Ammonium and nitrate uptake rates and rhizosphere pH in non-mycorrhizal roots of Norway spruce [Picea abies (L.) Karst.]

Summary Relationships between root zone temperature, concentrations and uptake rates of NH ₄ ⁺ and NO ₃ ^– were studied in non-mycorrhizal roots of 4-year-old Norway spruce under controlled environmental conditions. Additionally, in a forest stand NH ₄ ⁺ and NO ₃ ^– uptake rates along the root axis and changes in the rhizosphere pH were measured. In the concentration (C_min) range of 100–150 M uptake rates of NH ₄ ⁺ were 3–4 times higher than those of NO ₃ ^– The preference for NH ₄ ⁺ uptake was also reflected in the minimum concentration (C_min) values. Supplying NH₄NO₃, the rate of NO ₃ ^– uptake was very low until the NH ₄ ⁺ concentrations had fallen below about 100 M. The shift from NH ₄ ⁺ to NO ₃ ^– uptake was correlated with a corresponding shift from net H⁺ production to net H⁺ consumption in the external solution. The uptake rates of NH ₄ ⁺ were correlated with equimolar net production of H⁺. With NO ₃ ^– nutrition net consumption of H⁺ was approximately twice as high as uptake rates of NO ₃ ^– In the forest stand the NO ₃ ^– concentration in the soil solution was more than 10 times higher than the NH ₄ ⁺ concentration (<100 M), and the rhizosphere pH of non-mycorrhizal roots considerably higher than the bulk soil pH. The rhizosphere pH increase was particularly evident in apical root zones where the rates of water and NO ₃ ^– uptake and nitrate reductase activity were also higher. The results are summarized in a model of water and nutrient transport to, and uptake by, non-mycorrhizal roots of Norway spruce in a forest stand. Model calculations indicate that delivery to the roots by mass flow may meet most of the plant demand of nitrogen and calcium, and that non-mycorrhizal root tips have the potential to take up most of the delivered nitrate and calcium.     

Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction

Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.     

Phosphorylation of tau protein by recombinant GSK-3beta: pronounced phosphorylation at select Ser/Thr-Pro motifs but no phosphorylation at Ser262 in the repeat domain.

Glycogen synthase kinase-3beta (GSK-3beta) has been described as a proline-directed kinase which phosphorylates tau protein at several sites that are elevated in Alzheimer paired helical filaments. However, it has been claimed that GSK-3beta can also phosphorylate the non-proline-directed KXGS motifs in the presence of heparin, including Ser262 in the repeat domain of tau, which could induce the detachment of tau from microtubules. We have analyzed the activity of recombinant GSK-3beta and of GSK-3beta preparations purified from tissue, using two-dimensional phosphopeptide mapping, immunoblotting with phosphorylation-sensitive antibodies, and phosphopeptide sequencing. The most prominent phosphorylation sites on tau are Ser396 and Ser404 (PHF-1 epitope), Ser46 and Thr50 in the first insert, followed by a less efficient phosphorylation of other Alzheimer phosphoepitopes (antibodies AT-8, AT-270, etc). We also show that the non-proline-directed activity at KXGS motifs is not due to GSK-3beta itself, but to kinase contaminations in common GSK-3beta preparations from tissues which are activated upon addition of heparin.     

The GTP-binding peptide of beta-tubulin. Localization by direct photoaffinity labeling and comparison with nucleotide-binding proteins

The binding site of the guanine moiety of GTP on beta-tubulin was located within the peptide consisting of residues 63-77, AILVDLEPGTMDSVR. The result was obtained using direct photoaffinity labeling, peptide sequencing, and limited proteolysis. Peptides were identified by end-labeling with a monoclonal antibody against beta-tubulin whose epitope was located between 3 and 4 kDa from the C terminus. The sequence of the GTP-binding site is consistent with predictions from other GTP-binding proteins such as elongation factor Tu or ras p21.     

Human Rod Monochromacy: Linkage Analysis and Mapping of a Cone Photoreceptor Expressed Candidate Gene on Chromosome 2q11

We have performed linkage analysis in eight families with rod monochromacy, an autosomal recessively inherited condition with complete color blindness. Significant linkage was found with markers located at the pericentromeric region of chromosome 2. A maximum lod score of 5.36 was obtained for marker D2S2333 at θ = 0.00. Mapping of meiotic breakpoints localized the disease gene between markers D2S2187 and D2S2229. Homozygosity for a number of subsequent markers indicating identity by descent was found in two families and provides evidence for a further refinement of the locus proximal to D2S373. This defines an interval of ≈3 cM covering theACHM2locus for rod monochromacy. Radiation hybrid mapping of theCNGA3gene encoding the α-subunit of the cGMP gated cation channel in human cone photoreceptors resulted in a maximum lod score of 16.1 with marker D2S2311 combined with a calculated physical distance of 6.19cR_10,000. Screening of the CEPH YAC library and subsequent STS mapping indicated the physical order cen–D2S2222–D2S2175–(D2S2187/D2S2311)–qtel ofmarkers on 2q11 and showed that theCNGA3gene maps most closely to D2S2187 and D2S2311. These data indicate that theCNGA3gene maps within the critical interval of theACHM2locus for rod monochromacy and thus is a candidate gene for this disease.     

Molecular and cellular requirements for enhanced antigen cross-presentation to CD8 cytotoxic T lymphocytes

MHC class I-mediated cross-priming of CD8 T cells by APCs is critical for CTL-based immunity to viral infections and tumors. We have shown previously that tumor-secreted heat shock protein gp96-chaperoned peptides cross prime CD8 CTL that are specific for genuine tumor Ags and for the surrogate Ag OVA. We now show that tumor-secreted heat shock protein gp96-chaperoned peptides enhance the efficiency of Ag cross-priming of CD8 CTL by several million-fold over the cross-priming activity of unchaperoned protein alone. Gp96 also acts as adjuvant for cross-priming by unchaperoned proteins, but in this capacity gp96 is 1000-fold less active than as a peptide chaperone. Mechanistically, the in situ secretion of gp96-Ig by transfected tumor cells recruits and activates dendritic cells and NK cells to the site of gp96 release and promotes CD8 CTL expansion locally. Gp96-mediated cross-priming of CD8 T cells requires B7.1/2 costimulation but proceeds unimpeded in lymph node-deficient mice, in the absence of NKT and CD4 cells and without CD40L. Gp96-driven MHC I cross-priming of CD8 CTL in the absence of lymph nodes provides a novel mechanism for local, tissue-based CTL generation at the site of gp96 release. This pathway may constitute a critically important, early detection, and rapid response mechanism that is operative in parenchymal tissues for effective defense against tissue damaging antigenic agents.     

Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters

The yeast glucose transporters Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 and Gal2, individually expressed in an hxt1-7 null mutant strain, demonstrate the phenomenon of countertransport. Thus, these transporters, which are the most important glucose transporters in Saccharomyces cerevisiae, are facilitated diffusion transporters. Apparent K(m)-values from high to low affinity, determined from countertransport and initial-uptake experiments, respectively, are: Hxt6 0.9+/-0.2 and 1.4+/-0.1 mM, Hxt7 1.3+/-0.3 and 1.9+/-0.1 mM, Gal2 1.5 and 1.6+/-0.1 mM, Hxt2 2.9+/-0.3 and 4.6+/-0.3 mM, Hxt4 6.2+/-0.5 and 6.2+/-0.3 mM, Hxt3 28.6+/-6.8 and 34.2+/-3.2 mM, and Hxt1 107+/-49 and 129+/-9 mM. From both independent methods, countertransport and initial uptake, the same range of apparent K(m)-values was obtained for each transporter. In contrast to that in human erythrocytes, the facilitated diffusion transport mechanism of glucose in yeast was symmetric. Besides facilitated diffusion there existed in all single glucose transport mutants, except for the HXT1 strain, significant first-order behaviour.     

Holistic differential analysis of embryo-induced alterations in the proteome of bovine endometrium in the preattachment period

During the peri-implantation period, molecular signaling between embryo and endometrium (layer of tissue lining the uterus lumen) is supposed to be crucial for the maintenance of pregnancy. To investigate embryo-induced alterations in the proteome of bovine endometrium in the preattachment period (day 18), we used monozygotic cattle twins (generated by embryo splitting) as a model eliminating genetic variability as a source for proteome differences. One of the twins was pregnant after the transfer of two in vitro produced blastocysts, while the corresponding twin received a sham-transfer and served as a nonpregnant control. The two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of the endometrium samples of three twin pairs (pregnant/nonpregnant) revealed four proteins with significantly higher abundance (p < 10(-9)) in each sample derived from the pregnant animals: Rho GDP dissociation inhibitor beta; 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD); soluble NADP(+)-dependent isocitrate dehydrogenase 1; and acyl-CoA-binding protein. To verify the accuracy of the 2-D DIGE quantification, the abundances of 20 alpha-HSD were quantified by a targeted cleavable isotope-coded affinity tag (ICAT) approach. The mass spectrometry-based ICAT quantification matched perfectly the results obtained by 2-D DIGE quantification, demonstrating the accuracy of our data. These results demonstrate that our model (monozygotic twins) in combination with the appropriate analytical tools is particularly suitable for the detection of the proteins involved in the embryo-maternal interactions.