The physiology and biochemistry of cultivar-strain interactions in the white clover-Rhizobium symbiosis

Two white clover cultivars were inoculated with two Rhizobium leguminosarum bv. trifolii strains in a factorial series of experiments. Plants were grown in axenic conditions in nitrogen free nutrient solution in a controlled environment room. Variations in nitrogen fixation were dependent partly upon general strain effects, partly upon general cultivar effects but there were also substantial differences attributable to precise interactions between specific combinations. The physiological and biochemical basis of these differences was examined. There were variations in the onset of nodulation and nitrogenase (acetylene reduction) activity. The rate at which nitrogenase activity developed also differed between associations as did the average size and number of nodules but none of these effects correlated well with differences in plant dry matter accumulation. Studies on nodule biochemistry revealed that the major nitrogen fixation enzymes were present in all four associations. Nodule protein content and enzyme activity (on a g nodule fresh weight basis) were substantially greater in associations formed by the more effective strain but cannot explain the interactive effect on dry matter accumulation. The relevance of these data to our understanding of factors regulating variations in nitrogen fixation is discussed.     

Intracellular Regulation of Cross-Presentation during Dendritic Cell Maturation

We have investigated the effect of different maturation stimuli on the ability of mature dendritic cells (DCs) to cross-present newly acquired particulate antigens. Cross-presentation was impaired in DCs matured by treatment with TNF-α, CpG and LPS, but was less affected upon CD40L-induced maturation. The difference could not be explained by decreased antigen uptake or translocation into the cytosol, but decreased cross-presentation ability did correlate with increased phagosomal/lysosomal acidification. Nevertheless, intra-phagosomal degradation of OVA was not increased in matured samples, suggesting that decreasing phagosomal pH may also regulate cross-presentation by a mechanism other than enhancing degradation.     

Biochemical and growth regulatory activities of the HIN-200 family member and putative tumor suppressor protein, AIM2

The human HIN-200 family member AIM2 was originally identified in a screen for suppressors of melanoma tumorigenicity following introduction of chromosome 6 into the UACC903 human melanoma cell line. Although the AIM2 protein contained many of the conserved structural motifs common to other HIN-200 proteins, the biochemical characteristics of AIM2 and the ability of overexpressed AIM2 to phenocopy the effect of introduction of chromosome 6 in the UACC903 cells had not been assessed. Herein we demonstrated that AIM2 was localised within the nucleus of transfected or interferon-treated human cells. In addition, AIM2 could homodimerise via the amino-terminal (PAAD/DAPIN) region and heterodimerise with the related IFI 16 protein. However, overexpressed AIM2 did not significantly affect the growth or survival of UACC903 cells or another human melanoma cell line. These data indicate that AIM2 has many of the biochemical and structural characteristics of HIN-200 proteins, however, its expression is not sufficient to induce a tumor-suppressor-like phenotype.     

Intracellular surveillance: controlling the assembly of MHC class I-peptide complexes

MHC class I molecules bind with high affinity to peptides in the endoplasmic reticulum and display them on the cell surface. Here they are screened by CD8-positive T-lymphocytes for the presence of foreign, pathogen-derived peptides within the mass of self-peptides expressed. MHC class I assembly is a complicated process involving a number of accessory molecules, including specialized components as well as common chaperones. Our understanding of the mechanisms involved, while quite advanced, is far from complete.     

A role for calnexin in the assembly of the MHC class I loading complex in the endoplasmic reticulum

Heterodimers of MHC class I glycoprotein and beta(2)-microglobulin (beta(2)m) bind short peptides in the endoplasmic reticulum (ER). Before peptide binding these molecules form part of a multisubunit loading complex that also contains the two subunits of the TAP, the transmembrane glycoprotein tapasin, the soluble chaperone calreticulin, and the thiol oxidoreductase ERp57. We have investigated the assembly of the loading complex and provide evidence that after TAP and tapasin associate with each other, the transmembrane chaperone calnexin and ERp57 bind to the TAP-tapasin complex to generate an intermediate. These interactions are independent of the N:-linked glycan of tapasin, but require its transmembrane and/or cytoplasmic domain. This intermediate complex binds MHC class I-beta(2)m dimers, an event accompanied by the loss of calnexin and the acquisition of calreticulin, generating the MHC class I loading complex. Peptide binding then induces the dissociation of MHC class I-beta(2)m dimers, which can be transported to the cell surface.     

The occurrence of two species of <Emphasis Type="Italic">Macroramphosus</Emphasis> (Gasterosteiformes: Macroramphosidae) in Japan: morphological and ecological observations on larvae,juveniles, and adults

Earlier opinions that Macroramphosus is monotypic are refuted, with two species apparently occurring in Japan (tentatively identified as M. gracilis and M. scolopax). In postsettlement young and adults, the former is characterized by a dark slender body (vs. red-orange and deep) and short second dorsal fin spine with a smooth posterior margin (vs. long spine with a serrated margin). Food habits also differ between the two species, which are either plankton or benthos feeders. Two types of Macroramphosus larvae and juveniles occurring at the surface were recognized, one having a straight ventral body profile of the body (identified here as M. gracilis) and the other having a notch in the anal region. The dark body of postsettlement M. gracilis is considered to be a retention of the character suited to the neustonic distribution of the larval and juvenile stages, the species remaining to ca. 40mm in standard length (SL) in that habitat (vs. to ca. 12mm SL in M. scolopax).     

Identification of specific glycoforms of major histocompatibility complex class I heavy chains suggests that class I peptide loading is an adaptation of the quality control pathway involving calreticulin and ERp57

Glycosylation analysis was used to probe the sequence of events accompanying the binding of antigenic peptides to the major histocompatibility complex class I heavy chains. Free heavy chains were isolated from the beta(2)-microglobulin-negative cell line Daudi and from the B-lymphoblastoid cell line Raji. Heavy chains were also isolated from Raji cells in multimolecular complexes (peptide loading complexes) containing the transporter associated with antigen processing, tapasin and ERp57 with and without the lectin-like folding chaperone, calreticulin. Calreticulin is a soluble protein that recognizes primarily the terminal glucose of Glc(1)Man(7-9)GlcNAc(2) glycans. This paper shows that monoglucosylated glycoforms of heavy chain, which exist transiently in the endoplasmic reticulum in the initial stages of the glycosylation processing pathway, are present in the peptide loading complex. The data are consistent with a model in which the release of peptide-loaded major histocompatibility complex class I molecules from calreticulin, induced by deglucosylation of the heavy chain N-linked glycan, signals the dissociation of the complex. This is consistent with the hypothesis that the class I loading process is an adaptation of the quality control mechanism involving calreticulin and ERp57.     

Quality control of transmembrane domain assembly in the tetraspanin CD82

Retention of misfolded proteins in the endoplasmic reticulum (ER) is a primary mechanism of quality control. To discover whether quality control can monitor assembly inside the hydrophobic ER membrane, we characterized the folding and transport of the tetraspanin glycoprotein CD82. Truncated forms of CD82 that are missing one or more transmembrane segments remain in the ER. A construct (TM 2-4) that is missing the first transmembrane segment remains in the ER, even though its extracellular domain, which is facing the ER lumen, has folded to the native structure. Transport to the cell surface is restored by co-expressing the missing segment (TM 1) as a separate polypeptide. Prior to leaving the ER, CD82 transiently associates with the membrane-bound chaperone calnexin but not with its soluble homolog calreticulin. TM 2-4, in contrast, remains in a prolonged interaction with calnexin that is partially reversed by co-expressing TM 1. These findings establish a simple system to study transmembrane domain assembly, show that ER quality control can directly monitor assembly inside the lipid bilayer and suggest that calnexin may play a role in this process.     

Stem tissue phosphorus as an index of the phosphorus status of Banksia ericifolia L. f.

The effects of P fertilizer rate on shoot growth and the total P concentration of the whole shoot, new and mature leaves, symptom leaves and stems of Banksia ericifolia L. f., a P-sensitive species, were investigated in a six month greenhouse pot experiment. Shoot dry weight of plants growing in an Australian sedge peat, coarse sand and perlite potting mix (1:1:1) increased with up to 100 mg P L⁻¹ supplied as a six month controlled release P (0:18:0) fertilizer, but was reduced by toxicity at the highest application rate (200 mg P L⁻¹). Plants receiving this treatment developed chlorotic new and mature leaves. Leaf symptoms observed at rates of 60–100 mg P L⁻¹ were confined to old leaves and were related to the P concentration of the shoot. Growth was not affected at these rates. The P concentration of stems was strongly influenced by P supply. This tissue acted as a sink for excess P, helping to regulate the P concentration of leaves. The approximate range of P concentrations in stem tissue, associated with greater than 90% of maximum shoot dry weight, was 0.5–1.5 g P kg⁻¹ tissue dry weight. This was greater than that calculated for mature leaves (0.5–0.8 g kg⁻¹) or for whole shoots (0.5–1.2 g kg⁻¹). This wider range, and the capacity to store P in excess to requirement, makes the stem a better index tissue for plant P status than either leaves or whole shoots. This revised version was published online in June 2006 with corrections to the Cover Date.