Boundary region between coexisting lipid phases as initial binding sites for Escherichia coli alpha-hemolysin: A real-time study

Abstractα-Hemolysin (HlyA) is a protein toxin, a member of the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenic strands of Escherichia coli. The mechanism of action of this toxin seems to involve three stages that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin within the membrane. Since the influence of phase segregation on HlyA binding and insertion in lipid membranes is not clearly understood, we explored at the meso- and nanoscale—both in situ and in real-time—the interaction of HlyA with lipid monolayers and bilayers. Our results demonstrate that HlyA could insert into monolayers of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/16:0SM/Cho) and DOPC/24:1SM/Cho. The time course for HlyA insertion was similar in both lipidic mixtures. HlyA insertion into DOPC/16:0SM/Cho monolayers, visualized by Brewster-angle microscopy (BAM), suggest an integration of the toxin into both the liquid-ordered and liquid-expanded phases. Atomic-force-microscopy imaging reported that phase boundaries favor the initial binding of the toxin, whereas after a longer time period the HlyA becomes localized into the liquid-disordered (Ld) phases of supported planar bilayers composed of DOPC/16:0SM/Cho. Our AFM images, however, showed that the HlyA interaction does not appear to match the general strategy described for other invasive proteins. We discuss these results in terms of the mechanism of action of HlyA

ApoCIII-Enriched LDL in Type 2 Diabetes Displays Altered Lipid Composition, Increased Susceptibility for Sphingomyelinase, and Increased Binding to Biglycan

Objective- Apolipoprotein CIII (apoCIII) is an independent risk factor for cardiovascular disease, but the molecular mechanisms involved are poorly understood. Here, we investigated potential proatherogenic properties of apoCIII-containing LDL from hypertriglyceridemic patients with type 2 diabetes. Research design and methods - LDL was isolated from controls and subjects with type 2 diabetes, and from apoB transgenic mice. LDL-biglycan binding was analyzed with a solid-phase assay using immunoplates coated with biglycan. Lipid composition was analyzed with mass spectrometry. Hydrolysis of LDL by sphingomyelinase was analyzed after labeling plasma LDL with [(3)H]sphingomyelin. ApoCIII isoforms were quantified after isoelectric focusing. Human aortic endothelial cells were incubated with desialylated apoCIII or with LDL enriched with specific apoCIII isoforms. Results- We showed that enriching LDL with apoCIII only induced a small increase in LDL-proteoglycan binding, and this effect was dependent on a functional Site A in apoB100. Our findings indicated that intrinsic characteristics of the diabetic LDL other than apoCIII per se are responsible for further increased proteoglycan binding of diabetic LDL with high endogenous apoCIII, and we showed alterations in the lipid composition of diabetic LDL with high apoCIII. We also demonstrated that high apoCIII increased susceptibility of LDL to hydrolysis and aggregation by SMase. In addition, we demonstrated that sialylation of apoCIII increased with increasing apoCIII content, and that sialylation of apoCIII was essential for its proinflammatory properties. Conclusions- We have demonstrated a number of features of apoCIII-containing LDL from hypertriglyceridemic patients with type 2 diabetes that could explain the proatherogenic role of apoCIII

Pengembangan E-modul Model Eksperiental Jelajah Alam Sekitar (Ejas) pada Materi Plantae

. Penelitian pengembangan ini bertujuan untuk menguji kevalidan dan kepraktisan E-modul model Eksperiental Jelajah Alam Sekitar (EJAS) pada materi plantae. Produk yang dikembangkan menggunakan model pengembangan ADDIE. Model pembelajaran yang digunakan yaitu Model Eksperiental Jelajah Alam Sekitar (EJAS). Subjek penelitian adalah 30 siswa kelas X IPA B SMAN 01 Singosari. Hasil implementasi berupa data hasil validasi validator ahli dan data angket respon siswa sebagai pengguna E-modul. Hasil validasi e-modul oleh ahli media sebesar 99 persen, hasil validasi oleh ahli materi berupa hasil validasi komponen materi e-modul sebesar 100 persen. Hasil validasi oleh ahli perangkat pembelajaran dan praktisi lapangan berturut-turut menunjukkan persentase sebesar 98 persen dan 97 persen. Data kepraktisan setelah implementasi e-modul yakni sebesar 88 persen. Hasil analisis dari masing-masing uji menunjukkan bahwa produk pengembangan telah dinyatakan sangat valid dan sangat praktis. E-modul model Eksperiental Jelajah Alam Sekitar (EJAS) materi plantae yang dikembangkan telah memenuhi syarat kevalidan dan kepraktisan untuk digunakan dalam pembelajaran matei plantae

Clearly Detectable, Kinetically Restricted Solid-Solid Phase Transition in cis-Ceramide Monolayers

Sphingosine [(2S,3R,4E)-2-amino-4-octadecene-1,3-diol] is the most common sphingoid base in mammals. Ceramides are N-acyl sphingosines. Numerous small variations on this canonical structure are known, including the 1-deoxy, the 4,5-dihydro, and many others. However, whenever there is a Δ4 double bond, it adopts the trans (or E) configuration. We synthesized a ceramide containing 4Z-sphingosine and palmitic acid (cis-pCer) and studied its behavior in the form of monolayers extended on an air-water interface. cis-pCer acted very differently from the trans isomer in that, upon lateral compression of the monolayer, a solid-solid transition was clearly observed at a mean molecular area ≤44 Å2·molecule-1, whose characteristics depended on the rate of compression. The solid-solid transition, as well as states of domain coexistence, could be imaged by atomic force microscopy and by Brewster-angle microscopy. Atomistic molecular dynamics simulations provided results compatible with the experimentally observed differences between the cis and trans isomers. The data can help in the exploration of other solid-solid transitions in lipids, both in vitro and in vivo, that have gone up to now undetected because of their less obvious change in surface properties along the transition, as compared to cis-pCer.Fil: Fanani, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Busto, Jon V.. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Sot, Jesús. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; EspañaFil: Abad, José L.. Consejo Superior de Investigaciones Científicas. Instituto de Química Avanzada de Catalunya; EspañaFil: Fabrías, Gemma. Consejo Superior de Investigaciones Científicas. Instituto de Química Avanzada de Catalunya; EspañaFil: Saiz, Leonor. University of California; Estados Unidos. Massachusetts Institute of Technology; Estados UnidosFil: Vilar, Jose M. G.. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Goñi, Félix M.. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; EspañaFil: Maggio, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Alonso, Alicia. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; Españ