Showing papers on "Cellular compartment published in 1988"

Lipid sorting in epithelial cells.

Abstract: One of the challenges of contemporary cell biology is to unravel how the molecular composition of the different cellular compartments is generated and maintained during the cell cycle. In animal cells most of the efforts have been directed toward the study of how newly synthesized proteins are transported to their correct cellular destinations, whereas the lipids, which make up the framework of the membranes in the cell, have been given much less attention. Lipid biochemistry has remained a fairly esoteric branch of molecular cell biology. This situation is now gradually changing with the discovery of phosphoinositide involvement in signal transduction (Ber- ridge,

Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic β cell via two distinct site-specific endopeptidases

Abstract: Insulin is produced from an inactive precursor, proinsulin, through initial endoproteolytic cleavage at sites marked by pairs of basic amino-acid residues. We report here that lysates of insulin secretory granules contain two distinct Ca-dependent acidic endoproteases; one (type I) cleaving exclusively on the C-terminal side of Arg 31.Arg 32 (B-chain/C-peptide junction), the other (type II) preferentially on the C-terminal side of Lys 64.Arg 65 of proinsulin (C-peptide/A-chain junction). The Ca and pH requirements of these proteinases suggested that the type-II proteinase would be active in the Golgi apparatus and the secretory granule, whereas type-I activity would be compatible only with the intragranular environment. Kinetic analyses of (pro)insulin conversion intermediates in [35S]methionine-pulsed rat islets support this supposition. Our results suggest a simple mechanism whereby different dibasic sites can be cleaved in different cellular compartments. In conjunction with the regulation of the ionic composition of such compartments and the operation of post-Golgi segregation, our results also suggest how proteolytic conversion of diverse proproteins destined for different cellular sites can occur differentially and in a regulated manner.

A spatial-temporal model of cell activation.

Abstract: A spatial-temporal model of calcium messenger function is proposed to account for sustained cellular responses to sustained stimuli, as well as for the persistent enhancement of cell responsiveness after removal of a stimulus, that is, cellular memory. According to this model, spatial separation of calcium function contributes to temporal separation of distinct phases of the cellular response. At different cellular sites, within successive temporal domains, the calcium messenger is generated by different mechanisms and has distinct molecular targets. In particular, prolonged cell activation is brought about by the interaction of calcium with another spatially confined messenger, diacylglycerol, to cause the association of protein kinase C with the plasma membrane. Activity of the membrane-associated protein kinase C is controlled by the rate of calcium cycling across the plasma membrane. In some instances, a single stimulus induces both protein kinase C activation and calcium cycling and thus causes prolonged activation; but in others, a close temporal association of distinct stimuli brings about cell activation via interaction of these intracellular messengers. Persistent enhancement of cell responsiveness after removal of stimuli is suggested to be due to the continued association, or anchoring, of protein kinase C to the membrane.

Short-term pH regulation in plants

Abstract: Cellular pH regulation consists of two features: (i) Long-term pH homeostasis, which ensures that all H+ or OH− produced in excess is ultimately removed from the cell and which requires metabolic energy; (ii) short-term reactions of the cell(s) to sudden shifts in intracellular pH, in order to prevent acute disturbances of metabolism. Recent progress in measuring and understanding of mainly short-term cellular regulation is summarized, including cellular responses to pH loads that arise from different sources such as external pH, weak acids/bases, protonophores, metabolic inhibitors, H+/cotransport, light and phytohormones. Whereas the plasma membrane H+ pump and metabolic adjustments may serve both long- and short-term pH control, physico-chemical buffering and the translocation of H+ from and to cellular compartments render only time-limited capacity for the neutralization of pH loads and seem exhausted within minutes. In spite of the widespread opinion that, because of tight regulation, intracellular pH does not vary with time, there is good evidence for long-lasting pH changes in plant cells, i.e. after hormonal stimulation, light/dark changes or carboxylation during crassulacean acid metabolism (CAM). This emphasizes that cytoplasmic pH, besides being well regulated, is essential not only for the regulation of membrane transport but also as a cellular messenger.

Ultrastructural localization of steroid sulphatase in cultured human fibroblasts by immunocytochemistry: a comparative study with lysosomal enzymes and the mannose 6-phosphate receptor.

Abstract: Immunocytochemistry was used to study the subcellular localization of steroid sulphatase in cultured human fibroblasts. Ultra-thin cryosections were incubated with antibodies raised against steroid sulphatase purified from human placenta and immune complexes were visualized with gold probes as electron dense markers. Steroid sulphatase was found in rough endoplasmic reticulum, Golgi cisternae and in the trans-Golgi reticulum, where it co-distributes with lysosomal enzymes and the mannose 6-phosphate receptor. The enzyme was not detected in lysosomes. Steroid sulphatase was also found at the plasma membrane and in the endocytic pathway (i.e. coated pits, endosomes and multivesicular endosomes). These may be the sites where sulphated oestrogen precursors are hydrolysed. Also here, it co-localizes with lysosomal enzymes and the mannose 6-phosphate receptor. It is concluded that microsomal steroid sulphatase and lysosomal enzymes share several cellular compartments.

Distribution of elements in rat peripheral axons and nerve cell bodies determined by x-ray microprobe analysis.

Abstract: X-ray microprobe analysis was used to determine concentrations (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in cellular compartments of frozen, unfixed sections of rat sciatic and tibial nerves and dorsal root ganglion (DRG). Five compartments were examined in peripheral nerve (axoplasm, mitochondria, my-elin, extraaxonal space, and Schwann cell cytoplasm), and four were analyzed in DRG nerve cell bodies (cytoplasm, mitochondria, nucleus, and nucleolus). Each morphological compartment exhibited characteristic concentrations of elements. The extraaxonal space contained high concentrations of Na, Cl, and Ca, whereas intraaxonal compartments exhibited lower concentrations of these elements but relatively high K. contents. Nerve axoplasm and axonal mitochondria had similar elemental profiles, and both compartments displayed proximodistal gradients of decreasing levels of K, Cl, and, to some extent, Na. Myelin had a selectively high P concentration with low levels of other elements. The elemental concentrations of Schwann cell cytoplasm and DRG were similar, but both were different from that of axoplasm, in that K and Cl were markedly lower whereas P was higher. DRG cell nuclei contained substantially higher K levels than cytoplasm. The subcellu-lar distribution of elements was clearly shown by color-coded images generated by computer-directed digital x-ray imaging. The results of this study demonstrate characteristic elemental distributions for each anatomical compartment, which doubtless reflect nerve cell structure and function.

Immunogold Localization of the L3 Protein of Maize Lipid Bodies during Germination and Seedling Growth

Abstract: We have used antibodies directed against the 16.5 kilodalton protein L3, the most abundant integral protein of maize (Zea mays L. cv Mo 17) lipid bodies, to follow the fate of this protein in scutellar parenchyma cells of maize during germination and subsequent seedling growth. Using gel electrophoresis and immunoblotting as well as immunocytochemical electron microscopy, we found that the amount of L3 decreases gradually during the first 3 to 4 days of seedling growth and more rapidly over the course of the next several days. Immunogold localization of the protein on thin sections indicated that L3 is found exclusively in the surface phospholipid monolayer of lipid bodies. The density of L3 in the surface layer of individual lipid bodies does not change during seedling growth; therefore, the decrease in the amount of L3 can be attributed to a decrease in the number of lipid bodies rather than to selective removal of protein components from the surface of all lipid bodies. Thus, L3 is apparently degraded at the same time as the matrix lipid of each lipid body. Unlike lipase, L3 does not appear to be transferred to other cellular compartments such as vacuoles during late stages of seedling growth.

Avian sarcoma-leukosis virus pol-endo proteins expressed independently in mammalian cells accumulate in the nucleus but can be directed to other cellular compartments.

Abstract: Eucaryotic expression vectors have been used to study transient expression of the avian sarcoma-leukosis retrovirus pol-endo protein in COS cells. The constructs encode proteins with N termini identical to that of authentic viral pp32 endonuclease with the exception of a single met residue encoded by the initiator AUG. The C termini correspond to unprocessed viral pol protein, authentic processed pp32, or a derivative which includes eight amino acids from the unprocessed portion. All three proteins localize to the nucleus. However, when the pol-endo domain is fused to a secretory signal peptide, the protein is found in medium and appears also to localize in the Golgi bodies and the cell membrane. These and derivative vectors will make it possible to assess the consequence of retroviral pol gene expression in eucaryotic cells.

Immunocytochemical and biochemical evaluation of pancreatic lipase in acinar cells of control and streptozotocin-induced diabetic rats.

Abstract: Pancreatic lipase was revealed by immunocytochemistry and analyzed biochemically in pancreatic tissue from control, diabetic, and insulin-treated diabetic rats. In the three groups of animals, lipase antigenic sites were detected with high resolution in the acinar cells in the compartments involved in protein secretion: rough endoplasmic reticulum, Golgi apparatus, and secretory zymogen granules. The quantitative evaluation of the intensities of labeling has demonstrated that, in contrast to other pancreatic proteins, lipase is concentrated only at the transition between the Golgi apparatus and the condensing vacuoles. This indicates that, although sharing the same secretory pathway as amylase and chymotrypsinogen, lipase may in fact be processed differently. On the other hand, when compared with controls, lipase immunolabelings in tissues with diabetic condition were higher in all the cellular compartments. Treatment of diabetic animals with insulin was found to restore these levels to those obtained in control condition. The biochemical determination of lipase activities in pancreatic tissues confirmed the immunocytochemical data. These results, together with those obtained previously for amylase and chymotrypsinogen, indicate that in diabetic condition secretion from the acinar cells is significantly altered, which may influence intestinal digestion and absorption processes. These modifications, and the enhancement of lipase in particular, could play a role in the pathogenesis of the hyperlipidemic condition present in diabetes.

The role of peroxisomes in glycerol ether lipid metabolism.

Abstract: Peroxisomes (microbodies) are ubiquitous subcellular organelles whose functions in cellular metabolism are not clear. In recent years peroxisomes have been shown to play roles in the oxidation of long chain fatty acids and ether lipid biosynthesis. The key enzymes of the acyl DHAP pathway i.e. DHAP acyltransferase and alkyl DHAP synthase have been shown to be localized in peroxisomes indicating that these organelles are obligatory for the biosynthesis of cellular glycerol ether lipids. This is proved by the discovery that in the tissues of patients suffering from Zellweger cerebrohepatorenal syndrome, an autosomal recessive disorder where peroxisomes are known to be absent, the acyl DHAP pathway enzymes and the ether lipids were also found to be deficient. Based on such biochemical abnormalities a number of similar genetic disorders such as neonatal adrenoleukodystrophy, different forms of chondrodysplasia punctata, infantile Refsum disease etc. have been characterized as peroxisomal deficiency disorders. These and other findings show that peroxisomes have a regulatory role in membrane lipid biogenesis. It seems that the main role of peroxisomes is to compartmentalize biochemical reactions which cannot proceed in other cellular organelles either due to formation of toxic product (H2O2) or due to non-availability of crucial metabolite (DHAP). The products of the reactions catalyzed by the peroxisomal enzymes e.g. acetyl CoA or alkyl DHAP are then transported out from peroxisomes to other cellular compartments where they are utilized to produce fatty acids, cholesterol, glycerol ether lipids etc. for membrane biogenesis.

Post-translational processing of the murine third component of complement

Abstract: The biosynthesis and secretion of the third component of complement (C3) has been studied with the macrophage cell line J774.2. C3 is initially synthesized as a single polypeptide chain precursor termed pro-C3, of relative molecular weight (Mr) 170,000 that is post-translationally modified by proteolytic cleavage into two polypeptides linked by disulphide bonds. The larger polypeptide, termed the alpha chain, has an Mr of 110,000-115,000, while the smaller beta chain has an Mr of 55,000-60,000. Pulse-chase experiments indicate that the proteolytic processing of pro-C3 occurs intracellularly, just prior to secretion. Unlike human C3, which has carbohydrate on both the alpha and beta chains, only the alpha chain of murine C3 is glycosylated. The carboxylic ionophores monensin and nigericin totally inhibit the proteolytic processing of pro-C3 at a concentration of approximately 10(-6) M. This block on proteolytic processing was shown not to be mediated by changes in intracellular pH induced by the disruption of proton gradients. Rather, data from experiments using carboxylic ionophores and other perturbants of cellular physiology indicated that the enzyme(s) responsible for the proteolytic cleavage of pro-C3 either reside in a cellular compartment with a neutral pH or are proteinases active over a relatively broad pH range.

Molecular mechanisms of membrane fusion and applications of membrane fusion techniques.

Abstract: The functioning of all eukaryotic cells is critically dependent on a variety of membrane fusion phenomena. For example, membrane fusion plays a key role not only in such cell-cell fusion events as fertilization and myogenesis, but also in intracellular transport processes, involving specific fusion events between membrane vesicles derived from various cellular compartments. This vesicular fusion appears to be a general trafficking mechanism in the sorting of cellular components during membrane biogenesis and the assembly of cell organelles. Vesicular fusion also plays a central role in the internalization of compounds from the extracellular environment through endocytosis, and their subsequent intracellular processing, and in the exocytotic secretion of products synthesized by the cell. Finally, membrane fusion is a crucial step in the infectious entry of enveloped viruses into cells.

[61] High-affinity, calmodulin-dependent isoforms of cyclic nucleotide phosphodiesterase in rat testis

Abstract: Publisher Summary This chapter presents methods to examine changes in the pattern of isoforms during sexual development, and approaches to studying their localization within the various cellular compartments of the testis. To facilitate the latter, in addition to intact testes, methods for studies of Sertoli cell only (SCO) testes, Leydig cell tumors, and purified germ cell suspensions are also presented. The testis, like a wide range of other tissues, contains not one but several forms of cyclic nucleotide phosphodiesterase; three of these appear to be low-k m , calmodulin-dependent isoforms. That these isoforms are not simply artifacts of the chromatographic procedure but distinct isoenzymes is based on differences in substrate specificity, kinetics, thermal stability, and reactions to calmodulin and trifluoperazine. The relatively specific cGMP is prominent in both adult intact and SCO testes, especially in the latter, is the major isoform in the Leydig cell tumor, but is absent in round spermatids. These observations suggest that this phosphodiesterase is more characteristic of the endocrine tissue of the testis rather than the germinal elements, possibly the Leydig cells.

Use of Lipidotic Cultured Cells (From Wolman Disease and Multisytemic Lipid Storage Myopathy) for Studying the Metabolic Relations between the Cellular Compartments Containing Neutral Lipids

Abstract: Neutral lipids stored in cultured cells can be from extracellular (lipoproteins) or intracellular (cellular biosynthesis or recycling of lipids) origin.