Calorimetric studies of the structural transitions of the human erythrocyte membrane. Studies of the B and C transitions

Differential scanning calorimetry has been used to study several structural transitions of the human erythrocyte membrane. Earlier studies have shown that one of these transitions (the A transition) is due to the thermal unfolding of spectrin on the membrane. In this paper, it is shown that two of the other transitions (B and C) exhibit a high sensitivity to a local anesthetic, benzyl alcohol. Increasing the ionic strength of the suspending medium results in a splitting of the B transition into two independent transitions (B₁ and B₂). It is found that one of these (B₂) is associated with titrating groups, since the midpoint for the transitions shifts by about 20°C, with an apparent $\text{p}\text{K}$ near 7.5. Extensive bilateral proteolysis by papain causes a drastic decrease in the size of all transitions except the C transition, which remains unaltered. On the other hand, treatment with phospholipase A₂ largely affects the C transition, causing its disappearance. Because of the lack of sensitivity to proteolysis and the high sensitivity to phospholipase, it appears that the C transition has a large extent of ‘lipid involvement’. It might result from the melting of a small fraction of phospholipid which exists in a crystalline state under physiological conditions. Alternatively, the C transition could arise from changes in protein-lipid interactions or from lipid-dependent changes in protein-protein interactions, providing one assumes that only protease-resistant portions of membrane proteins are participating.     

Interaction of lipopolysaccharide with detergents and its possible role in the detergent resistance of the outer membrane of gram-negative bacterua

In the presence of MgCl₂, amounts of detergents which disrupted phospholipid vesicles caused lipopolysaccharide I from Proteus mirabilis to aggregate and form vesicular, membrane-like structures. Vesicle formation with P. mirabilis lipopolysaccharide II containing longer O-polysaccharide chains was extremely poor. Lipopolysaccharides of Salmonella minnesota R mutants (chemotypes Ra, Rc and Re) displayed a growing tendency for vesicle formation with increasing deficiency of the R core polysaccharide. Lipopolysaccharides of chemotypes Rc and Re produced vesicles even in the absence of MgCl₂ and detergent. Spherical aggregates consisting of P. mirabilis lipopolysaccharide I, MgCl₂ and detergent were unable to either entrap or retain [¹⁴C]-sucrose, [³H]inulin or [³H]dextran. On the other hand, S. minnesota R mutant lipopolysaccharides of chemotypes Rc and Re could entrap all three saccharides and retain them for at least short periods of time. Leakage of [³H]-inulin out of Re-lipopolysaccharide vesicles was greatly retarded by addition of MgCl₂ to the vesicle system. Incorporation of P. mirabilis lipopolysaccharide I or S. minnesota Rc lipopolysaccharide into phospholipid vesicles protected these model membranes from disruption by detergent. This suggested a similar protective function of lipopolysaccharide in the outer membrane of enteric bacteria against the action of surfactants occuring in their normal intestinal habitat.     

Thymidylate synthetase: Studies on the peptide containing covalently bound 5-fluoro-2′-deoxyuridylate and 5,10-methylenetetrahydrofolate

Studies are reported on the FdUMP-CH₂-H₄ folate-peptide obtained upon proteolysis of the complex formed from thymidylate synthetase, FdUMP and 5,10-CH₂-H₄folate. Contrary to a previous report from this laboratory, the peptide does contain a cysteine residue. The sequence of the largest peptide obtained is Ala-Leu-Pro-Pro-(His,Cys)-Thr. Quantitative modification of the histidine residue with the Pauly reagent indicates that imidazole is not directly linked to the nucleotide. The stability of the peptide indicates the covalent bond to the cofactor involves its 5-nitrogen; from this, it may be concluded that the reactive form of the cofactor is the 5-iminium ion.     

Patterns of divergence of a large family of nodule cysteine‐rich peptides in accessions of Medicago truncatula

The nodule cysteine‐rich (NCR) groups of defensin‐like (DEFL) genes are one of the largest gene families expressed in the nodules of some legume plants. They have only been observed in the inverted repeat loss clade (IRLC) of legumes, which includes the model legume Medicago truncatula. NCRs are reported to play an important role in plant–microbe interactions. To understand their diversity we analyzed their expression and sequence polymorphisms among four accessions of M. truncatula. A significant expression and nucleotide variation was observed among the genes. We then used 26 accessions to estimate the selection pressures shaping evolution among the accessions by calculating the nucleotide diversity at non‐synonymous and synonymous sites in the coding region. The mature peptides of the orthologous NCRs had signatures of both purifying and diversifying selection pressures, unlike the seed DEFLs, which predominantly exhibited purifying selection. The expression, sequence variation and apparent diversifying selection in NCRs within the Medicago species indicates rapid and recent evolution, and suggests that this family of genes is actively evolving to adapt to different environments and is acquiring new functions.     

Pharmacological rescue of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) detected by use of a novel fluorescence platform

Numerous human diseases arise because of defects in protein folding, leading to their degradation in the endoplasmic reticulum. Among them is cystic fibrosis (CF), caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR ), an epithelial anion channel. The most common mutation, F508del, disrupts CFTR folding, which blocks its trafficking to the plasma membrane. We developed a fluorescence detection platform using fluorogen-activating proteins (FAPs) to directly detect FAP-CFTR trafficking to the cell surface using a cell-impermeant probe. By using this approach, we determined the efficacy of new corrector compounds, both alone and in combination, to rescue F508del-CFTR to the plasma membrane. Combinations of correctors produced additive or synergistic effects, improving the density of mutant CFTR at the cell surface up to ninefold over a single-compound treatment. The results correlated closely with assays of stimulated anion transport performed in polarized human bronchial epithelia that endogenously express F508del-CFTR. These findings indicate that the FAP-tagged constructs faithfully report mutant CFTR correction activity and that this approach should be useful as a screening assay in diseases that impair protein trafficking to the cell surface.     

Effector and memory CD8+ T cells can be generated in response to alloantigen independently of CD4+ T cell help

There is now considerable evidence suggesting that CD8(+) T cells are able to generate effector but not functional memory T cells following pathogenic infections in the absence of CD4(+) T cells. We show that following transplantation of allogeneic skin, in the absence of CD4(+) T cells, CD8(+) T cells become activated, proliferate, and expand exclusively in the draining lymph nodes and are able to infiltrate and reject skin allografts. CD44(+)CD8(+) T cells isolated 100 days after transplantation rapidly produce IFN-gamma following restimulation with alloantigen in vitro. In vivo CD44(+)CD8(+) T cells rejected donor-type skin allografts more rapidly than naive CD8(+) T cells demonstrating the ability of these putative memory T cells to mount an effective recall response in vivo. These data form the first direct demonstration that CD8(+) T cells are able to generate memory as well as effector cells in response to alloantigen during rejection in the complete absence of CD4(+) T cells. These data have important implications for the design of therapies to combat rejection and serve to reinforce the view that CD8(+) T cell responses to allografts require manipulation in addition to CD4(+) T cell responses to completely prevent the rejection of foreign organ transplants.     

Methyl β-cyclodextrin reduces accumulation of reactive oxygen species and cell death in yeast

Stabilized F-actin structures have been shown to be detrimental to both mammalian and yeast cells. In yeast, stabilization of actin caused by addition of jasplakinolide, by point mutations in the act1 gene, or by deletion of certain genes that regulate F-actin leads to cell death with hallmarks of apoptosis. In particular, there is an elevation in the levels of reactive oxygen species, and we have shown the importance of the Ras/cAMP pathway for this effect. Here we show that in yeast cells deleted for end3, which functions to regulate actin organization during endocytosis, treatment of cells with methyl β-cyclodextrin reduces levels of reactive oxygen species and inhibits cell death progression. Methyl β-cyclodextrin is widely used to disrupt lipid rafts that contain cholesterol. The mechanism through which the rescue is achieved was investigated and we demonstrate that methyl β-cyclodextrin reduces accumulation of Ras2 at the plasma membrane in Δend3 cells. We use FRAP and live cell imaging to determine the possible mechanism through which methyl β-cyclodextrin functions to elicit this effect on Ras2 localization. Finally, we demonstrate that addition of methyl β-cyclodextrin to wild-type cells can act to protect cells from acute oxidative stress caused by addition of hydrogen peroxide.