Intracellular Transport Mechanisms Research Articles

Extrasynaptic accumulations of acetylcholinesterase in the rat sternocleidomastoid muscle after neonatal denervation. Light and electron microscopic localization and molecular forms.

Denervated neonatal rat sternocleidomastoid muscle has decreased levels of total AChE when compared to control muscle. Denervated versus control values of total muscle AChE present a three-phase curve in function of time after denervation. There is a rapid initial fall 0-3 days after denervation, an increase during about 2 weeks, then again a decrease in total AChE. Thus, there is a transitory net accumulation of AChE after the initial fall of activity in denervated developing muscle. Extrasynaptic areas of high AChE activity develop between 1 and 2 weeks after denervation and remain visible up to 1 month after denervation before vanishing. An electron microscope study shows that these accumulations are internal to the muscle fiber, close to a limited number of muscle nuclei and associated to the sarcoplasmic reticulum and nuclear envelope, but not to the T-tubule system. As found in adult rat muscle, the initial fall in AChE affects first the 16 S AChE form, and soon after, the 4 S and 10 S AChE forms. A main difference with adult muscle is the sudden increase and predominance over other forms of 10 S AChE 2 weeks after denervation at birth. Later, the decrease in AChE affects 16 S and 4 S AChE before 10 S AChE. The regions rich in extrasynaptic sites of AChE accumulation possess a very high proportion of 10 S AChE. Thus, the mechanisms of biosynthesis, intracellular transport and/or secretion of AChE may be very different in young, developing muscle compared to adult muscle.

Comparative Ultrastructure and Physiology of Chromatophores, with Emphasis on Changes Associated with Intracellular Transport

Recent advances in our understanding of the mechanism of intracellular transport in unicellular chromatophores are reviewed. Emphasis is placed on the possible role(s) of the three major cytoskeletal components microtubules, intermediate filaments, and actin filaments. A comparative analysis of different chromatophore types suggests that these cytoskeletal fiber systems may play different roles, or at least seem to be involved to a varying degree, in intracellular pigment translocation. Ultrastructure, transport characteristics, and responses to pharmacological agents of a wide variety of vertebrate and invertebrate chromatophores do show sufficient variation to make one consider the possibility that the underlying mechanisms also vary to some extent. It is hoped that the increased use of new methodological approaches, including microinjection of single cells and the development of reactivatable cell models, will greatly aid us in our exploration of the physical-chemical basis of chromatophore motility.

Biosynthesis and transport of plasma membrane glycoproteins in the rat intestinal epithelial cell: studies with sucrase-isomaltase.

Sucrase-isomaltase (SI), an integral heterodimeric glycoprotein of the intestinal microvillus membrane, is synthesized as a single enzymically active precursor protein (pro-SI) of high relative molecular mass. After glycosylation in the Golgi complex pro-SI is transferred to the microvillus membrane where it is cleaved into the two subunits by pancreatic elastase. Pro-SI was purified by monoclonal antibody-affinity chromatography from microvillus membranes of fetal intestinal transplants in which SI is found exclusively in the non-cleaved precursor form. The N-terminal amino acid sequence of pro-SI was identical to that of the isomaltase subunit of SI which anchors the mature enzyme complex to the lipid bilayer, but it differed from the N-terminal sequence of the sucrase subunit of SI. This structural comparison indirectly gave insight into the mechanisms of membrane insertion and assembly of pro-SI during its biosynthesis. Subcellular fractionation studies indicate transient structural association of newly synthesized pro-SI with the basolateral membrane on its transfer from the Golgi complex to the microvillus membrane, suggesting that part of the basolateral membrane or its associated structures might be involved in the sorting-out processes of microvillar membrane proteins. This concept may have general relevance for the mechanisms of membrane insertion, intracellular transport and sorting of other microvillar membrane glycoproteins in the intestinal epithelial cell.

Physiology of epithelial transport in the human eye.

Epithelia of the eye are located in the cornea, the lens, ciliary body, and pigment epithelium. In this review the relevant aspects of electrolyte transport properties in the isolated human cornea and lens are described. The main physiological function of both the cornea and the lens is the conservation of the transparency of these structures. (1) In the isolated human cornea sodium and chloride ions are pumped towards the tear side. Both net transports are sensitive to pH-changes and can be stimulated by cyclic AMP, epinephrine, and norepinephrine. The response to adrenergic stimulation is mainly mediated by β2 receptors. Propranolol, a β-blocking agent widely used for treatment of glaucoma, hypertension, and angina pectoris, inhibits net sodium transport. By measuring intracellular membrane potentials and ion activities active transport mechanisms can be localized within the epithelium. In addition, an active transport across the endothelial cell layer has to be postulated. The discrepancy between the measured net ion flux and the short circuit current indicates that other ions but Na+ and Cl− ions are transported across the human cornea. (2) In the human lens the epithelial layer is localized on the anterior side of the tissue. In the isolated human lens short circuit current can only be inhibited by ouabain when the glycoside is added to the anterior side of the lens. From the effect of ouabain and ion substitution experiments it can be concluded that about 30% of the translenticular ion transport is maintained by Na+ transport. In addition, short-circuit current is also dependent on K+, Cl−, and HCO3 −. Measurements of intracellular membrane potential and potassium activity reveal that the interior of the lens is an electrically well connected tissue. Translenticular electrolyte transport, ionic content, and bioelectric properties of the whole lens are controlled by the epithelial monolayer of the anterior lens side. (3) Some disorders of cornea and lens in patients with chronic renal failure are discussed.

Studies on rat liver catalase. XI. Site of synthesis and segregation by stripped ER membranes.

We reinvestigated the site of synthesis of rat liver catalase, and it has been reconfirmed that catalase is synthesized not only by free polysomes but also by membrane-bound polysomes. Considerable amounts of nascent catalase on rough microsomes were released from the membrane into the medium upon incubation with puromycin, not transported directly into the intracisternal cavity of microsomes. On the other hand, catalase newly synthesized in vitro was shown to be segregated by stripped rat liver microsomal membranes in a state resistant to proteolysis. Since this segregation occurred without coupled protein synthesis, catalase appears to be transported by a mechanism different from co-translational transfer. A hypothesis is presented regarding the mechanism of intracellular transport of liver catalase.

Evidence for rapid and concerted turnover of membrane phospholipids in MOPC 41 myeloma cells and its possible relationship to secretion.

Newly synthesized microsomal membrane phospholipids of actively secreting myeloma cells all appear to decay rapidly, with initial half-lives of less than 1 h. Their rates of decay are similar to each other and to the rates observed earlier for the decay of the protein components of the membrane and also for the intracellular turnover of immunoglobulin light chain, the predominant secretory product of these cells. This evidence, which suggests that many different components of the membrane and, therefore, presumably the membrane itself turn over rapidly, provides support for the hypothesis that unidirectional membrane flow, accompanied by rapid synthesis of the membranes at the origin of flow and their rapid degradation at or near the terminus of flow, is the mechanism of intracellular transport of secretory product by the myeloma cell. An attractive feature of this mechanism is that rapid synthesis and degradation of the membranes could provide the driving force for membrane flow.

A study for hypercholesterolaemia and regulation of cholesterol metabolism in livers--with special reference to disorder of intracellular transport mechanism of cholesterol (author's transl)

A study for hypercholesterolaemia and regulation of cholesterol metabolism in livers--with special reference to disorder of intracellular transport mechanism of cholesterol (author's transl)

Inhibitory effect of cholephilic anions on fatty acid uptake by the perfused rat liver

The mechanisms of uptake and intracellular transport of plasma free fatty acids by the liver cell are poorly known. A cytosolic protein, termed Z protein, has been identified in the liver, intestine and other tissues; in vitro studies show a high affinity of fatty acid and organic anions for this protein and suggest that it could be involved in the cellular transport of fatty acids in vivo. The present experiments demonstrate an inhibitory effect of various organic cholephilic anions (B.S.P., D.B.S.P., flavaspidic acid, bilirubin, etc) on fatty acid uptake by the isolated perfused rat liver. This inhibition is rapidly reversible when the liver is perfused with a medium lacking cholephilic anions, which together with the absence of any functional or ultrastructural damage to the organ, excludes any hepatotoxic effect. Such an inhibition of fatty acid uptake could be due to a competition for binding either to membrane sites and/or to intracellular carriers common to both fatty acids and cholephilic anions. These data are consistent with the involvement of carrier-mediated processes in fatty acid transport in the liver. Finally, simultaneous uptake of radioactive fatty acid and cholephilic anions does not interfere with further fatty acid utilization by the cell.

In vitro synthesis, transport, and assembly of ribulose 1,5-bisphosphate carboxylase subunits.

It is well established that the chloroplast synthesizes only a limited number of its own proteins. Many stromal enzymes and thyla-koid membrane polypeptides are made in the cytosol and must be transferred subsequently from their sites of synthesis to their final locations inside the chloroplast (1). The mechanism of intracellular transport of proteins across the two chloroplast envelope membranes and the events associated with it are unknown.

Mechanism of intracellular energy transport in heart muscle

Mechanism of intracellular energy transport in heart muscle

Effects of colchicine on phagosome-lysosome interaction in retinal pigment epithelium

The retinal pigment epithelium (RPE) from young albino rats was examined under light and electron microscope 2 1/2 to 16 h after intravenous injections of colchicine (0.5 mg per 100 gm body weight). One of the primary effects of colchicine treatment on the RPE is the appearance of extensive acPase-positive dense body aggregates in the Golgi area. The size of the aggregates and the disappearance of microtubules appear to be related to the length of time allowed for colchicine to react. Acid phosphatase (acPase) activity is localized on the dense body aggregates and is particularly evident at a light microscopic level, where it can be observed as darkly stained clumps. Dense bodies are only rarely seen in the immediate proximity of acPase-positive phagosomes in colchicine-treated animals. The results of these experiments suggest the existence of an intracellular transport mechanism for acPase-carrying dense bodies. This intracellular transport is inhibited by colchicine, being probably related to a block of microtubular assembly.

Pathogenesis of Reye's Syndrome: A Working Hypothesis

The brief history of Reye's syndrome has been one of progressive familiarity with a deepening mystery. It is possible that the syndrome reflects a variety of etiologically unrelated conditions with similar clinicopathological manifestations. In this view, subclinical defects in ammonia metabolism may constitute a distinct, identifiable group among disorders responsible for encephalopathy with fatty liver. Awareness of the possible role of mutant urea cycle enzymes and deficient substrates in the pathogenesis of this important illness should stimulate investigations of enzyme kinetics and intracellular transport mechanisms which may reveal a spectrum of abnonmalities in ammonia metabolism-and a specific therapy for each.

Purification and characterization of mouse kidney beta-glucuronidase.

Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.

Estradiol: alterations in the mechanism of intracellular transport.

Abstract (1) Uptake of estradiol into cell nuclei of the anterior hypothalamus and uterus (females) was studied in normal male, normal female, and neonatally androgen treated (androgenized) adult female rats. (2) In the uterus, the addition of excess cytosol to homogenates of similar tissue in incubation increased nuclear uptake of estradiol in normal but not in androgenized females. (3) In the hypothalamus, the addition of excess cytosol to homogenates of similar tissue in incubation increased nuclear uptake of estradiol to similar levels in normal females, normal males and androgenized females. (4) The addition of excess cytosol from normal uterus or hypothalamus to homogenates of androgenized uterus or hypothalamus (respectively) consistently increased nuclear uptake of estradiol during incubation. (5) The data suggest that androgens act during a critical period of hypothalamic differentiation to alter the mechanism for intracellular transport of estradiol.

Radioimmunoassay for proparathyroid hormone.

A sensitive radioimmunoassay has been developed to bovine proparathyroid hormone which employs an antiserurm produced by immunization of rabbits with a synthetic 18-amino acid peptide fragment of the bovine prohormone. The synthetic prohormone fragment was prepared by solid-phase synthesis and consists of the amino-terminal 12 residues of bovine parathyroid hormone plus the hexapeptide sequence of the bovine prohormone. The antiserum is uniquely specific to detection of the prohormone or synthetic fragments that preserve the peptide linkage between the prohormone region and amino-terminal hormonal sequences. Bovine proparathyroid hormone of natural origin, as well as synthetic fragments incorporating the prohormone hexapeptide sequence and amino-terminal sequences of bovine parathyroid hormone, are bound by the antiserum, but the hexapeptide, intact bovine or human hormone and amino-terminal hormonal fragments alone are not detected. The assay readily measures prohormone in extracts of bovine but not human parathyroids, a finding that may be explained by the fact that human proparathyroid hormone differs from bovine by a substitution of serine for alanine at position number one and leucine for phenylalanine at position seven of the prohormone sequence. Thus, the assay should be particularly useful in studies of the intracellular localization of bovine proparathyroid hormone and the mechanism of intracellular transport and conversion of proparathyroid hormone to parathyroid hormone. In preliminary studies, it was found that proparathyroid hormone was undetectable in peripheral or parathyroid effluent blood from calves, a finding which reflects either a low rate of prohormone secretion or a rapid cleavage of the prohormone in blood once secreted.

Morphology and development of dengue-2 virus employing the freeze-fracture and thin-section techniques

The freeze-fracturing of purified dengue-2 virus has shown at least three distinct fracture planes: a grazing fracture removing at most some surface projections; a fracture apparently splitting the membrane; and a cross fracture through the nucleocapsid of the virus. Morphogenesis in LLC-MK2 cell culture was studied by use of the freeze-fracture technique and also by thin sectioning. Freeze-fracturing has been helpful in determining the sites and extent of virus development before the virus appears in vacuoles, and in determining related phenomena of infection. There is no direct evidence from freeze-fracturing for the “budding” of virus into these vacuoles, and their release by reverse pinocytosis, both of which have been previously proposed. However, evidence from thin sections herein suggests that release of mature virus from the cell may yet be by a reverse pinocytosis from small vesicles involved with the normal mechanisms of intracellular transport.

Intracellular mechanisms of oxygen transport in flowing blood

As a side effect deformation of red cells in the microvasculature produces intracellular convection. Therefore, beside the conventionally assumed diffusive intracellular oxygen transport an additional convective transport must definitely be taken into consideration, which increases the rate of gas exchange by the blood distinctly. In this paper an attempt is made to determine quantitatively the relative role of the four possible mechanisms of intracellular oxygen transport, i.e. diffusion and convection of both dissolved and chemical bound oxygen. They can be detached from each other by measuring the oxygen release of resting and flowing blood before and after inhibiting the HbO 2 system by carbon monoxide. Under the present experimental conditions (packed cells in viscometric flow) intracellular convective oxygen transport is of greater significance than the diffusive; its relative role increases with time of deoxygenation.