Cisternal Contents Research Articles

Giant Cisterna Chyli: MRI Depiction with Gadolinium-DTPA Enhancement

AIM: To demonstrate the use of MRI with Gadolinium-DTPA enhancement in the diagnosis of giant cisterna chyli.MATERIALS AND METHODS: The study consisted of contrast enhanced MRI with ultrasound and CT correlation in three patients with a giant cisterna chyli. An analysis of the morphology, location and post-contrast MR signal characteristics in relation to time was performed.RESULTS: Cisterna chyli has a characteristic lobulated morphology and location but the unenhanced MRI appearances are not specific. The post-Gadolinium-DTPA MRI appearances are critically dependent on the time elapsed after injection of contrast medium. Within the first 5min, there is no enhancement at all, but by 10min there is early layering of contrast medium evident, which by 30min produces a clear fluid-fluid level. Delayed images at 4–5h demonstrate a uniform enhancement of the cisternal contents producing a homogeneous intermediate signal. All of these features are best visualized on T1 fast saturation sequences.CONCLUSION: MRI with Gadolinium-DTPA enhancement is valuable in confirming the nature of the lymphatic ducts in the retroperitoneal space and helps to differentiate these normal structures from alternative lesions such as lymphadenopathy and tumour recurrence.Lee, K. C. Y., Cassar-Pullicino, V. N. (2000).Clinical Radiology55 , 51–55.

The identity of mystery organelles in Arabidopsis plants expressing GFP

The identity of mystery organelles in Arabidopsis plants expressing GFP

Determination of the intracellular distribution and pool sizes of apolipoprotein B in rabbit liver.

We have investigated the intracellular distribution of apolipoprotein B (apo B) in rabbit liver by immunoblotting, radioimmunoassay (r.i.a.) and enzyme-linked immunoassay (e.l.i.s.a.). Apo B100 was detected in total microsomes, rough microsomes, smooth microsomes, trans-enriched Golgi and cis-enriched Golgi and membrane and cisternal-content subfractions prepared from these fractions. There was also evidence of degradation of apo B100 in the Golgi membrane fractions. The amount of apo B in the subcellular fractions detected by competitive r.i.a. or e.l.i.s.a. ranged from 1.5 micrograms/mg of protein in the rough endoplasmic reticulum to 13 micrograms/mg of protein in the trans-Golgi fraction. Using internal standards (NADPH-cytochrome c reductase for the endoplasmic reticulum and galactosyltransferase for the Golgi membranes) it was calculated that all the apo B of liver is recovered within the secretory compartment, with 63% of the total apo B in the endoplasmic reticulum and the remainder in the Golgi. When the subcellular fractions were separated into membranes and cisternal contents, 60%, 50%, 60% and 30% of the total apo B was recovered in the membrane of the rough microsomes, smooth microsomes, cis-Golgi and trans-Golgi respectively. Using competitive e.l.i.s.a. we found that the membrane-bound form of the apo B was exposed at the cytosolic surface of the intact subcellular fractions. These observations are consistent with a model for assembly of very-low-density lipoproteins (VLDL) in which newly synthesized apo B is incorporated into a membrane-bound pool and a lumenal pool. The membrane-bound pool not used for VLDL assembly may be degraded, possibly in the Golgi region.

The distribution of apolipoprotein B in endoplasmic reticulum and Golgi subfractions of rabbit liver

The liver is the site of synthesis and secretion of very-lowdensity lipoproteins (VLDL), the vehicle for the transport of endogenous triacylglycerol (TAG) and the precursor of lowdensity lipoproteins (LDL). Elevated plasma levels of VLDL and LDL are recognized risk factors in the development of atherosclerosis. The production of VLDL by the liver is considered to be the most appropriate therapeutic target for the reduction of circulating lipoprotein levels. An understanding of the molecular and topographical events in the assembly of VLDL is thus of fundamental importance. Apolipoprotein B (apo B) is the major non-exchangeable protein of VLDL, and is essential for their secretion. Studies in HEP-G2 cells have indicated that apo B is synthesized by membrane-bound polysomes [I]. Newly synthesized apo B remains associated with the endoplasmic reticulum (ER) membrane for a period of time before being transferred to the cisternal space [2]. Consistent with this, we have previously shown that apo B does not associate with TAG-richVLDL precursors in the ER cisternae, and that the main site of packaging of VLDL lipid and protein is the Golgi cisternae [3]. To examine the intracellular events in apo B synthesis and translocation in more detail, we have determined the intracellular location and amount of apo B within the secretory pathway. The intracellular location of apo B was determined by SDS/PAGE and immunoblotting of subcellular fractions. ER and Golgi fractions were prepared from rabbit liver, and separated into membrane and cisternal content fractions by sodium carbonate treatment 14, 51. Fractions were delipidated and separated on 3-20% (w/v) polyacrylamidegradient gels [6], and then electroblotted on to nitrocellulose membrane [7]. Apo B- 100, the only apparent species of apo B secreted by rabbit liver, was detected by immunoblotting using sheep anti-rabbit apo B (primary antibody), and biotinylated anti-sheep IgG (secondary antibody). The secondary antibody was detected colorimetrically by the highly sensitive Vector ABC-alkaline phosphatase system. Apo €3- 100 was detectable in both ER and Golgi fractions (Fig. 1 ). When ER was separated into membranes and cisternal contents, apo B-100 was found in both fractions at apparently similar levels. When Golgi was separated into membranes and cisternal contents, although apo B- 100 could be detectcd in membranes, most of the protein appeared to be located in the contents. Apo €3- 100 in subcellular fractions was quantified by radioimmunoassay. LDL were isolated from rabbit plasma (d 1 .O 19-d 1.063). and radiolabclled with lz51 [ 8, 01. Apo B-I00 was the only protein detectable in LDL by SDS/ PAGE. and was found to be 'z51-labelled. Using a fixed concentration of labelled LDL with varying concentrations of

Fasting induces modifications of the endoplasmic reticulum in intestinal cells

The polymorphism of the endoplasmic reticulum (ER) in epithelial cells with different transport functions such as the enterocyte suggests that the ER may be involved in some way in molecular transport. To further access this possibility, we examined the ER from the intestine of winter flounder, Pseudopleuronectes americanus, a species which undergoes an annual fast of approximately 6 months' duration, a time during which previous work indicates nutrient carrier number does not change. Fish from June (feeding) and January (8-10 weeks fasted) were sampled. Tissues from the pyloric caeca, foregut, midgut, and hindgut were prepared for electron microscopy using two techniques of staining. Cell height was unaltered in any section, although microvillar length shortened variably. Cellular organization, including position of nuclei, number and distribution of mitochondria, and presence of basolateral membranes, did not change. The ER appeared equally abundant in June and January. However, use of the osmium impregnation technique, which is specific for ER cisternal contents, revealed a change in the impregnation of ER, from a heavily impregnated network in summer to little or no impregnation in winter. These results suggest that a shift in function of the ER had occurred when nutrient transport ceased, and supports a role of the ER in nutrient transport.

An improved purification of glycosomes from the procyclic trypomastigotes of Trypanosoma brucei

The glycosomes of in vitro grown procyclic trypomastigote forms of Trypanosoma brucei were purified by three different procedures and the results compared by electron microscopy, enzyme assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Centrifugation on a self-forming Percoll gradient followed by a sucrose gradient centrifugation resulted in the least enriched glycosomal preparation. Centrifugation on a pre-formed Nycodenz gradient gave an improved preparation but the most homogeneous preparation of intact glycosomes was obtained after centrifugation on two successive sucrose gradients. Glycosomes purified by both the Nycodenz and double sucrose gradient procedures appeared larger than in situ glycosomes presumably due to an osmotic effect resulting from disruption of the granular matrix of the organelles. Nevertheless, there appears to be no loss of cisternal contents due to the swelling of the organelles. The glycosomes of the bloodstream from trypomastigotes purified by the same procedures show, however, no sign of swelling. A comparison of glycosomes purified from procyclic trypomastigotes and bloodstream form trypomastigotes prepared by the same double sucrose procedure demonstrated that in the glycosome of procyclic trypomastigotes: (1) activities of hexokinase, phosphoglucose isomerase, phosphofructose kinase, aldolase and phosphoglycerate kinase and diminished by 80–100%; (2) activities of glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase and glycerol-3-phosphate dehydrogenase remain unchanged or are only slightly reduced; (3) there is an appearance of four major new proteins, among which could be phosphoenol pyruvate carboxykinase and malate dehydrogenase. These observations are in basic agreement with those by Hart et al. (Mol. Biochem. Parasitol. 12, 25–35, 1984).

Plasma membrane specialization at the discharge site of the excretory poreless vacuole of the ciliate Euplotes raikovi

The contractile vacuole of Euplotes raikovi consists basically of a membranedelimited cistern that has no direct communication with the exterior of the cell. Contiguous alveoli interpose between the cisterna and the plasma membrane and probably receive the cisternal contents before being expelled. The area of the plasma membrane that interacts with the outer membrane of the alveoli overlying the cistern has been found to carry a special region of intramembranous particles organized in 100–200 strands, showing varied sizes and a tendency to align parallel with one another. The region is also characterized by a smooth central core and by characteristic deformations that have been interpreted as focal sites of discharge of the contractile vacuole.

Glycogen—membrane complexes in denervated human skeletal muscle

We describe an unusual intracellular complex of glycogen with smooth, cisternal, cytoplasmic membranes (glycogen-membrane complexes, GMC) in denervated human skeletal muscle. Glycogen particles were always intimately associated with these structures, although the staining intensity varied markedly with different fixation conditions. This may account for previous investigations of similar structures in which an association with glycogen was not recognized. Electron micrographs of tannic acid-enhanced specimens, and of freeze-fracture replicas, showed similarities between the GMCs and the terminal cisternae of the sarcoplasmic reticulum: (i) GMCs resembled the terminal cisternae of the sarcoplasmic reticulum in the size and asymmetric distribution of intramembranous particles seen in freeze-fracture replicas; (ii) tannic acid-enhanced thin sections of GMCs showed intense staining of the cisternal contents and irregular staining of the cytoplasmic leaflet, similar to the appearance of the sarcoplasmic reticulum terminal cisternae; and (iii) triad-like junctions were seen between the glycogen-membrane complexes and elements of the transverse tubule system.

Lipid composition of the golgi apparatus of rat kidney and liver in comparison with other subcellular organelles

Golgi apparatus isolated from both rat liver and rat kidney have been characterized with respect to their neutral and phospholipid content and their phospholipid composition and compared with mitochondria, rough endoplasmic reticulum and plasma membranes. In addition, the distribution of sulfatide in the subcellular fractions of rat kidney was determined. The Golgi apparatus from both sources has the highest content of neutral lipids which are rich in cholesterol esters and ubiquinone. Removal of about 75% of the cisternal contents of rat liver Golgi reduced its content of cholesterol esters but not of ubiquinone. The Golgi complex of liver most closely resembles endoplasmic reticulum in its phospholipid composition except for a higher content of sphingomyelin. Removal of most of the contents of the Golgi cisternae did not appreciably alter the phospholipid composition of the Golgi apparatus of liver. Golgi apparatus from kidney has a phospholipid composition which resembles liver Golgi much more closely than it does any other cell fraction from kidney. The sulfatide content of kidney Golgi, the cell fraction richest in this glycolipid, is about 14% of the total lipid present in this fraction. Sulfatide was present in plasma membranes, mitochondria and rough microsomes, but at about one-third the level found in Golgi. Sulfatide is the main glycosphingolipid present in all the cell fractions from kidney which were studied.

Changes in the fine structure of tadpole ( Rana catesbeiana) liver during thyroxine-induced metamorphosis

Summary Alterations in the fine structure of tadpole ( Rana catesbeiana ) liver cells have been studied during the period of extensive biochemical modification caused by l -thyroxine. The nuclei, euchromatic in control cells, become heterochromatic. The mitochondria increase significantly in size with slight alterations in the cristae. The rough endoplasmic reticulum proliferates, and the staining density of the cisternal lumens increases. The Golgi complex and its cisternal contents increase in staining density, and the number of Golgi regions increases. These changes provide cytological criteria for the differentiation of liver cells during thyroxine-induced metamorphosis.

Electron microscope study on primary liquor folliculi secretion in the mouse ovary

This paper reports a study by light and electron microscopy on changes observed in the granulosa cells of the primary ovarian follicle of the mouse. These changes are, apparently, concomitant with primary follicular fluid secretion. The staining of thick sections of OSO4-fixed, Epon-embedded specimens with Azure B and periodic acid-Schiff reagent has revealed the presence of follicular fluid between granulosa cells and in the follicular spaces at an early phase of follicular development. Electron microscope observations have shown within the granulosa cells a sequence of changes which appear to be associated with cellular secretory functions. These changes begin with the enlargement of some of the Golgi canaliculi and are followed by an apparent discontinuity of their course. In the next phase smooth-walled cisternae appeared and were concentrated in the cortical area of the cells. The ultimate sequence was the discharge of the cisternal contents into the intercellular spaces. The follicular fluid appeared electron opaque within the intercellular spaces where it appeared to fill the spaces between thin, somewhat more electron dense, fibrillar micelles. Call-Exner bodies were observed as cytoplasmic droplets.