Purification and electron microscopic characterization of the membrane subunit (IICB(Glc)) of the Escherichia coli glucose transporter

The glucose transporter of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It consists of a cytoplasmic subunit IIA(Glc) and the transmembrane subunit IICB(Glc). IICB(Glc) was purified to homogeneity by urea/alkali washing of membranes and nickel-chelate affinity chromatography. About 1.5 mg highly pure IICB(Glc) representing 77% of the total activity present in the membranes was obtained from 8g (wet weight) of cells. IICB(Glc) was reconstituted into lipid bilayers by temperature-controlled dialysis to yield small 2D crystals and by a rapid detergent-dilution procedure to yield densely packed vesicles. Electron microscopy and digital image processing of the negatively stained 2D crystals revealed a trigonal lattice with a unit cell size of a = b = 14.5 nm. The unit cell morphology exhibited three dimers of IICB(Glc) surrounding the threefold symmetry center. Single particle analysis of IICB(Glc) in proteoliposomes obtained by detergent dialysis also showed predominantly dimeric structures.     

Residue 3 of β2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody

 Previous studies of class I MHC molecules have shown that the owl monkey (Aotus) possesses at least two variants of the β₂-microglobulin (β₂m) protein. These two variants have different isoelectric points, and exhibit differential reactivity with the monoclonal antibody W6/32. We report cDNA sequences of the B2m gene, from W6/32-positive and W6/32-negative Aotus cell lines. The two β₂m variants we identified exhibit a single amino acid difference at position three. An arginine residue at position 3 was correlated with W6/32 reactivity, whereas histidine was associated with non-reactivity. W6/32 reactivity was conferred to a W6/32-negative Aotus cell line when it was transfected with the B2m from the W6/32-positive cell line. Residue 3 of β₂m is located at the surface of the class I molecule. It is also close to position 121 of the MHC class I heavy chain, which has previously been shown to influence W6/32 antibody binding. We conclude that W6/32 binds a compact epitope on the class I molecule that includes both residue 3 of β₂m and residue 121 of the heavy chain. We examined the distribution of the two β₂m motifs in a sample Aotus population using an allele-specific polymerase chain reaction assay. The pattern of β₂m segregation we observed matches that which was defined previously by serology. Additionally, we identified laboratory-born hybrid animals who possess both variants of β₂m. Received: 1 April 1998 / Received: 3 July 1998     

Combined effects of selected Penicillium mycotoxins on in vitro proliferation of porcine lymphocytes

The in vitro effect of combinations of the Penicillium mycotoxins citrinin (CIT), cyclopiazonic acid (CPA), ochratoxin A (OTA), patulin (PAT), penicillic acid (PIA) and roquefortine C (RQC) on mitogen induced lymphocyte proliferation was determined using purified lymphocytes from six piglets. Dose–response curves for each mycotoxin and mycotoxin combinations were generated. The combined effects of toxin pairs based on IC₂₀ were illustrated in isobole diagrams and statistically calculated. OTA and CIT elicited a synergistic effect. Four toxin pairs elicited additive effects, four pairs less–than–additive effects and six pairs independent effects. Thus, the majority of toxin pairs tested produced lower combined effects than an additive effect. The results indicate that the sum effect of all toxins is less than that from the summation of concentrations of the individual compounds, adjusted for differences in potencies.     

AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis

For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K(0.5) = 416 microm) for Ala-Asp compared with Ala-Ala (K(0.5) = 54 microm) and Ala-Lys (K(0.5) = 112 microm). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.     

Covalent binding of the 13C-labeled skin sensitizers 5-chloro-2-methylisothiazol-3-one (MCI) and 2-methylisothiazol-3-one (MI) to a model peptide and glutathione

The reactivity of 4-[13C]- and 5-[13C]-5-chloro-2-methylisothiazol-3-one (MCI) and 2-methylisothiazol-3-one (MI) towards a model peptide and glutathione was followed by 13C and 1H[13C] NMR spectroscopy. Both molecules were found to react with GSH but in addition MCI was found to react with histidine and lysine to form adducts of a different nature. Reaction with histidine led to stable substitution adducts through an addition-elimination reaction at position 5 while reaction with lysine led to the formation of open adducts of the thioamide or amide type.     

Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair

Chromosome stability depends on accurate chromosome segregation and efficient DNA double-strand break (DSB) repair. Sister chromatid cohesion, established during S phase by the protein complex cohesin, is central to both processes. In the absence of cohesion, chromosomes missegregate and G2-phase DSB repair fails. Here, we demonstrate that G2-phase repair also requires the presence of cohesin at the damage site. Cohesin components are shown to be recruited to extended chromosome regions surrounding DNA breaks induced during G2. We find that in the absence of functional cohesin-loading proteins (Scc2/Scc4), the accumulation of cohesin at DSBs is abolished and repair is defective, even though sister chromatids are connected by S phase generated cohesion. Evidence is also provided that DSB induction elicits establishment of sister chromatid cohesion in G2, implicating that damage-recruited cohesin facilitates DNA repair by tethering chromatids.     

Analysis of kavalactones from Piper methysticum (kava-kava)

The chemical analysis and quality control of both Piper methysticum G. Forster (kava-kava) and extracts obtained by aqueous acetone or aqueous methanol as well as supercritical fluid extraction are reviewed. In the last two decades various procedures concerning the separation and detection of kavalactones have been routinely carried out by gas chromatography (without previous derivatization of kavalactones) and high performance liquid chromatography but most of them are not validated or only partially validated. Recently, analyses by supercritical fluid chromatography and micellar electrokinetic chromatography have also been reported. Both gas chromatography and high performance liquid chromatography can be used for the analysis of kavalactones with some advantages and disadvantages for each method. Using gas chromatography analysis, methysticin and yangonin, which are two of the major components, are generally not separated. In addition, the high temperature of the injection port caused the decomposition of methysticin. Concerning high performance liquid chromatography analyses, the reversed-phase is generally better because highly reproducible with a very low detection limit for all compounds even if the quantitative analysis of the kavalactones by liquid chromatography needs to be carried out in the absence of light to prevent the cis/trans isomerisation of yangonin.     

Reloading of atrophied rat soleus muscle induces tenascin-C expression around damaged muscle fibers

The hypothesis was tested that mechanical loading, induced by hindlimb suspension and subsequent reloading, affects expression of the basement membrane components tenascin-C and fibronectin in the belly portion of rat soleus muscle. One day of reloading, but not the previous 14 days of hindlimb suspension, led to ectopic accumulation of tenascin-C and an increase of fibronectin in the endomysium of a proportion (8 and 15%) of muscle fibers. Large increases of tenascin-C (40-fold) and fibronectin (7-fold) mRNA within 1 day of reloading indicates the involvement of pretranslational mechanisms in tenascin-C and fibronectin accumulation. The endomysial accumulation of tenascin-C was maintained up to 14 days of reloading and was strongly associated with centrally nucleated fibers. The observations demonstrate that an unaccustomed increase of rat soleus muscle loading causes modification of the basement membrane of damaged muscle fibers through ectopic endomysial expression of tenascin-C.