Phenotypic changes associated with RNA interference silencing of chalcone synthase in apple (Malus × domestica)

We have identified in apple (Malus × domestica) three chalcone synthase (CHS) genes. In order to understand the functional redundancy of this gene family RNA interference knockout lines were generated where all three of these genes were down‐regulated. These lines had no detectable anthocyanins and radically reduced concentrations of dihydrochalcones and flavonoids. Surprisingly, down‐regulation of CHS also led to major changes in plant development, resulting in plants with shortened internode lengths, smaller leaves and a greatly reduced growth rate. Microscopic analysis revealed that these phenotypic changes extended down to the cellular level, with CHS‐silenced lines showing aberrant cellular organisation in the leaves. Fruit collected from one CHS‐silenced line was smaller than the ‘Royal Gala’ controls, lacked flavonoids in the skin and flesh and also had changes in cell morphology. Auxin transport experiments showed increased rates of auxin transport in a CHS‐silenced line compared with the ‘Royal Gala’ control. As flavonoids are well known to be key modulators of auxin transport, we hypothesise that the removal of almost all flavonoids from the plant by CHS silencing creates a vastly altered environment for auxin transport to occur and results in the observed changes in growth and development.     

Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy

Gut-associated lymphoid tissue (GALT) harbors the majority of T lymphocytes in the body and is an important target for human immunodeficiency virus type 1 (HIV-1). We analyzed longitudinal jejunal biopsy samples from HIV-1-infected patients, during both primary and chronic stages of HIV-1 infection, prior to and following the initiation of highly active antiretroviral therapy (HAART) to determine the onset of CD4(+) T-cell depletion and the effect of HAART on the restoration of CD4(+) T cells in GALT. Severe depletion of intestinal CD4(+) T cells occurred during primary HIV-1 infection. Our results showed that the restoration of intestinal CD4(+) T cells following HAART in chronically HIV-1-infected patients was substantially delayed and incomplete. In contrast, initiation of HAART during early stages of infection resulted in near-complete restoration of intestinal CD4(+) T cells, despite the delay in comparison to peripheral blood CD4(+) T-cell recovery. DNA microarray analysis of gene expression profiles and flow-cytometric analysis of lymphocyte homing and cell proliferation markers demonstrated that cell trafficking to GALT and not local proliferation contributed to CD4(+) T-cell restoration. Evaluation of jejunal biopsy samples from long-term HIV-1-infected nonprogressors showed maintenance of normal CD4(+) T-cell levels in both GALT and peripheral blood. Our results demonstrate that near-complete restoration of mucosal immune system can be achieved by initiating HAART early in HIV-1 infection. Monitoring of the restoration and/or maintenance of CD4(+) T cells in GALT provides a more accurate assessment of the efficacy of antiviral host immune responses as well as HAART.     

Dietary oxidized fatty acids may enhance intestinal apolipoprotein A-I production

Apolipoprotein (apo)A-I, the major protein component of HDL, is synthesized principally in the small intestine and liver. Recently we observed an increase in plasma apoA-I level in humans who were on an oxidized fat diet. To test whether oxidized fatty acids could affect apoA-I synthesis, we incubated day 4 (undifferentiated) and day 14 (differentiated) Caco-2 cells with varying concentrations of oxidized linoleic acid (ox-linoleic acid) (5, 10, and 25 microM) and unoxidized linoleic acid for 24 h. Ox-linoleic acid caused a dose-dependent increase in the levels of apoA-I protein in both differentiated and undifferentiated Caco-2 cells as assessed by ELISA and Western blot analysis. Whereas apoB production was not increased by ox-linoleic acid in both day 4 and day 14 Caco-2 cells. The mRNA expression for apoA-I paralleled the protein expression when measured by RT-PCR. We also found that both day 4 and day 14 Caco-2 cells did express peroxisomal proliferator-activated receptor-gamma (PPAR-gamma). mRNA and PPAR-gamma ligand could increase apoA-I secretion in these cells.Therefore we propose that the mechanism for the induction of apoA-I might include PPAR-gamma for which oxidized fatty acid is a ligand.     

Effect of denaturants on the structure and activity of 3-hydroxybenzoate-6-hydroxylase

The effect of denaturants such as urea, sodium dodecylsulphate (SDS), guanidinium hydrochloride (Gu.HCl) on the structure of enzyme 3-hydroxybenzoate-6-hydroxylase was studied using intrinsic fluorescence and far and near-UV-CD spectroscopic techniques. Also, activity profiles of the enzyme, as a function of increasing concentrations of denaturants were studied. The far-UV CD spectrum of the enzyme did not show appreciable alterations in the presence of urea, SDS or Gu.HCl, thereby suggesting that the protein does not undergo gross conformational changes in its alpha-helical secondary structure. The treatment of enzyme with 2 M urea resulted in almost complete loss of catalytic activity, accompanied by the reduction of emission fluorescence of enzyme. Similarly, treatment with 0.01% SDS also caused almost complete loss of activity and quenching of enzyme fluorescence as well as a red shift in the emission peak. In addition, reduction in the intensity of near-UV-CD spectrum, especially at 280 nm was observed. About 70% of the activity was lost by treatment with 20 mM Gu.HCl, accompanied by quenching of intrinsic fluorescence of the enzyme. The change in intrinsic fluorescence of the enzyme in the presence of 5 mM-100 mM Gu.HCI could be correlated to progressive loss of catalytic activity. Thus, intrinsic fluorescence (due to tryptophan residues) could be used as an effective probe to provide an insight into the relation between the activity and subtle conformational changes of the enzyme. The results suggested that denaturants caused very slight conformational changes in the enzyme that perturbed the microenvironment of aromatic amino acid residues such as tryptophan accompanied by reduction or loss of catalytic activity.     

Interaction of 3-hydroxybenzoate-6-hydroxylase with cibacron blue

Cibacron blue is a potent inhibitor of 3-HBA-6-hydroxylase at a concentration < 1 microM. Kinetic analyses revealed that at a concentration below 0.5 microM the dye behaves as an uncompetitive inhibitor with respect to 3-HBA and competes with NADH for the same site on the enzyme. The alteration of the near-UV CD spectrum and quenching of the emission fluorescence of the enzyme by cibacron blue indicates a significant alteration in the environment of aromatic amino acid residues due to a stacking interaction and subtle conformatiodnal changes in the enzyme. The concentration-dependent quenching of the intrinsic fluorescence of the enzyme by cibacron blue was employed to determine the binding parameters such as association constant (Ka) and stoichiometry (r) for the enzyme-dye complex.     

Antiviral therapy during primary simian immunodeficiency virus infection fails to prevent acute loss of CD4+ T cells in gut mucosa but enhances their rapid restoration through central memory T cells

Gut-associated lymphoid tissue (GALT) is an early target of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) and a site for severe CD4+ T-cell depletion. Although antiretroviral therapy (ART) is effective in suppressing HIV replication and restoring CD4+ T cells in peripheral blood, restoration in GALT is delayed. The role of restored CD4+ T-cell help in GALT during ART and its impact on antiviral CD8+ T-cell responses have not been investigated. Using the SIV model, we investigated gut CD4+ T-cell restoration in infected macaques, initiating ART during either the primary stage (1 week postinfection), prior to acute CD4+ cell loss (PSI), or during the chronic stage at 10 weeks postinfection (CSI). ART led to viral suppression in GALT and peripheral blood mononuclear cells of PSI and CSI animals at comparable levels. CSI animals had incomplete CD4+ T-cell restoration in GALT. In PSI animals, ART did not prevent acute CD4+ T-cell loss by 2 weeks postinfection in GALT but supported rapid and complete CD4+ T-cell restoration thereafter. This correlated with an accumulation of central memory CD4+ T cells and better suppression of inflammation. Restoration of CD4+ T cells in GALT correlated with qualitative changes in SIV gag-specific CD8+ T-cell responses, with a dominance of interleukin-2-producing responses in PSI animals, while both CSI macaques and untreated SIV-infected controls were dominated by gamma interferon responses. Thus, central memory CD4+ T-cell levels and qualitative antiviral CD8+ T-cell responses, independent of viral suppression, were the immune correlates of gut mucosal immune restoration during ART.