Vezivanje lipida za hemoglobin pod dejstvom insulina

Under hypoglycemic conditions, concomitant hyperinsulinism causes an apparent modification of hemoglobin (Hb) which is manifested by its a aggregation (Niketic et al.. Clin. Chim. Acia 197 (1991) 47). In the present work the causes and mechanisms underlying this Hb modification were Studied. Hemoglobin isolated from normal erythrocytes incubated with insulin was analyzed by applying P-31-spectrometry and lipid extraction and analysis. To study the dynamics of the plasma membrane during hperinsulinisra a fluorescent lipid-analog was applied. In the presence of insulin phosphatidylserine (PS). phosphatidylethanolamine (PE) and cholesterol were found to bind to Hb. Lipid binding resulted in Hb aggregation, a condition that can be reproduced when phospholipids arc incubated with Hb in vitro. Using a fluorescent lipid-analog, it was also shown that exposing crythrocytes to supraphysiological concentrations of insulin in vitro resulted in the internalization of lipids. The results presented in this work, may have relevance to cases of diabetes mellitus and hypoglycemia.Uranijim radovima je pokazano da hiperinsulinizam u uslovima hipoglikemije izaziva modifikaciju molekula hemoglobina koja se manifestuje njegovim agregiranjem (Niketić et al., Clin. Chim. Acta 197 (1991) 47). U ovom radu ispitivana je ova modifikacija molekula hemoglobina, kao i mehanizam njenog nastajanja. Primenom 31P-spektrometrije i analizom lipidnog ekstrakta utvrđeno je da u normalnim eritrocitima inkubiranim sa insulinom dolazi do vezivanja fosfatidil-serina, fosfatidil-etanolmina i holesterola za molekul hemoglobina. Vezivanje fosfolipida za hemoglobin dovodi do njegovog agregiranja što je potvrđeno eksperimentima u kojima je hemoglobin inkubiran sa fosfolipidima in vitro. Primenom fluorescentnog lipidnog analoga pokazano je da pri izlaganju eritrocita suprafiziološkim koncentracijama insulina dolazi do internalizacije membranskih lipida. Dobijeni rezultati mogu biti od značaja za pacijente obolele od šećerne bolesti i hipoglikemije

Comparative Lipidomics in Clinical Isolates of Candida albicans Reveal Crosstalk between Mitochondria, Cell Wall Integrity and Azole Resistance

Prolonged usage of antifungal azoles which target enzymes involved in lipid biosynthesis invariably leads to the development of multi-drug resistance (MDR) in Candida albicans. We had earlier shown that membrane lipids and their fluidity are closely linked to the MDR phenomenon. In one of our recent studies involving comparative lipidomics between azole susceptible (AS) and azole resistant (AR) matched pair clinical isolates of C. albicans, we could not see consistent differences in the lipid profiles of AS and AR strains because they came from different patients and so in this study, we have used genetically related variant recovered from the same patient collected over a period of 2-years. During this time, the levels of fluconazole (FLC) resistance of the strain increased by over 200-fold. By comparing the lipid profiles of select isolates, we were able to observe gradual and statistically significant changes in several lipid classes, particularly in plasma membrane microdomain specific lipids such as mannosylinositolphosphorylceramides and ergosterol, and in a mitochondrial specific phosphoglyceride, phosphatidyl glycerol. Superimposed with these quantitative and qualitative changes in the lipid profiles, were simultaneous changes at the molecular lipid species levels which again coincided with the development of resistance to FLC. Reverse transcriptase-PCR of the key genes of the lipid metabolism validated lipidomic picture. Taken together, this study illustrates how the gradual corrective changes in Candida lipidome correspond to the development of FLC tolerance. Our study also shows a first instance of the mitochondrial membrane dysfunction and defective cell wall (CW) in clinical AR isolates of C. albicans, and provides evidence of a cross-talk between mitochondrial lipid homeostasis, CW integrity and azole tolerance

Sendai virus-erythrocyte membrane interaction: quantitative and kinetic analysis of viral binding, dissociation, and fusion.

A kinetic and quantitative analysis of the binding and fusion of Sendai virus with erythrocyte membranes was performed by using a membrane fusion assay based on the relief of fluorescence self-quenching. At 37 degrees C, the process of virus association displayed a half time of 2.5 min; at 4 degrees C, the half time was 3.0 min. The fraction of the viral dose which became cell associated was independent of the incubation temperature and increased with increasing target membrane concentration. On the average, one erythrocyte ghost can accommodate ca. 1,200 Sendai virus particles. The stability of viral attachment was sensitive to a shift in temperature: a fraction of the virions (ca. 30%), attached at 4 degrees C, rapidly (half time, ca. 2.5 min) eluted from the cell surface at 37 degrees C, irrespective of the presence of free virus in the medium. The elution can be attributed to a spontaneous, temperature-induced release, rather than to viral neuraminidase activity. Competition experiments with nonlabeled virus revealed that viruses destined to fuse do not exchange with free particles in the medium but rather bind in a rapid and irreversible manner. The fusion rate of Sendai virus was affected by the density of the virus particles on the cell surface and became restrained when more than 170 virus particles were attached per ghost. In principle, all virus particles added displayed fusion activity. However, at high virus-to-ghost ratios, only a fraction actually fused, indicating that a limited number of fusion sites exist on the erythrocyte membrane. We estimate that ca. 180 virus particles maximally can fuse with one erythrocyte ghost