Increased hexose transport in the roots of tomato plants submitted to prolonged hypoxia

We investigated the effects of prolonged hypoxia on the sugar uptake in tomato (Solanum lycopersicum L. var. MP-1) roots. Hydroponic cultures of whole tomato plants were submitted to hypoxic treatment for 1 week, and the roots were analyzed for sugar concentrations, hexose uptake and hexose transporter expression level. Contrary to what has been observed after anoxic shock or short-term hypoxic treatment, we show that sugar concentrations increase and hexose uptake is up-regulated in the roots after 1 week of hypoxic treatment. Increased hexose transport is concomitant with the induction of the hexose transporter gene LeHT2. These responses may be due either to a direct effect of low O₂ supply, or to a secondary effect associated with the increase in sugar concentrations, which, typically, develops in most hypoxic plants.     

Structural characterization and anti-fatigue activity of polysaccharides from the roots of Morinda officinalis

Three polysaccharides MP-1, MP-2, and MP-3 were isolated from hot water extract of Chinese medicine Morinda officinalis through 95% ethanol precipitation and gel-filtration chromatography (DEAE-Sepharose CL-6B column and Sephadex G-75 or G-100 column). MP-1 was identified as an inulin-type fructan with simple linear (2 → 1)-linked structure. Both MP-2 and MP-3 were acidic polysaccharides which consisted predominantly of galacturonic acid, arabinose and galactose. Partial structure characterization of MP-3 was carried out by partial acid hydrolysis and periodate oxidation. The total polysaccharides of the herb were tested in mice weight-loaded swimming model and were found to have anti-fatigue activity.     

Myelopeptide MP-5 and fluorescent derivatives: Synthesis and biological activity

The Val-Val-Tyr-Pro-Asp bone marrow peptide (MP-5) and its analogue (MP-5-Lys) were synthesized. Fluorescent derivatives, Ftc-MP-5 and MP-5-Lys(Ftc), were prepared. The iological activity of MP-5 and MP-5-Lys was studied in vitro and in vivo. The MP-5 peptide caused 60–84% inhibition of growth of the following mouse cancers: lymphatic leukemia P388, melanoma B-16, and cervical carcinoma CUC-5. These peptides also restored functional activity of T-lymphocytes that was inhibited by metabolic products of the HL-60 leukemic cell line. MP-5-Lys(Ftc) was shown to preserve the functional properties of MP-5 towards T-lymphocytes, but Ftc-MP-5 was practically inactive.     

抗HIV-1活性的大环多胺类化合物与RNA的识别作用及其对细胞凋亡的影响(英)

研究了具有抗HIV-1活性的大环多胺类化合物与RNA的识别作用，以及其对cos-7细胞凋亡的影响，以进一步探讨其抗HIV-1的作用机理.实验采用琼脂糖凝胶电泳方法, 观察化合物与RNA的识别作用；通过流式细胞计数法探讨其对cos-7细胞凋亡的影响；运用计算机分子模型, 从理论上Docking计算化合物与TAR RNA结合的可能性.结果表明, 大环多胺类化合物MP-1、MP-2和MP-3不仅具有断裂RNA的作用，并可抑制Tat-RNA的相互作用, 还可影响cos-7细胞亚二倍体的含量; 理论化学计算数据与实验结果基本一致.这一结果提示化合物的抗HIV-1活性可能通过作用于病毒基因组RNA而发挥作用，是多靶作用的结果.     

Helix-stabilizing effects of the pentapeptide KIFMK and its related peptides on the sodium channel inactivation gate peptides.

We have previously found by NMR and CD spectroscopic studies that the helical content of the sodium channel inactivation gate-related peptide (Ac-GGQDIFMTEEQK-NH2; MP-1A) in 80% trifluoroethanol solutions was increased by adding a pentapeptide, KIFMK. In order to study in further detail whether the presence of the IFM motif and the two lysine residues is a prerequisite for stabilizing the helical conformation, we examined interactions between various oligopeptides (RIFMR, KIFMTK, KIQMK, KAFAK, KIIIK) and MP-1A and its related peptides; that is, MP-2A in which Phe was replaced by Gln, MP-1MMA in which Thr was replaced by Met, MP-1TA in which Thr was removed from MP-1A, and MP-1A' in which L-Phe was replaced by D-Phe. It was found that the IFM motif was absolutely necessary in both the oligopeptide and the inactivation gate peptide. This finding means that hydrophobic interactions are operative between KIFMK and MP-1A. In contrast, KIFMK destabilized the helical structure of MP-1MMA, MP-1TA, and MP-1A', showing that the conformation around the IFM motif in the inactivation gate peptides is an important factor. It was concluded that the IFM motif and the two Lys residues are a prerequisite for effectively stabilizing the alpha-helix of MP-1A.     

Myelopeptides: Bone marrow regulatory mediators

Bone marrow cells of various animal species and men produce a group of bioregulatory peptides called myelopeptides (MPs). A highly purified MP fraction and some individual molecules have been isolated from the supernatant of porcine bone marrow cell cultures by reverse phase chromatography.MPs have a wide spectrum of functional activities: immunoregulatory, differentiating and opiate-like. They evoke 2–5-fold stimulation of antibody production to various antigens. They correct some immune defects in MRL/lpr mice with spontaneous autoimmune disorders that results in 2-fold prolongation of the life span of these mice. MPs influence the differentiation of bone marrow and peripheral blood cells derived from healthy and leukemic donors. They induce terminal differentiation in the leukemic human HL-60 cell line. MPs also show an effect on pain sensitivity.A new immunocorrective drug Myelopid has been developed on the basis of MP mixtures. This drug is effectively used in Russia both in medicine and veterinary practice for prophylaxis and treatment of diseases accompanied by immunodeficiency.Two individual MPs were isolated and identified: Phe-Leu-Gly-Phe-Pro-Thr (MP-1) and Leu-Val-Val-Tyr-Pro-Trp (MP-2). MP-1 displays immunoregulatory activity; MP-2 abolishes the inhibitory effect of leukemic cells on T-lymphocyte functional activity.MPs seem to provide not only immunoregulation but also to participate in complex interactions between different systems in the organism.     

Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4'-hydroxylase

A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4'-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using non-overlapping 5' and 3' portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage lambda gt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4'-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3' noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs--individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the mephenytoin 4'-hydroxylase activity. These results indicate that several closely related P-450 genes are all expressed in individual human livers. The MP-4/MP-8 gene products are proposed to be the ones most likely involved in mephenytoin 4'-hydroxylation, and much of the variation in catalytic activity among individuals is not a result of differences in levels of P-450MP-1 or mRNA but may be due to base differences in the structural gene(s).     

Effect of myelopeptide-1 on the functional activity of phagocytes from mice treated with cyclophosphane

The capacity of the bone marrow-derived myelopeptide-1 (MP-1) to affect in vivo and in vitro the functional activity of phagocytes of intact mice and mice treated with a cytostatic agent (cyclophosphane) has been studied. It was found that MP-1 produces a correcting effect on the functional activity of bone marrow and peripheral blood phagocytes. An optimal scheme of the injection of MP-1 to mice with the cyclophosphane-induced immunodeficiency was developed, which provides a maximum immunocorrecting action. MP-1 had the most pronounced effect on the quantitative characteristics and the functional activity of phagocytes of different localization when introduced prior to the cytostatic; under these conditions, the pep tide affects peripheral blood neutrophils. The results obtained enable one to consider MP-1 as a preparation protecting the peripheral blood phagocytes from the damaging action of cyclophosphane.     

Isolation and partial characterization of membrane protein constituents of human neutrophil receptors for chemotactic formylmethionyl peptides

Plasma membranes of human neutrophils were solubilized in buffer containing a nonionic detergent and applied to a formylmethionylleucylphenylalanine (fMet-Leu-Phe)-Sepharose column that was washed and eluted with the chemotactic peptide fMet-Leu-Phe. Analysis of the eluate by filtration on Bio-Gel P150 in sodium dodecyl sulfate (NaDodSO4) buffer and by NaDodSO4-polyacrylamide gel electrophoresis revealed three predominant membrane proteins of approximate molecular weight 94 000 (MP-1), 68 000 (MP-2), and 40 000 (MP-3), of which MP-2 accounted for 74--93% of the total protein. Purified MP-1 and MP-2 contained an above average content of hydrophobic amino acids, while MP-2 and MP-3 had an above average content of acid and/or amide amino acids and a below average content of basic amino acids. MP-2 and MP-3, but not MP-1, bound [3H]fMet-Leu-Phe in equilibrium dialysis chambers. Both MP-2 and MP-3 exhibited high-affinity sites with a valence of 0.2--0.3 and mean KA values of 9 x 10(8) and 2 x 10(7) M-1, respectively, and low-affinity sites with a valence of 0.3--0.5 and mean KA values of 3 x 10(7) and 2 x 10(6) M-1 (n = 3). The specificity of the binding of fMet-Leu-Phe was suggested by the failure of MP-2 and MP-3 to bind lipid chemotactic factors and to adhere to a Sepaharose column to which had been coupled chemotactic fragments of the fifth component of complement. A series of synthetic formylmethionyl peptides exhibited the same rank order of potency as inhibitors of the binding of [3H]fMet-Leu-Phe by MP-2 and as stimuli of neutrophil chemotaxis. Membrane proteins isolated by fMet-Leu-Phe-Sepharose affinity chromatography may represent constituents of specific human neutrophil receptors for chemotactic peptides.     

抗HIV—1活性的大环多胺类化合物与RNA的识别作用及其对细胞凋亡的影响

研究了具有抗HIV－1活性的大环多胺类化合物与RNA的识别作用,以及其对cos-7细胞凋亡的影响,以进一步探讨其抗HIV－1的作用机理。实验采用琼脂糖凝胶电泳方法。观察化合物与RNA的识别作用。通过流式细胞计数法探讨其对cos-7细胞凋亡的影响。运用计算机分子模型,从理论上Docking计算化合物与TAR RNA结合的可能性。结果表明,大环多胺类化合物MP－1、MP－2和MP－3不仅具有断裂RNA的作用,并可抑制Tat-RNA的相互作用,还可影响cos-7细胞亚二倍体的含量;理论化学计算数据与实验结果基本一致,这一结果提示合物的抗HIV－1活性可能通过作用于病毒基因组RNA而发挥作用,是多靶作用的结果。     

1H-NMR and circular dichroism spectroscopic studies on changes in secondary structures of the sodium channel inactivation gate peptides as caused by the pentapeptide KIFMK.

The pentapeptide KIFMK, which contains three clustered hydrophobic amino acid residues of isoleucine, phenylalanine, and methionine (IFM) in the sodium channel inactivation gate on the cytoplasmic linker between domains III and IV (III-IV linker), is known to restore fast inactivation to the mutant sodium channels having a defective inactivation gate or to accelerate the inactivation of the wild-type sodium channels. To investigate the docking site of KIFMK and to clarify the mechanisms for restoring the fast inactivation, we have studied the interactions between KIFMK and the fragment peptide in the III-IV linker GGQDIFMTEEQK (MP-1A; G1484-K1495 in rat brain IIA) by one- and two-dimensional (1)H-NMR and circular dichroism (CD) spectroscopies. KIFMK was found to increase the helical content of MP-1A in 80% trifluoroethanol (TFE) solution by approximately 11%. A pentapeptide, KIFMT, which can restore inactivation but less effectively than KIFMK, also increased the helical content of MP-1A, but to a lesser extent ( approximately 6%) than did KIFMK. In contrast, KDIFMTK, which is ineffective in restoring inactivation, decreased the helical content ( approximately -4%). Furthermore, we studied the interactions between KIFMK and modified peptides from MP-1A, that is, MP-1NA (D1487N), MP-1QEA (E1492Q), or MP-1EQA (E1493Q). The KIFMK was found to increase the helical content of MP-1EQA to an extent nearly identical to that of MP-1A, whereas it was found to decrease those of MP-1NA and MP-1QEA. These findings mean that KIFMK, by allowing each of the Lys residues to interact with D1487 and E1492, respectively, stabilized the helical structure of the III-IV linker around the IFM residues. This helix-stabilizing effect of KIFMK on the III-IV linker may restore and/or accelerate fast inactivation to the sodium channels having a defective inactivation gate or to wild-type sodium channels.     

Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction

Two forms of cytochrome P-450 (P-450), designated P-450MP-1 and P-450MP-2, were purified to electrophoretic homogeneity from human liver microsomes on the basis of mephenytoin 4-hydroxylase activity. Purified P-450MP-1 and P-450MP-2 contained 12-17 nmol of P-450/mg of protein and had apparent monomeric molecular weights of 48,000 and 50,000, respectively. P-450MP-1 and P-450MP-2 were found to be very similar proteins as judged by chromatographic behavior on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE- and CM-cellulose columns, spectral properties, amino acid composition, peptide mapping, double immunodiffusion analysis, immunoinhibition, and N-terminal amino acid sequences. In vitro translation of liver RNA yielded polypeptides migrating with P-450MP-1 or P-450MP-2, depending upon which form was in each sample, indicating that the two P-450s are translated from different mRNAs. When reconsituted with NADPH-cytochrome-P-450 reductase and L-alpha-dilauroyl-sn-glyceryo-3-phosphocholine, P-450MP-1 and P-450MP-2 gave apparently higher turnover numbers for mephenytoin 4-hydroxylation than did the P-450 in the microsomes. The addition of purified rat or human cytochrome b5 to the reconstituted system caused a significant increase in the hydroxylation activity; the maximum stimulation was obtained when the molar ratio of cytochrome b5 to P-450 was 3-fold. Rabbit anti-human cytochrome b5 inhibited NADH-cytochrome-c reductase and S-mephenytoin 4-hydroxylase activities in human liver microsomes. In the presence of cytochrome b5, the Km value for S-mephenytoin was 1.25 mM with all five purified cytochrome P-450s preparations, and Vmax values were 0.8-1.25 nmol of 4-hydroxy product formed per min/nmol of P-450. P-450MP is a relatively selective P-450 form that metabolizes substituted hydantoins well. Reactions catalyzed by purified P-450MP-1 and P-450MP-2 preparations and inhibited by anti-P-450MP in human liver microsomes include S-mephenytoin 4-hydroxylation, S-nirvanol 4-hydroxylation, S-mephenytoin N-demethylation, and diphenylhydantoin 4-hydroxylation. Thus, at least two very similar forms of human P-450 are involved in S-mephenytoin 4-hydroxylation, an activity which shows genetic polymorphism.     

Peculiarities of immunocorrective effects of the bone marrow regulatory peptides (myelopeptides)

Myelopeptides (MPs) are low-molecular-weight immunoregulatory peptides of bone marrow origin. The peculiarities of their immunoregulatory effects are demonstrated with two of the six synthesized MPs, MP-1 (Phe-Leu-Gly-Phe-Pro-Thr) and MP-2 (Leu-Val-Val-Tyr-Pro-Trp). It is shown that MP action is directed to the damaged links of immunity. MP-1 enhances a decreased level of antibody production in cyclophosphamide (Cy)-treated mice, but does not influence the antibody formation in normal animals. MP-2 inhibits the tumor growth more in a tumor-bearing organism as the tumor size gets larger, insofar as MP-2 antitumor effect is concerned, by its ability to recover functional activity of T lymphocytes suppressed by tumor products. Selective immunocorrective effects of MPs are based on ligand-receptor interactions. Using FITC-labeled MP-1 and [3H]-labeled MP-2, specific binding of these peptides with appropriate cell populations is shown. The cytofluorimetric analysis revealed a target cell for MP-1--CD4+ T lymphocyte (T helper). The data obtained suggest that MPs are endogenic immunoregulators which participate in the maintenance of immune homeostasis.     

Assessment of transgene flow in tomato and potential effects of genetically modified tomato expressing Cry3Bb1 toxins on bumblebee feeding behaviour

One of the concerns surrounding the commercial release of genetically modified (GM) crops is the escape of transgenes into agricultural or semi‐natural habitats through vertical gene flow, as this may cause environmental or economic problems. There is also the concern that GM crops may affect pollinators and the pollination services they provide. Despite the growing commercial interest of GM tomato (Solanum lycopersicum), gene flow has been assessed only sparsely in tomato. To evaluate the likelihood of gene flow from GM tomato plants to sexually compatible plants, and to assess whether bumblebee activity is affected by GM tomato, three experiments were conducted under greenhouse conditions, using a Bt‐tomato expressing the insecticidal Cry3Bb1 protein as model system: (a) artificial crosses between a GM tomato line, two wild tomato relatives (Solanum hirsutum and Solanum nigrum) and a non‐GM tomato variety; (b) bumblebee‐mediated crosses between GM and non‐GM tomato plants and (c) visual observations of bumblebees' feeding behaviour. No hybrids were obtained between the GM tomato line and S. hirsutum and S. nigrum. In an experimental design where non‐GM receptor plants outnumbered GM plants by approximately 3:1, the bumblebee‐mediated cross‐fertilisation rate between GM and non‐GM tomato plants was measured at 4.3 ± 5.47%. No significant differences in feeding behaviour of bumblebees foraging on GM and non‐GM tomato plants were observed. Therefore, we conclude that: (a) the probability of transgene introgression between the GM tomato line used in this study and its wild relatives S. hirsutum and S. nigrum is negligible; (b) bumblebee activity can mediate cross‐fertilisation between GM and non‐GM tomato and (3) the Cry3Bb1‐expressing tomato line tested does not adversely affect the feeding behaviour of bumblebees.     

Peroxidase activity and isoenzymes in the culture medium of NaCl adapted tomato suspension cells

The medium of tomato (Lycopersicon esculentum) cells adapted to grow in the presence of 15 g l^–1 NaCl had a higher peroxidase activity than the medium of an unadapted tomato cell line. When the adapted cells were cultured in a medium without NaCl, the value found for peroxidase activity was intermediate. The increase in peroxidase activity was parallel to an increase of lignin-like compounds in the cell walls, as well as to an increased content or appearance of neutral and basic peroxidase isoenzymes. Apparently, the high values of peroxidase activity in the medium of the salt-adapted cells reflect the changed mechanical properties of the cell wall which, in turn, could be related to the salt adaptation process.Abbreviations LO Control tomato cell line unable to grow in the presence of 15 g 1^–1 of NaCl - L15 tomato cell line adapted to 15 g 1^–1 of NaCl and growing in this salt concentration - L15-0 tomato cell line adapted to 15 g 1^–1 of NaCl and growing in the absence of this salt - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PBS phosphate buffer saline     

Regulation of the peritoneal macrophage functional activity by the MP-5 and MP-6 myelopeptides under stress

In mice, two-hour immobilization stress inhibited zymosan-induced production by macrophages of the oxygen radicals and cytokine IL-1β. After myelopeptides MP-5 and MP-6 were administered into mice, the stress-induced inhibition of the reactive oxygen species (ROS) and IL-1β was abrogated. MP-5 peptide stimulated spontaneous ROS production by macrophages and reduced IL-10 production under stress. Thus, under in vivo conditions and under stress, the effect of MP-5 and MP-6 myelopeptides modulates the peritoneal macrophage activity.     

Modified expression of alternative oxidase in transgenic tomato and petunia affects the level of <Emphasis Type="Italic">tomato spotted wilt virus</Emphasis> resistance

Background

Tomato spotted wilt virus (TSWV) has a very wide host range, and is transmitted in a persistent manner by several species of thrips. These characteristics make this virus difficult to control. We show here that the over-expression of the mitochondrial alternative oxidase (AOX) in tomato and petunia is related to TSWV resistance.

Results

The open reading frame and full-length sequence of the tomato AOX gene LeAox1au were cloned and introduced into tomato 'Healani' and petunia 'Sheer Madness' using Agrobacterium-mediated transformation. Highly expressed AOX transgenic tomato and petunia plants were selfed and transgenic R1 seedlings from 10 tomato lines and 12 petunia lines were used for bioassay. For each assayed line, 22 to 32 tomato R1 progeny in three replications and 39 to 128 petunia progeny in 13 replications were challenged with TSWV. Enzyme-Linked Immunosorbent Assays showed that the TSWV levels in transgenic tomato line FKT4-1 was significantly lower than that of wild-type controls after challenge with TSWV. In addition, transgenic petunia line FKP10 showed significantly less lesion number and smaller lesion size than non-transgenic controls after inoculation by TSWV.

Conclusion

In all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower TSWV levels than non-transgenic plants after challenge with TSWV, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of AOX in tomato and petunia can affect the levels of TSWV resistance.

     

Genome Sequence of the Psychrophilic Deep-Sea Bacterium Moritella marina MP-1 (ATCC 15381)

Moritella marina MP-1 is a bacterial species known for its production of docosahexaenoic acid. We present the draft genome sequence of the type strain Moritella marina MP-1 (ATCC 15381), having 4,636,778 bp with a G+C content of 40.5% and consisting of 83 contigs.     

Identification of a short rDNA spacer sequence highly specific of a tomato line containing Tm-1 gene introgressed from Lycopersicon hirsutum

Summary We studied rDNA restriction fragment length polymorphism between two tomato lines used for F₁ hybrid seed production: line A, containing the Tm-1 gene responsible for tobacco mosaic virus tolerance introgressed from the wild species Lycopersicon hirsutum, and line B, a tobacco mosaic virus sensitive line. Hybridization patterns led to distinct rDNA maps with two size classes, 10.4 and 10.7 kb, in line A and a single, 8.9-kb class in line B. Size differences were located in the intergenie sequence (IGS). A highly specific 54-bp TaqI fragment was cloned from the line A IGS and used in dot blot experiments to probe total DNA from line A, line B, and their F₁ hybrid. It proved capable of discriminating B from A and the hybrid. This probe could thus serve to screen inbreds in commercial seed lots where line A is used as male. This fragment showed 80–90% sequence homology with the 53-bp subrepeats previously characterized in a region of the tomato IGS close to the 25S rRNA gene. Preliminary comparison of rDNA in line A and several wild related species indicated that the L. hirsutum H₂ genotype was the closest to line A. rDNA variations between line A and this wild genotype could be explained by recombination during the introgression process involving numerous backcrosses or by an important intraspecific polymorphism. Our results strongly suggest that Tm-1 and the rDNA were introgressed together into tomato from L. hirsutum through linkage drag.