signal transduction, particularly at the neuronal synapse. To understand the interaction of mGluR with PSD-Zip45, a Homer/vesl family member, we designed a series of chimeric receptor proteins, consisting of C-terminal mGluR1alpha sequences that were fused to endothelin B receptors (ET(B)Rs). In vitro and in vivo studies revealed that an extended 20 amino acid long C-terminal mGluR1alpha peptide, including the proline-rich core motif PPXXF, is sufficient to induce clustering of chimeric ET(B)R/mGluR1alpha receptors by PSD-Zip45. This result is especially important because it constitutes the basis for a new approach to form two-dimensional crystals of membrane proteins in situ, which may render unstable membrane proteins amenable to electron crystallographic structure determination.     

Characteristics of the end-joining of DNA double-strand breaks by the ataxia-telangiectasia nuclear extract

A double-strand break was introduced in plasmid pZErO-2 at a specific site within the ccdB gene that is lethal to Escherichia coli cells and treated with nuclear extracts from human cells. The efficiency of rejoining was monitored by Southern blot analysis and the fidelity of rejoining was measured by expressing the ccdB gene after bacterial transformation. The efficiency of rejoining in the nuclear extract from an ataxia-telangiectasia (A-T) cell line was comparable to that from a control cell line. However, the accuracy of rejoining was much lower for the A-T cell extract than for the control cell extract. All mutations were deletions, most of which contained short direct repeats at the breakpoint junctions. The deletion spectrum caused by the A-T nuclear extract was distinct from that of the control extract. These results indicate that the ccdB gene is useful for analysis of mis-rejoining and that A-T cells have certain deficiencies in end-joining of double-strand breaks in DNA.