[show abstract][hide abstract] ABSTRACT: Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860). In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN), the outer nuclear membrane (ONM), the inner nuclear membrane (INM) and the cell cytosol (CC). In contrast to Endoplasmic Reticulum (ER) which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM) of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA). The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of the lipids in the ER, in the region adjacent to nucleus, is defining nuclear outer and inner biomembrane composition, is responsible for transport of the cytosolic protein into the nucleus and, replenishment of ER membrane used for vesicular transport.
International journal of biological sciences 02/2006; 2(4):216-26. · 3.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: The outcome of chronic ethanol consumption recorded in liver by in situ staining of the genomic DNA in fragmented nuclei indicates the course of cellular events that has been coined as apoptosis or programmed cell death. Hence, we designed the study to determine which ethanol-induced modification of the cellular make-up is responsible for the hastening the cell damage. The in vitro assays were performed with cellular organelles and cytosol prepared from hepatocytes derived from rats subjected to 9 weeks of chronic alcohol consumption. The results were compared with the pair-fed controls receiving isocaloric liquid diet. In the initial phase of the studies, we established that the process of apoptosis was not triggered by the aberrant activity of neutral or acidic sphingomyelinase. The hepatocytes derived from alcohol and control diets manifested equal enzymatic activity. The de novo synthesis of sphingoid bases and ceramides in the alcohol-derived sample of endoplasmic reticulum was reduced, but the in situ apoptosis was up to 36-fold higher than in the control. Also, the isolated hepatocytes contained a 2- to 4-fold higher amount of nucleosomal fragments in the cytosolic extracts. The endosomes from liver hepatocytes of ethanol-consuming rats, in the presence of the cytosol and mitochondria from pair-fed controls, disclosed 2 to 3 times higher apoptotic potential than sample consisting of ethanol-derived fractions only, and 3 to 5 times higher than the control-derived fractions. The substantial increase in apoptosis, as recorded in the amount of DNA fragments released to the cytosol from the fresh nuclei, was also recorded when the microsomal membranes of endoplasmic reticulum and Golgi were incubated in the conditions with preserved intracellular transport. The maximal 20-fold increase of apoptotic activity was recorded in the incubation mixtures of ethanol-derived endoplasmic reticulum-Golgi membranes with control-derived cytosol in the presence of the ATP generating system. Results infer that the intracellular transport vesicles, generated from ethanol-modified membranes in the presence of the substrates that are available in the cytosol of the control hepatocytes, activate the apoptotic activity in the in vitro system. This interpretation is supported by the results of analysis of the clathrin-coated transport vesicles that, in contrast to nonclathrin transport vesicles, contain a sizable accumulation of ceramides that are known to induce apoptosis.
Alcoholism Clinical and Experimental Research 01/1999; 23(2):334 - 343. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/μg of protein and 80–90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39–55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.
Archives of Biochemistry and Biophysics 01/1992; 298(1):167-175. · 3.37 Impact Factor