Occurrence,location and development of anthocyanoplasts

Intensely pigmented organelles (anthocyanoplasts) have been found in anthocyanin-producing cells of more than 70 species representing at least 33 families of angiosperms. When fully developed these structures are typically spherical and normally only one is present in each pigmenented cell. The development of the anthocyanoplast has been studied in both light and dark-grown red cabbage seedlings and the location of the mature organelle has been shown, by the use of isolated protoplasts and vacuoles, to be within the main cell vacuole. Evidence is presented which suggests that the anthocyanoplast is membrane-bounded and that it is the site of anthocyanin biosynthesis.     

Immunoblotting of hydrophobic integral membrane proteins

For diagnosis and research purposes it is frequently desirable to measure by immunoblotting small amounts of proteins in complex mixtures such as tissue biopsy homogenates. Standard immunoblot procedures that give excellent results for soluble proteins unexpectedly gave low and irreproducible signals with some hydrophobic membrane proteins. We found that this was due to inefficient electrophoretic transfer to nitrocellulose, which could be corrected by modification of the transblot buffer. Hydrophobic integral membrane proteins of peroxisomes as well as other rat and human liver proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. The nitrocellulose-bound proteins were detected both by staining and by immunoblotting with an antiserum against the 22-kDa integral membrane protein of peroxisomes plus 125I-labeled protein A. A modified transblot buffer with 0.7 M glycine and 25 mM Tris (pH 7.7) but no methanol allowed use of a much shorter transfer time and strikingly improved the electrophoretic transfer of membrane proteins such that a peroxisomal integral membrane protein could be easily detected in human liver biopsy homogenates.     

Cholesterol content of red blood cells and low-density lipoproteins in hypertriglyceridemia

The red blood cells and the low-density lipoproteins in hypertriglyceridemia have a lower ratio of unesterified cholesterol to phospholipid than normal. The low-density lipoproteins are also smaller and more dense in hypertriglyceridemia, and contain only 45% of the normal unesterified cholesterol mass. The phase behavior of the lipids shows that normal red cells and low-density lipoproteins are close to saturation with cholesterol, whereas in hypertriglyceridemia less cholesterol is present. Because newly secreted triacylglycerol-rich lipoproteins are poor in cholesterol, their excess production and transport in hypertriglyceridemia may prevent maintenance of the normal cholesterol content of blood cells and low-density lipoproteins. Partitioning of cholesterol into triacylglycerol-rich lipoproteins is able to account for significant fluxes of unesterified cholesterol in the plasma compartment.     

The inhibitory action of Mycobacterium ulcerans soluble factor on monocyte/T cell cytokine production and NF-kappa B function.

Buruli ulcer is a chronic and progressive necrotizing ulcer for which there is no medical treatment. Historically, a soluble toxin (factor) derived from the causative Mycobacterium ulcerans was found to induce the massive necrosis of skin and s.c. tissue seen in this condition. However, the persistence of the disease is thought to be caused by a lack of any immune response. We therefore investigated whether the factor was related to immunosuppression. A protocol to partially purify the factor was developed, and its effects on immune competent cells were tested. The factor produced >95% inhibition of LPS-induced release of TNF and IL-10 from human monocytes and caused a loss of adherence of these cells without cell death. The factor also blocked the production of IL-2 from activated T lymphocytes. The factor had no effect on TNF-induced cytotoxicity, but abrogated TNF-induced NF-kappa B activation. Surprisingly, a synergy was observed between the factor and phorbol ester-directed NF-kappa B activation. The factor had no effect on IL-1- or LPS-induced NF-kappa B activity, indicating selective activity of the factor. The factor did not inhibit the degradation of I kappa B alpha induced by TNF, indicating that the target for its activity lies within an undefined part of the TNF signaling mechanism. The data indicate that the localized immunosuppression associated with Buruli ulcer relates to the activity of the released factor, and this may provide a target for future therapeutic strategies for this intractable disease.     

Oleic acid allows more apoprotein A-1 to bind with higher affinity to large emulsion particles saturated with cholesterol

Apoprotein (apo) A-1 binding to large triolein-rich emulsion particles saturated with cholesterol has been examined as a function of the oleic acid content. Six emulsion systems were formed containing 0.3-1.0% (by weight) oleic acid, 82.9-86.3% triolein, 10.6-7.2% egg yolk phosphatidylcholine, and 6.7-5.5% cholesterol. The average emulsion particle diameters calculated from these lipid compositions ranged between 84 and 116 nm. Negative stain electron microscopy of an emulsion containing 1% oleic acid showed a polydisperse population of only large spherical particles with a mean diameter of 116 +/- 54 nm. The calculated cholesterol concentrations of the particles surface and core for the six emulsions were 43.3 +/- 1.1 and 5.6 +/- 0.2 mol%, respectively, and were rather constant. Therefore, when the surface oleic acid concentrations increased from 2.6 to 10.1 mol%, the phospholipid concentration decreased from 55.1 to 45.9 mol%. In the core, oleic acid increased at the expense of triolein. In the range studied a nearly 4-fold increase in the surface oleic acid content produces a similar increase in the binding capacity (N) and reduces the dissociation constant (Kd). The changes in the Kd and N values were linearly dependent on the surface oleic acid concentration. These data show that oleic acid allows more apoA-1 to bind with higher affinity to large emulsion particles saturated with cholesterol.     

pH-induced structural changes of apo-lactoferrin and implications for its function: a molecular dynamics simulation study

The minor protein in milk, lactoferrin (Lf), is known for a variety of biological functions, and has been investigated as a protective encapsulant for probiotic bacteria in health-promoting food products. Lf is likely to be exposed to extreme pH conditions which are known to have disruptive influences on its functionality. The molecular mechanisms underlying these pH-dependent changes are not well-understood. To explore the potential of Lf as an encapsulant, molecular dynamics (MD) simulations were applied to study its conformational changes under extreme acidic (pH 1.0) or basic (pH 14.0) conditions, relative to neutral pH. Simulations indicate that the structure of apo-Lf is relatively stable at neutral pH, while acidic and basic pH result in substantially greater flexibility, partly induced by the loss of contacts between the N- and C-terminal lobes, causing them to undergo extensive relative bending and twisting motions. Basic pH causes greater structural disruption compared to acidic exposure. The latter has greater influence on the N-terminus, with increased fluctuations and disruptions of inter-residue contacts compared to those at neutral pH; while basic pH was found to more prominently disrupt contacts at the C-terminus. These results help elucidate possible functional consequences on Lf of exposure to extreme pH conditions.     

Compound Heterozygous Mutation of Rag1 Leading to Omenn Syndrome

Omenn syndrome is a primary immunodeficiency disorder, featuring susceptibility to infections and autoreactive T cells and resulting from defective genomic rearrangement of genes for the T cell and B cell receptors. The most frequent etiologies are hypomorphic mutations in “non-core” regions of the Rag1 or Rag2 genes, the protein products of which are critical members of the cellular apparatus for V(D)J recombination. In this report, we describe an infant with Omenn syndrome with a previously unreported termination mutation (p.R142*) in Rag1 on one allele and a partially characterized substitution mutation (p.V779M) in a “core” region of the other Rag1 allele. Using a cellular recombination assay, we found that while the p.R142* mutation completely abolished V(D)J recombination activity, the p.V779M mutation conferred a severe, but not total, loss of V(D)J recombination activity. The recombination defect of the V779 mutant was not due to overall misfolding of Rag1, however, as this mutant supported wild-type levels of V(D)J cleavage. These findings provide insight into the role of this poorly understood region of Rag1 and support the role of Rag1 in a post-cleavage stage of recombination.     

Predictable Alteration of Sequence Recognition by RNA Editing Factors from Arabidopsis

RNA editing factors of the pentatricopeptide repeat (PPR) family show a very high degree of sequence specificity in the recognition of their target sites. A molecular basis for target recognition by editing factors has been proposed based on statistical correlations but has not been tested experimentally. To achieve this, we systematically mutated the pentatricopeptide motifs in the Arabidopsis thaliana RNA editing factor CLB19 to investigate their individual contribution to RNA recognition. We find that the motifs contributing significantly to the specificity of binding follow the previously proposed recognition rules, distinguishing primarily between purines and pyrimidines. Our results are consistent with proposals that each motif recognizes one nucleotide in the RNA target with the protein aligned parallel to the RNA and contiguous motifs aligned with contiguous nucleotides such that the final PPR motif aligns four nucleotides upstream of the edited cytidine. By altering S motifs in CLB19 and another editing factor, OTP82, and using the modified proteins to attempt to complement the respective mutants, we demonstrate that we can predictably alter the specificity of these factors in vivo.