Showing papers in "Sub-cellular biochemistry in 1988"
TL;DR: Improvements in the sensitivity of fluorescence detection, and advances in two-di-mensional detectors have resulted in increased emphasis on the use of fluo-rescence microscopy to obtain a more detailed understanding of cellular phenomena.
Abstract: Fluorescence spectroscopy is often used to study the dynamic and hydrodynamic properties of proteins, membranes, and nucleic acids (Cundall and Dale, 1980; Lakowicz, 1983, 1986; Visser, 1985; Demchenko, 1986). More recently, the sensitivity of fluorescence detection, and advances in two-di-mensional detectors have resulted in increased emphasis on the use of fluo-rescence microscopy to obtain a more detailed understanding of cellular phenomena (Taylor et al., 1986). An unfavorable characteristic of fluorescence is the relatively low degree of specificity. The emission spectra of fluorophores often overlap on the wavelength scale, and the emission spectra of different fluorophores are often similar in shape. A further complication is that the emission may be complex due to the presence of multiple environments in a membrane, several fluorophores in a macromolecule, or the intrinsically complex emission of macromolecules or even simple molecules like tryptophan.
79 citations
TL;DR: The two types of ChE (generic abbreviation for any Cholinesterase) are readily distinguished not only by their substrate specificity but also by their response to selective inhibitors.
Abstract: Acetylcholinesterase (acetylcholine acetylhydrolase, AChE: EC 3.1.1.7) and butyrylcholinesterase (acylcholine acylhydrolase, BuChE: EC 3.1.1.8) both possess the capacity to hydrolyze choline esters, although the latter accepts a much wider variety of substrates. These two enzymes are found in a large number of excitable and nonexcitable tissues in most species, including humans (for review see Silver, 1974). The two types of ChE (generic abbreviation for any Cholinesterase) are readily distinguished not only by their substrate specificity but also by their response to selective inhibitors.
43 citations
TL;DR: The ABH and Lewis antigens are carbohydrate structures that occur on the surface of certain hemopoietic and epithelial cells and in many body fluids and the biochemical pathways are shown in summary form.
Abstract: The ABH and Lewis antigens are carbohydrate structures that occur on the surface of certain hemopoietic and epithelial cells and in many body fluids. The role of the ABO, Se, H, and Le gene loci and their glycosyltransferase products in determining the structures and the genetic polymorphisms of these terminal carbohydrate antigens has been reviewed in detail elsewhere (Oriol et al., 1986; Watkins, 1980). The biochemical pathways are shown in summary form in Figure 1.
32 citations
TL;DR: In this article, the positions of the S 1 and S 2 bands in the Jablonski diagram correspond to maxima in the absorption spectrum, which is a plot of the absorbance versus wave number, proportional to transition energy.
Abstract: Fluorescence is the phenomenon that certain molecules emit light with longer wavelength than the light with which they were illuminated. Such molecules are called fluorophores. Incident photons can excite a fluorophore from its electronic ground state, S 0, to a vibrational level of the electronic state S 1 or S 2. Vibrational energy is lost thermally after excitation within picoseconds, and the molecule drops to the ground vibrational state of the excited electronic state. The molecule then returns to one of the levels of S 0 after a short period of the order of nanoseconds, by emission of fluorescent light. This process is depicted in Figure 1. The absorption and emission of light is also illustrated in the Jablonski diagram of Figure 2, where the energy levels are depicted as vertical lines with energy increasing to the right. [Solvent relaxation and intersystem crossing can also be included in such diagrams (Lakowicz, 1983) but are not shown here.] The positions of the S 1 and S 2 bands in the Jablonski diagram correspond to maxima in the absorption spectrum, which is a plot of the absorbance versus wave number, proportional to transition energy (see the upper left part of Figure 2) or wavelength (see the upper right-hand side of Figure 2). The corresponding fluorescence spectra, plots of fluorescence intensity versus wave number (lower left of Figure 2) or wavelength (lower right of Figure 2), are also shown.
25 citations
TL;DR: The brush border membrane (also designated microvillar, luminal, or apical membrane) is a highly ordered surface domain of transporting epithelial cells like small-intestinal enterocytes, large-intestinal colonocytes, or kidney proximal tubular cells.
Abstract: The brush border membrane (also designated microvillar, luminal, or apical membrane) is a highly ordered surface domain of transporting epithelial cells like small-intestinal enterocytes, large-intestinal colonocytes, or kidney proximal tubular cells In the small intestine, the brush border membrane faces the intestinal lumen Its microvilli provide a surface enlargement of approximately 20-fold as compared to a flat luminal cell surface This membrane amplification serves the purpose of increasing the capacity of membrane-mediated digestion and carrier-mediated uptake
24 citations
TL;DR: Mammalian cell membranes basically consist of a bilayer of lipid molecules interacting with each other and with proteins that have either a transmem-branous or a superficial position that determine the structure and molecular motions in the membrane.
Abstract: Mammalian cell membranes basically consist of a bilayer of lipid molecules interacting with each other and with proteins that have either a transmem-branous or a superficial position. These physical interactions determine the structure and molecular motions in the membrane.
12 citations
TL;DR: Jaundice of newborn infants was first thought to be similar to that of adults, which often was due to occlusion of the bile ducts, but the fact that there often were several cases in the same family argued against this view.
Abstract: Already in the seventeenth century jaundice of newborn infants had been described. The condition was first thought to be similar to that of adults, which often was due to occlusion of the bile ducts. The fact, however, that there often were several cases in the same family argued against this view (Clarke, 1982).
8 citations
TL;DR: This chapter is concerned primarily with the design, application, and advantages of immunoaffinity purification using a solid matrix.
Abstract: A wide variety of techniques have been developed for the subfractionation of mammalian cell membranes and organelles. The most widely used are density gradient centrifugation, free-flow electrophoresis, polymer phase partition, fluorescent activated “cell-sorting,” and immunoaffinity purification. While the immunoaffinity approach relies totally on antibody-antigen reactions, the specificity of the other systems, which depend largely on physical differences between particles, can be increased by the selective use of affinity procedures. This chapter, after discussing the relative merits of other membrane separation techniques, is concerned primarily with the design, application, and advantages of immunoaffinity purification using a solid matrix.
6 citations
TL;DR: Molecules in living organisms, as well as those in dead matter, undergo continual, irregular motions, which are thermal fluctuations, or the Brownian motions.
Abstract: Molecules in living organisms, as well as those in dead matter, undergo continual, irregular motions. It is thermal fluctuations, or the Brownian motions.
5 citations
TL;DR: There is clearly a need for assays that might provide earlier, accurate diagnoses and that reflect tumor burden, and their use in immunoassays offers the possibilities for the measurement of these clinical parameters and for optimizing therapy.
Abstract: The development of monoclonal antibodies against malignant cells is projected to have a number of significant applications related to the clinical management of breast cancer. These include the early diagnosis of tumors, monitoring patients for the detection of recurrence or effects of therapy, and therapeutic modalities, whereby antibodies are employed to target cytotoxic agents (drugs, toxins, or radionuclides) to the site of the tumor. Since in Western countries, breast cancer will afflict in the region of one out of every 14 women, it is essential that the potential of these agents should be explored fully. Conventional wisdom indicates that aids to earlier diagnoses would have an important impact on prognosis. Present methods of diagnosis by palpation and mammography permit detection of tumor masses with diameters as small as 1 cm. However, even at this apparently early stage, tumors may be sufficiently established for the dissemination of tumor cells to have already occurred (reviewed by Tagnon, 1986). Thus, if breast cancer is only rarely a localized disease when it is clinically evident, there is clearly a need for assays that might provide earlier, accurate diagnoses and that reflect tumor burden. Given the sensitivity and specificity that can be obtained with monoclonal antibodies, their use in immunoassays offers the possibilities for the measurement of these clinical parameters and for optimizing therapy.
4 citations
TL;DR: A brief summarization of a number of known interactions between the plasma membrane and other cellular components forms the basis of evaluations of membrane fluidity using fluorescence polarization.
Abstract: Fluorescence polarization measurements assess movement of membrane constituents under near-normal conditions, as those membranes are challenged with toxic and/or nontoxic ions. Data interpretation of fluorescence polarization measurements are contingent upon an understanding of the interactions between the plasma membrane and other cellular constituents. Therefore, a brief summarization of a number of known interactions between the plasma membrane and other cellular components forms the basis of evaluations of membrane fluidity using fluorescence polarization.
TL;DR: Every step in thyroid metabolism is activated by TSH (thyroid stimulating hormone, thyrotropin), which evokes intracellular metabolic responses by recognition and binding to the cell surface, transmission of information across the cell membrane, and activation of internal metabolic pathways.
Abstract: The main function of the thyroid cell is the secretion of thyroid hormones (T4, thyroxine; T3, triiodothyronine). Thyroid hormones are first synthesized as part of thyroglobulin. Newly synthesized thyroglobulin is iodinated and vectorially transported to the apical region of the cell and released for storage in the lumen by exocytosis. When hormones are needed, thyroglobulin is removed from the luminal content by endocytosis and transferred to the lysosomes, where it is hydrolysed by cathepsin D liberating the hormones. Nonhormonal active iodotyrosines (3-monoiodotyrosine; 3,5-diiodotyrosine) are also formed. The hormones are released in the bloodstream and the iodotyrosine deiodinated. The iodide formed is partly reutilized by the cell (Taurog, 1978). This bidirectional transport of thyroglobulin is compartmented and implies concomitant transfer of membrane (Herzog, 1981). Every step in thyroid metabolism is activated by TSH (thyroid stimulating hormone, thyrotropin). It evokes intracellular metabolic responses by (1) recognition and binding to the cell surface, (2) transmission of information across the cell membrane, and (3) activation of internal metabolic pathways.
TL;DR: Animal models have been used extensively in the search for clues to the etiology of human autoimmune thyroiditis and to investigate basic mechanisms in the regulation of autoimmunity.
Abstract: Autoimmune thyroiditis is characterized by mononuclear cell infiltration of the thyroid gland, with accompanying tissue damage, and associated cellular and humoral autoimmunity to thyroid antigens such as thyroglobulin (Tg) and microsomal antigen (for reviews see Bigazzi and Rose, 1985; Rose et al., 1981; Weetman and McGregor, 1984; Weigle, 1980; Wick et al., 1985). Since the seminal studies by Rose and Witebsky (1956) demonstrating the experimental induction of autoimmune thyroiditis, animal models have been used extensively, both in the search for clues to the etiology of human autoimmune thyroiditis and to investigate basic mechanisms in the regulation of autoimmunity. Thyroid autoimmunity arises spontaneously in a number of species (Bigazzi and Rose, 1975), but most work has concentrated on the obese strain chicken (Wick et al., 1982, 1985), buffalo rats (Bigazzi and Rose, 1975), and the diabetes-prone BB/W rat (Sternthal et al., 1981). Thyroiditis may also be induced experimentally in many species by injecting Tg and an appropriate adjuvant (Weigle, 1980). A different model of thyroiditis can be induced in rats by a combination of thymectomy and irradiation (Penhaie et al., 1973, 1975). Studies of these various models have provided valuable information regarding the mechanisms involved in thyroid autoimmunity. More recently, we and others have begun to use a variety of in vitro techniques to gain insight into the molecular basis of thyroid autoimmunity.
TL;DR: Hybridoma technology and consequently the ability to generate monoclonal antibodies have provided cell biologists with means, hitherto unavailable to them, to probe the uniqueness of cells and tissues and map the distribution of their antigenic components.
Abstract: The impact of a new concept or a new technological advance usually results in accelerated pace in the application of the newly acquired concept or technique that produces advances of knowledge in the field. In cell biology in general, the repercussions in cell research have been spectacular following Abbe’s theory of image formation and following development of commercial transmission electron microscopes. These advances in microscopy have had parallel advances in preparative techniques suitable for examination of biological specimens by light microscopy and electron microscopy (Palade, 1967). Such advances in microscopy coupled with coincidental advances in biochemical and biophysical approaches for cell research have been instrumental in bringing our knowledge of the cell to its current phenomenal level. The functioning of cell organelles and of cells themselves, their interactions with other cells and with matrix of tissues are determined by the molecular architecture. Toward this end, hybridoma technology and consequently the ability to generate monoclonal antibodies (Mabs) (Kohler and Milstein, 1975) have provided cell biologists with means, hitherto unavailable to them, to probe the uniqueness of cells and tissues and map the distribution of their antigenic components. Monoclonal antibodies enable us to study functional homologies in cells and tissues between diverse species. They enable us to detect sparsely distributed and unknown antigens that could not be discovered by routine biochemical techniques.
TL;DR: Fusion of the pH-dependent virions with the plasma membrane can be triggered by lowering the pH of the medium containing the virus-associated cells and leads to the introduction of the viral content into the intracellular space.
Abstract: Enveloped virions penetrate eukaryotic cells by two alternative routes (Chop-pin and Scheid, 1980; White et al., 1983). Envelopes of viruses belonging to the paramyxovirus group fuse with cells’ plasma membranes at pH 7.4 and consequently microinject their content, the viral nucleocapsid, directly into the cell cytoplasm (Choppin and Scheid, 1980; Loyter and Volsky, 1982; White et al., 1983). A different way of entry has been described for most other enveloped virions such as those belonging to the orthomyxovirus, toga, rhabdo, and herpes groups. Such viruses are taken into cells by endocyticlike processes. Fusion of the viral envelopes with the endosomal or lysosomal membranes is triggered by the intraorganelle low-pH environment and leads to the introduction of the viral content into the intracellular space (Chopin and Scheid, 1980; White et al., 1983). Fusion of the pH-dependent virions with the plasma membrane can be triggered by lowering the pH of the medium containing the virus-associated cells.
TL;DR: The high affinity of α-neurotoxins for AchR, combined with their extreme specificity, has greatly facilitated the purification and characterization of AchR from different sources.
Abstract: The acetylcholine receptor (AchR) was the first neurotransmitter receptor to be identified and purified in an active form. It is a complex transmembrane glycoprotein present in the postsynaptic side of the neuromuscular junctions. When an action potential reaches the motor nerve terminals, acetylcholine is released into the synaptic cleft, where its local concentration can rise transiently to 10-4 to 10-3 M. Binding of Ach to specific sites located on the extracellular domains of the AchR molecules triggers the opening of short-lived cation channels, thus increasing the permeability of the postsynaptic membrane and causing the muscle fiber membrane to be depolarized beyond a critical threshold. The final result of this chain of events is muscle contraction. The AchR is present in high amounts in the electric organ of certain fishes. Using Torpedo (electric ray) electroplax as the starting material, one can purify milligram quantities of active protein, as well as substantial amounts of its constituent subunits. Moreover, a group of closely related protein toxins (α-neurotoxins) have been isolated from the venom of several Elapid snakes, which bind to the AchR with dissociation constants in the nanomolar to subnanomolar range [for review see Karlsson (1979) and Low (1979)]. The high affinity of α-neurotoxins for AchR, combined with their extreme specificity, has greatly facilitated the purification and characterization of AchR from different sources.
TL;DR: The refined picture shows a dynamic membrane in which molecular associations are tightly controlled, and in which long-range lateral motions are surprisingly restricted.
Abstract: Cell membranes regulate a variety of cellular processes ranging from permeability, transport, and excitability to intercellular interaction, morphological differentiation, and fusion. Numerous models have been advanced to characterize the organization of lipids and proteins in cell membranes. Today, there is substantial agreement on the “fluid mosaic” model (Singer and Nicholson, 1972), which emphasizes the dynamic behavior of the membrane components. Both lipids and proteins can undergo a variety of motions: rotational motion around the axis perpendicular to the plane of the membrane; lateral diffusion in the plane of the membrane; in addition, lipids can “flip-flop” (exchange from one monolayer to the other) and undergo trans-gauche conformational changes in the phospholipid acyl chains, which give rise to an increased segmental mobility toward the center of the bilayer. Since the fluid mosaic model has been proposed, its rather crude and generalizing picture has been filled with some details. The refined picture shows a dynamic membrane in which molecular associations are tightly controlled, and in which long-range lateral motions are surprisingly restricted.
TL;DR: Some of the more common techniques used to study the physical properties of biological membranes include electron microscopy, x-ray and neutron diffraction, Raman spectroscopy, electron spin resonance (ESR), nuclear magnetic resonance (NMR), infrared spectroscopic, fluorescence spectroscope, and differential scanning calorimetry (DSC).
Abstract: Some of the more common techniques used to study the physical properties of biological membranes include electron microscopy, x-ray and neutron diffraction, Raman spectroscopy, electron spin resonance (ESR), nuclear magnetic resonance (NMR), infrared spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry (DSC). The uses and limitations of these various analytical tools were examined in a number of recent reviews (Ax-elrodet al., 1976; Andersen, 1978; Seelig and Seelig, 1980; Jacobs and Oldsfield, 1981; Yguerabide and Foster, 1981; Amey and Chapman, 1983; Bach, 1983; Davis, 1983; Devaux, 1983,1985; Hoffmann and Restall, 1983; Verma and Wallach, 1983; Chapman and Benga, 1984; Makowski and Li, 1984; Bergelson et al., 1985; Blaurock, 1985; Bloom and Smith, 1985; Muhlethaler and Jay, 1985; McElhaney, 1986; Restall and Chapman, 1986).
TL;DR: Application of time-resolved fluorescence depolarization techniques to the study of biological membrane structure and dynamics requires selective signal monitoring of specifically located intrinsic or extrinsic fluorophores.
Abstract: Application of time-resolved fluorescence depolarization techniques to the study of biological membrane structure and dynamics requires selective signal monitoring of specifically located intrinsic or extrinsic fluorophores. In such systems, at least to date, the fluorescence characteristics of the naturally occurring chromophores (e.g., aromatic amino acid residues, enzymatic reactional cofactors, polyenic fatty acids and sterols, chlorophyll, and retinal) are of limited use because of low and/or reabsorbed emitted intensities, low signal-to-noise ratio (e.g., light scattering), and so on.
TL;DR: The reductionist approach to cell biology aims to explain the functioning of the whole cell in terms of the properties of its individual components, and has resorted to microanalytical methods to obtain this information.
Abstract: The reductionist approach to cell biology aims to explain the functioning of the whole cell in terms of the properties of its individual components. The central place of proteins in all aspects of cell activity justifies the theoretical and experimental attempts that have been made to relate amino acid sequence to molecular structure and function. Since many proteins are present in the cell in small amounts, the biochemist has resorted to microanalytical methods to obtain this information. Recent developments in instrumentation and computer technologies have greatly aided this process.
TL;DR: The complex machinery of the eukaryotic cell cytoplasm coordinates multiple simultaneous events of vesicular transport, the best characterized are those pertaining to transport along the secretory and endocytic paths.
Abstract: The complex machinery of the eukaryotic cell cytoplasm coordinates multiple simultaneous events of vesicular transport. The best characterized are those pertaining to transport along the secretory and endocytic paths.
TL;DR: Bacteriorhodopsin is the only protein found in the purple membrane, a specific patch in the plasma membrane of Halobacterium halobium, and has retinal as chromophore with an absorption maximum around 570 nm, hence the name purple membrane.
Abstract: Bacteriorhodopsin (bR) is the only protein found in the purple membrane, a specific patch in the plasma membrane of Halobacterium halobium. Bacteriorhodopsin accounts for 75% of the mass of the purple membrane and forms a two-dimensional crystalline lattice. It has retinal as chromophore with an absorption maximum around 570 nm, hence the name purple membrane. Light energy absorbed by the retinal chromophore in bR drives the protons actively across the purple membrane from the cytoplasmic to extracellular side. Although the mechanism of this light-driven proton pump has been the subject of a considerable number of investigations, the relation between the mechanism and tertiary structure of bR is still obscure [for reviews see Henderson (1977), Stoeckenius et al. (1979), Ottolenghi (1980), and Stoeckenius and Bogomolni (1982)1.