Nucleotide sequence of psbQ gene for 16-kDa protein of oxygen-evolving complex from Arabidopsis thaliana and regulation of its expression.

The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected.     

Microbial production of d-amino acids

The production of d-aminoacylase by Alcaligenes denitrificans and Alcaligenes faecalis has been studied. The enzyme was inducibly produced and N-acetyl-d-leucine and N-acetyl-d-valine were the most effective inducers. d-methionine, d-valine, d-phenylalamine and d-leucine were produced by the enzymic hydrolysis of the appropriate N-acetyl-d-amino-acids with whole cell biomass. The hydrolysis of N-acetyl-d-methionine by A. denitrificans and N-acetyl-d-valine by A. faecalis was preferential. Maximum yields of d-methionine and d-valine were 94.3 and 84.7% at a specific product formation rate of 20.10 and 19.19 μmol min⁻¹ mg⁻¹ of wet cells at 20 mM substrate concentration and 5 mg ml⁻¹ of cell density.     

Nucleolar organizer region heteromorphism associated with trisomy-21: a risk factor for non-disjunction?

An unusual nucleolar organizer region (double NOR) on chromosome 13 was observed in a Down syndrome child [47, XY, +21, dNOR(13)]. The variant chromosome was inherited from the mother [46, XX, dNOR(13)]. The extra chromosome 21 in the proband was maternal origin. The frequency of NOR chromosome association showed relatively high frequency in the mother and proband as compared to the controls. The result suggest that chromosome variants involving extra copies of NOR may indeed be involved in the meiotic nondisjunction of chromosome-21.     

The superoxide-generating respiratory burst oxidase of human neutrophil plasma membrane. Phosphatidylserine as an effector of the activated enzyme

The superoxide-generating respiratory burst oxidase is an integral membrane enzyme found in the plasma membrane of polymorphonuclear leukocytes (neutrophils). NADPH-dependent superoxide generation is seen in isolated plasma membranes and in their detergent extracts following activation of the intact cells with phorbol myristate acetate. We have herein examined the effects of phospholipids on the activity of the solubilized oxidase. Solubilization of plasma membranes with 0.5% each of Tween 20 plus deoxycholate resulted in an approximately 2-fold enhancement of activity. Inclusion of phospholipids in the extraction medium resulted in further activation. At 1.0 mg/ml the order of effectiveness was phosphatidylserine (PS) greater than cardiolipin greater than phosphatidylethanolamine greater than phosphatidylinositol; phosphatidylcholine and phosphorylated inositol lipids were not effective. The concentrations required for half-maximal activation by PS and phosphatidylethanolamine were 85 and 200 micrograms/ml, respectively. When PS was used at a maximally activating concentration (0.5 mg/ml), the activity was enhanced 3-5-fold. Detergent solubilization alone elevated the Km of the oxidase for NADPH from 68 microM in intact plasma membranes to 123 microM, but inclusion of PS with detergent restored the Km to near or below that seen in intact membranes. PS also increased the Vmax by a factor of 2-3, but had no effect on the pH optimum. A plot of the activity versus enzyme concentration was linear when membranes were used, but activity showed a quadratic dependence on concentration in solubilized membrane, with lower than expected activity at lower enzyme concentration. PS restored linearity of the concentration-activity plot. The activation by PS was not influenced by the addition of Ca2+, EGTA, or dioctanoylglycerol, indicating that activation was not dependent on protein kinase C. These results implicate phosphatidylserine as a direct effector of the NADPH-oxidase.     

Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: effects of guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile.

We reported previously that diacylglycerol (diC8) and GTP gamma S synergize with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to produce high rates of superoxide generation in a cell-free system consisting of neutrophil plasma membrane plus cytosol [Burnham, D. N., Uhlinger, D. J., & Lambeth, J. D. (1990) J. Biol. Chem. 265, 17550-17559]. Here we investigate the effects of these activating factors on the plasma membrane association in an in vitro translated radiolabeled recombinant p47-phox protein. Apparent translocation, assayed by cosedimentation with plasma membranes, required the presence of excess cytosol and an anionic amphiphile, was enhanced by both GTP gamma S and diC8, and was inhibited by high salt, correlating qualitatively with activation; up to 70% cosedimentation was observed with the combination of activators (compared with less than 20% in their absence). Similar results were obtained using heat-inactivated cytosol, wherein another oxidase component, p67-phox, has been inactivated. Unexpectedly, from 50 to 80% of the apparent translocation occurred in the absence of membranes, indicating that protein aggregation accounted for a significant part of the observed translocation. Nevertheless, the percent translocation was increased in all cases by the presence of membranes, indicating some degree of protein-membrane interaction. While a control in vitro translated protein failed to translocate, cosedimentation of p47-phox occurred equally well when red blood cell or neutrophil plasma membranes lacking cytochrome b558 were used. Also, the peptide RGVHFIF, which is contained within the C-terminus of the large subunit of cytochrome b558, failed to inhibit translocation/aggregation of p47-phox, despite its ability to inhibit cell-free activation of the oxidase. The data are consistent with the following: (a) SDS, diC8, and GTP gamma S all act on cytosolic components to alter protein-protein and/or protein-membrane associations, and these changes are necessary (but not sufficient) for activation; (b) these altered associations are likely to function by increasing the local concentration of p47-phox and other components at the plasma membrane; (c) a high background of nonspecific associations in the cell-free activation system is likely to obscure any specific, functionally relevant associations (e.g., with cytochrome b558); and (d) the mechanism of translocation in the cell-free system differs from that seen in intact neutrophils.     

The Residue Threonine 82 of DevR (DosR) Is Essential for DevR Activation and Function in Mycobacterium tuberculosis Despite Its Atypical Location

The DevR (DosR) response regulator initiates the bacterial adaptive response to a variety of signals, including hypoxia in in vitro models of dormancy. Its receiver domain works as a phosphorylation-mediated switch to activate the DNA binding property of its output domain. Receiver domains are characterized by the presence of several highly conserved residues, and these sequence features correlate with structure and hence function. In response regulators, interaction of phosphorylated aspartic acid at the active site with the conserved threonine is believed to be crucial for phosphorylation-mediated conformational change. DevR contains all the conserved residues, but the structure of its receiver domain in the unphosphorylated protein is strikingly different, and key threonine (T82), tyrosine (Y101), and lysine (K104) residues are placed uncharacteristically far from the D54 phosphorylation site. In view of the atypical location of T82 in DevR, the present study aimed to examine the importance of this residue in the activation mechanism. Mycobacterium tuberculosis expressing a DevR T82A mutant protein is defective in autoregulation and supports hypoxic induction of the DevR regulon only very weakly. These defects are ascribed to slow and partial phosphorylation and the failure of T82A mutant protein to bind cooperatively with DNA. Our results indicate that the T82 residue is crucial in implementing conformational changes in DevR that are essential for cooperative binding and for subsequent gene activation. We propose that the function of the T82 residue in the activation mechanism of DevR is conserved in spite of the unusual architecture of its receiver domain.     

Comparisons of Allergenic and Metazoan Parasite Proteins: Allergy the Price of Immunity

Allergic reactions can be considered as maladaptive IgE immune responses towards environmental antigens. Intriguingly, these mechanisms are observed to be very similar to those implicated in the acquisition of an important degree of immunity against metazoan parasites (helminths and arthropods) in mammalian hosts. Based on the hypothesis that IgE-mediated immune responses evolved in mammals to provide extra protection against metazoan parasites rather than to cause allergy, we predict that the environmental allergens will share key properties with the metazoan parasite antigens that are specifically targeted by IgE in infected human populations. We seek to test this prediction by examining if significant similarity exists between molecular features of allergens and helminth proteins that induce an IgE response in the human host. By employing various computational approaches, 2712 unique protein molecules that are known IgE antigens were searched against a dataset of proteins from helminths and parasitic arthropods, resulting in a comprehensive list of 2445 parasite proteins that show significant similarity through sequence and structure with allergenic proteins. Nearly half of these parasite proteins from 31 species fall within the 10 most abundant allergenic protein domain families (EF-hand, Tropomyosin, CAP, Profilin, Lipocalin, Trypsin-like serine protease, Cupin, BetV1, Expansin and Prolamin). We identified epitopic-like regions in 206 parasite proteins and present the first example of a plant protein (BetV1) that is the commonest allergen in pollen in a worm, and confirming it as the target of IgE in schistosomiasis infected humans. The identification of significant similarity, inclusive of the epitopic regions, between allergens and helminth proteins against which IgE is an observed marker of protective immunity explains the ‘off-target’ effects of the IgE-mediated immune system in allergy. All these findings can impact the discovery and design of molecules used in immunotherapy of allergic conditions.