bluuebunny/crossref_metadata_2025 huggingface dataset:
Human platelet-derived growth factor: radioimmunoassay and discovery of a specific plasma-binding protein.
Authors: Huang J S , Huang S S , Deuel T F
Published: May 2004

The platelet-derived growth factor (PDGF) is the principal mitogen in serum for cultured cells of mesenchymal origin. PDGF also is a potent chemotactic protein for inflammatory cells and for cells required for wound repair. Because activity levels of PDGF in biological fluids are difficult to measure, we attempted to develop a radioimmunoassay for PDGF. Rabbits were immunized with purified PDGF; the antiserum obtained was monospecific for PDGF in immunodiffusion analysis against concentrated platelet lysates, serum, and plasma. A radioimmunoassay for PDGF was developed with a sensitivity of congruent to 0.2 ng/ml. Levels of PDGF in plasma/serum were measured and compared with PDGF levels determined by a receptor-competition assay and by a standard biological assay measuring incorporation of [3H]thymidine into 3T3 cells. Radioimmunoassay showed apparent PDGF levels of 50 ng/ml in human plasma and 103 ng/ml in serum. The 50 ng/ml PDGF in plasma was unexpected because the plasma samples contained little or no platelet release products as determined by very low levels of platelet factor 4. We therefore sought an immunologically reactive PDGF molecule in human plasma. No immunologically reactive protein was detected by immunodiffusion analysis or when plasma was treated with an immunoaffinity gel. Subsequently, a 125I-PDGF-binding protein was identified; the 125I-PDGF-plasma-binding protein complex was not reactive with anti-PDGF immunoglobulin. Correction for 125I-PDGF bound by the plasma-binding protein established serum levels of PDGF of congruent to 50 ng/ml; congruent to 50 ng/ml PDGF was found in serum by radioreceptor-competition assays and by mitogenic assays as well. The plasma-binding protein may serve to clear PDGF released in the circulation, thereby limiting PDGF activity to its local interactions at the site of blood-vessel injury.
Monoclonal antibodies specific for tight-binding human chromatin antigens reveal structural rearrangements within the nucleus during the cell cycle.
Authors: Bhorjee J S , Barclay S L , Wedrychowski A , Smith A M
Published: May 2004

The class of nonhistone chromosomal proteins that remains bound to DNA in chromatin in the presence of 2.5 M NaCl-5 M urea has proven refractile to biochemical analysis. In order to study its role in chromatin organization, we have produced monoclonal antibodies that are specific for the HeLa DNA-protein complex that remains after extraction of chromatin with high salt and urea. The antibody-producing clones were identified with an ELISA assay. Of the six clones selected, five were stabilized by limiting dilution. All clones are IgG producers. None cross-react significantly with native DNA, core histones, or the high-mobility group nonhistone proteins. All antibodies are specific for nuclear or juxtanuclear antigens. Indirect immunofluorescence shows that three antibodies, which are nonidentical, stain three different nuclear networks. Available evidence indicates that two of these networks are the nuclear matrix. A fourth antibody reveals structures reminiscent of chromocenters. A fifth antibody, AhNA-1, binds to interphase HeLa chromatin and specifically decorates metaphase chromosomes. AhNA-1 similarly recognizes rat chromosomes. Each of these monoclonal antibodies also reveals a changing pattern of nuclear staining as cells progress through the cell cycle. Presumably, this reflects the rearrangement of the cognate antigens.
Factors influencing the expression of stress fibers in vascular endothelial cells in situ.
Authors: White G E , Gimbrone M A , Fujiwara K
Published: May 2004

The organization of actin and myosin in vascular endothelial cells in situ was studied by immunofluorescence microscopy. Examination of perfusion-fixed, whole mounts of normal mouse and rat descending thoracic aorta revealed the presence of axially oriented stress fibers containing both actin and myosin within the endothelial cells. In both species, the proportion of cells containing stress fibers varied from region to region within the same vessel. Some endothelial cells in mouse mesenteric vein and in rat inferior vena cava also contained stress fibers. Quantitative studies of the proportion of endothelial cells containing stress fibers in the descending thoracic aorta of age-matched normotensive and spontaneously hypertensive rats revealed significant differences. When animals of the same sex of the two strains were compared, the proportion was approximately two times greater in the spontaneously hypertensive rats. The proportion of endothelial cells containing stress fibers was about two times greater in males than in females of both strains. These observations suggest that multiple factors, including anatomical, sex, and hemodynamic differences, influence the organization of the endothelial cell cytoskeleton in situ.
Insulin as a surface marker on isolated cells from rat pancreatic islets.
Authors: Kaplan D R , Colca J R , McDaniel M L
Published: May 2004

Immunoreactive insulin was shown to exist as a surface molecule in the plasma membrane of dispersed rat pancreatic islet cells. The intact cells were stained by immunofluorescence with a guinea pig antisera specific for insulin. The hormone on the cell surface could not be accounted for by insulin bound to specific receptors or nonspecifically absorbed to cells. Thus, surface insulin was demonstrated to be a specific membrane antigen for islet cells. Furthermore, the proportion of islet cells with insulin on the cell surface was directly correlated with insulin secretion in several different settings. This correspondence was demonstrated by varying the glucose concentration in the medium, by withholding Ca2+, which inhibits secretion, and by adding theophylline, which potentiates secretion. Consequently, these results suggested that insulin as a membrane protein was a marker for cells that actively secreted the hormone and may have been derived in the fusion process of secretory granules with the plasma membrane.
Control of rat C6 glioma cell proliferation: uncoupling of the inhibitory effects of hydrocortisone hormone in suspension and monolayer cultures.
Authors: Armelin M C , Stocco R C , Armelin H A
Published: May 2004

We undertook a comparative study of the effects of the hormone hydrocortisone (Hy) on C6 glioma cells grown in monolayer and in suspension in cultures. We found Hy reversibly renders C6 cells anchorage- and serum-dependent for their growth. In monolayer cultures, Hy was found to inhibit cell cycle traversing exclusively at G1 phase. In agarose suspension, Hy was found to block colony development. Hy-resistant variants were selected and isolated in agarose suspension. Examination of these variants showed that cells selected for Hy-resistance in suspension can be Hy sensitive when anchored to a solid substrate. We conclude that resistance to Hy in suspension and resistance to it in monolayer culture are two independent phenotypes.
Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells.
Authors: Hopkins C R , Trowbridge I S
Published: May 2004

The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using 125I-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 125I and gold colloid. Using 125I-transferrin we have shown that, whereas at 37 degrees C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37 degrees C, the intracellular half-life of the most rapidly recycled transferrin was 7.5 min. 125I-ATR displayed the same kinetics of uptake but following its internalization at 37 degrees C, it was partially degraded. At 22 degrees C and below, the intracellular degradation of 125I-ATR was selectively inhibited and as a result it accumulated intracellularly. Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR-gold colloid complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22 degrees C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22 degrees C, no other compartments became labeled but if they were warmed to 37 degrees C the gold complexes were transferred to lysosome-like elements. Extracting ATR-gold complexes with Triton X after a 30-min chase at 22 degrees C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.
Elongation of the fertilization tubule in Chlamydomonas: new observations on the core microfilaments and the effect of transient intracellular signals on their structural integrity.
Authors: Detmers P A , Goodenough U W , Condeelis J
Published: May 2004

Experimental manipulations of gametes of Chlamydomonas reinhardi and ultrastructural observation were used to examine the composition of the microfilaments in the fertilization tubule, their probable mode of formation, and their interaction with intracellular signals. Decoration with myosin subfragment-1 was used to demonstrate that the microfilaments in the fertilization tubule were actin filaments having uniform polarity: Myosin subfragment-1 arrowheads pointed away from the membrane at the tip of the process. Filaments were attached to the cone-shaped "doublet zone" at the base of the process by their pointed ends. Discrete attachment sites for filaments on the surface of the doublet zone were seen in stereo view. To test whether actin polymerization might accompany elongation of the fertilization tubule, mating gametes were exposed to cytochalasin D in an attempt to block actin polymerization. Treatment of mating type "plus" gametes with cytochalasin D prior to and during mating inhibited the appearance of actin filaments in fertilization tubules, suppressed fertilization tubule outgrowth, and lowered mating efficiency from 90 to 15%. The role of signals generated by flagellar adhesion in maintaining the structural integrity of the microfilament-doublet zone complex was examined by correlating flagellar disadhesion with the kinetics of breakdown of the complex. In zygotes, where flagellar disadhesion occurred after cell fusion, the complex disassembled within 3 h after mating. In gametes that had been agglutinated by isolated mating type "minus" flagella, microfilaments and fertilization tubules progressively disassembled over a 3-h time course following flagellar disadhesion. Disassembly of microfilaments was inhibited by maintaining flagellar agglutination, suggesting that signals generated by flagellar adhesion were necessary to maintain microfilaments intact.
Measurements of the force produced by the mitotic spindle in anaphase.
Authors: Nicklas R B
Published: May 2004

The force the spindle exerts on a single moving chromosome in anaphase was measured with a flexible glass needle calibrated in dynes per micron of tip deflection. The needle was used to produce a force on the chromosome, which opposed that produced by the spindle and was measurable from needle tip deflection. The measurements were made in intact grasshopper spermatocytes after proving that the presence of materials such as the cell surface did not interfere. The results from 12 experiments in seven cells are as follows: Chromosome velocity was not affected until the opposing force reached approximately 10(-5) dyn, and then fell rapidly with increasing force. The opposing force that caused chromosome velocity to fall to zero--the force that matched the maximum force the spindle could produce--was of order 7 X 10(-5) dyn. This directly measured maximum force potential is nearly 10,000 times greater than the calculated value of 10(-8) dyn for normal chromosome movement, in which only viscous resistance to movement must be overcome. The spindle's unexpectedly large force potential prompts a fresh look at molecular models for the mitotic motor, at velocity-limiting governors, and at the possibility that force may sometimes affect microtubule length and stability.
Angiotensinogen production by rat hepatoma cells in culture and analysis of its regulation by techniques of somatic cell genetics.
Authors: Bouhnik J , Cassio D , Coezy E , Corvol P , Weiss M C
Published: May 2004

Angiotensinogen was synthesized by cells derived from the Reuber H35 rat hepatoma. Independent clones produced similar amounts of angiotensinogen, which corresponded to about four times more than expected for normal hepatocytes. The protein was secreted rapidly but could be visualized within cells using immunofluorescence. For one clone, it is shown that maximal angiotensinogen synthesis occurred during mid-exponential growth. Somatic cell genetics techniques have been used to investigate the regulation of angiotensinogen expression. Eleven clones of dedifferentiated variant hepatoma cells that failed to produce most or all of the liver specific proteins analyzed including albumin fell into two groups: Seven clones produced only 1-3% as much angiotensinogen as the differentiated clones, and four showed a reduction to 10-30%. Clones of the latter class were the only ones among the eleven analyzed that retained the potential to give rise to revertants, showing restoration of the differentiated state. All revertants fully restored angiotensinogen production, but only some of them re-expressed albumin. Somatic hybrids between differentiated hepatoma cells and one of the variants showed a substantial reduction in angiotensinogen production, whereas for some clones, albumin synthesis was fully maintained. These results show that regulation of the expression of angiotensinogen and of a second serum protein, albumin, was independent and that angiotensinogen synthesis was a faithful indicator of the general differentiation profile of all classes of clones.
Co-migration and internalization of transferrin and its receptor on K562 cells.
Authors: Enns C A , Larrick J W , Suomalainen H , Schroder J , Sussman H H
Published: May 2004

The incorporation of iron into human cells involves the binding of diferric transferrin to a specific cell surface receptor. We studied the process of endocytosis in K562, a human erythroid cell line, by using tetramethylrhodamine isothiocyanate-labeled transferrin (TRITC-transferrin) and fluorescein isothiocyanate-labeled Fab fragments of goat antireceptor IgG preparation (FITC-Fab-antitransferrin receptor antibody). Because the antireceptor antibody and transferrin bind to different sites on the transferrin receptor molecule it was possible to simultaneously and independently follow ligand and receptor. At 4 degrees C, the binding of TRITC-transferrin or FITC-Fab antitransferrin receptor antibody exhibited diffuse membrane fluorescence. At 20 degrees C, the binding of TRITC-transferrin was followed by the rapid formation of aggregates. However, the FITC-Fab antitransferrin receptor did not show similar aggregation at 20 degrees C unless transferrin was present. In the presence of transferrin, the FITC-Fab antitransferrin receptor antibody formed aggregates at the same sites and within the same time period as TRITC transferrin, indicating co-migration. Although the diffuse surface staining of either label was removed by proteolysis, the larger aggregates were not susceptible to enzyme degradation, indicating that they were intracellular. The internal location of the aggregates was also demonstrated using permeabilized cells that had been preincubated with transferrin and fixed with 4% paraformaldehyde. These cells showed aggregated receptor in the interior of the cell when reacted with fluorescein-labeled antibody to the receptor. This indicated that the transferrin and the transferrin receptor co-internalize and migrate to the same structures within the cell.
Uptake of lipoproteins by in situ perfused rat ovaries: identification of binding sites for high density lipoproteins.
Authors: Paavola L G , Strauss J F
Published: May 2004

We have examined the uptake and distribution of 125I-labeled human high density lipoprotein, apolipoprotein E-free (hHDL3), 125I-rat high density lipoprotein (HDL), and human HDL (hHDL) reconstituted with [3H]cholesteryl linoleate after their in situ vascular perfusion to ovaries of gonadotropin-primed immature rats on days 6-9 post human chorionic gonadotropin (hCG)-injection. Some rats were treated with 4-aminopyrazolopyrimidine to reduce plasma lipoproteins and ovarian cholesteryl ester stores. Perfused ovaries were analyzed biochemically and autoradiographically, and progestin content of the ovarian effluent was quantified. Infusion of ovine luteinizing hormone and hHDL increased ovarian progestin secretion severalfold, indicating that the perfused ovary was functional. After perfusion with HDL reconstituted with [3H]cholesteryl linoleate, radioactive progestin appeared in the effluent; thus, sterol carried by exogenous HDL was converted to steroid. At 37 degrees C, uptake of 125I-hHDL3 was greatest after 15 min of perfusion with label. This was decreased by 80% when the perfusion was carried out at 4 degrees C and by 70-95% when excess unlabeled hHDL, but not human low density lipoprotein (hLDL), was included in the perfusate with 125I-hHDL. Aminopyrazolopyrimidine treatment enhanced 125I-hHDL uptake twofold. After perfusion for 15 min with 125I-hHDL3, radioactivity in the ovary was high for 3-30 min of HDL-free wash, then declined 75% by 30-60 min. With light and electron microscope autoradiography, 125I-hHDL3 was localized to corpora lutea, both along luteal cell surfaces and over their cytoplasm. The plasma membrane grains appeared to be associated with segments that lacked bristle coats. Perfusion with 125I-rat HDL produced a similar pattern of labeling. In ovaries perfused with 125I-BSA, silver grains were concentrated over macrophage-like cells but were sparse over luteal cells. We conclude that the in situ perfused rat ovary takes up 125I-hHDL3 by a temperature-dependent, lipoprotein-specific process, and that this lipoprotein is accumulated by luteal cells.
Transcytosis in thyroid follicle cells.
Authors: Herzog V
Published: May 2004

Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species.
Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. I. Morphological evidence.
Authors: Matlin K , Bainton D F , Pesonen M , Louvard D , Genty N , Simons K
Published: May 2004

The G protein of vesicular stomatitis virus was implanted in the apical plasma membrane of Madin-Darby canine kidney cells by low pH-dependent fusion of the viral envelope with the cellular membrane. The amount of fusion as determined by removal of unfused virions, either by tryptic digestion or by EDTA treatment at 0 degree C, was 22-24% of the cell-bound virus radioactivity. Upon incubation of cells after implantation, the amount of G protein as detected by immunofluorescence diminished on the apical membrane and appeared within 30 min on the basolateral membrane. At the same time some G protein fluorescence was also seen in intracellular vacuoles. The observations by immunofluorescence were confirmed and extended by electron microscopy. Using immunoperoxidase localization, G protein was seen to move into irregularly shaped vacuoles (endosomes) and multivesicular bodies and to appear on the basolateral plasma membrane. These results suggest that the apical and basolateral domains of Madin-Darby canine kidney cells are connected by an intracellular route.
Modulation of glycosylation and transport of viral membrane glycoproteins by a sodium ionophore.
Authors: Alonso-Caplen F V , Compans R W
Published: May 2004

Analysis of viral glycoprotein expression on surfaces of monensin-treated cells using a fluorescence-activated cell sorter (FACS) demonstrated that the sodium ionophore completely inhibited the appearance of the vesicular stomatitis virus (VSV) G protein on (Madin-Darby canine kidney) MDCK cell surfaces. In contrast, the expression of the influenza virus hemagglutinin (HA) glycoprotein on the surfaces of MDCK cells was observed to occur at high levels, and the time course of its appearance was not altered by the ionophore. Viral protein synthesis was not inhibited by monensin in either VSV- or influenza virus-infected cells. However, the electrophoretic mobilities of viral glycoproteins were altered, and analysis of pronase-derived glycopeptides by gel filtration indicated that the addition of sialic acid residues to the VSV G protein was impaired in monensin-treated cells. Reduced incorporation of fucose and galactose into influenza virus HA was observed in the presence of the ionophore, but the incompletely processed HA protein was cleaved, transported to the cell surface, and incorporated into budding virus particles. In contrast to the differential effects of monensin on VSV and influenza virus replication previously observed in monolayer cultures of MDCK cells, yields of both viruses were found to be significantly reduced by high concentrations of monensin in suspension cultures, indicating that cellular architecture may play a role in determining the sensitivity of virus replication to the drug. Nigericin, an ionophore that facilitates transport of potassium ions across membranes, blocked the replication of both influenza virus and VSV in MDCK cell monolayers, indicating that the ion specificity of ionophores influences their effect on the replication of enveloped viruses.
Estrogen action at endometrial membranes: alterations in luminal surface detectable within seconds.
Authors: Rambo C O , Szego C M
Published: May 2004

The morphological effects of estrogen on the luminal surfaces of rat endometrial cells were investigated by scanning electron microscopy. Ovariectomized rats were injected intravenously with estradiol-17 beta (E2 beta), 0.5 micrograms/0.25 ml per 100 g body wt. At various intervals thereafter, the lumen of a uterine horn was flushed with buffered 2% glutaraldehyde and then prepared for scanning electron microscopy by conventional methods. In control rats that had received an equivalent volume of placebo vehicle, the luminal cell surface was characterized by short, sparse microvilli (MV) and, in most cells, a single, central cilium. At 30 s after E2 beta injection, the number of MV was significantly increased. By 1 min, MV density was further increased and MV were frequently clustered; also, the central cilium of many cells was no longer evident. Similar results were obtained after exposure to diethylstilbestrol for 30 s to 1 min, whereas neither a subthreshold dose of E2 beta nor a dose of the relatively inactive congener E2 alpha equivalent to a saturating concentration of E2 beta gave statistically significant responses in surface changes by the present criteria. After 3-7 min of E2 beta exposure, MV had increased greatly in length and density. These effects underwent dramatic regression by 15-30 min after E2 beta treatment, with distinct diminution of microvillar lengths and numbers, reduction of clustering, and reappearance of the central cilium in many cells. This was succeeded at 1 h by a renewed surge of surface activity. These results are consistent with cumulative evidence for rapid alterations of the surface membrane of estrogen-sensitive cells in response to physiological levels of active hormone. Whether these responses in the luminal surfaces are primary, or are secondary reflections of receptor-mediated membrane alterations at the basolateral blood-front, remains to be determined.
Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina.
Authors: Bunt-Milam A H , Saari J C
Published: May 2004

The recent discovery and characterization of several proteins that purify with endogenous, bound retinoid have given rise to the suggestion that these proteins, which are abundant in retina, perform a role in transport and function of vitamin A. Immunocytochemical techniques were used to localize two retinoid-binding proteins in the retina of four species. Antisera to cellular retinal-binding protein (CRALBP) and an interphotoreceptor retinoid-binding protein (IRBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antibodies from each antiserum reacted with a single component in retinal homogenates and supernatants which corresponded to the molecular weight and charge of the respective antigen (non-SDS and SDS PAGE, electrophoretic transfer to nitrocellulose, immunochemical staining). Immunocytochemistry controls were antibodies from nonimmune serum and antibodies absorbed with purified antigen. Antigens were localized on frozen-sectioned bovine, rat, monkey, and human retina using immunofluorescence and the peroxidase-antiperoxidase technique. Specific staining with anti-IRBP was found in the space that surrounds photoreceptor outer segments, with heaviest labeling in a line corresponding to the retinal pigment epithelium (RPE) apical surface. Cone outer segments were positive. Staining with anti-CRALBP was found in two cell types in all species: the RPE and the Müller glial cell. Within the RPE, labeling filled the cytoplasm and was heaviest apically, with negative nuclei. Labeling of Müller cells produced Golgi-like silhouettes with intense staining of all cytoplasmic compartments. Staining of the external limiting membrane was heavy, with labeled microvilli projecting into the interphotoreceptor space. Localization of IRBP to this space bordered by three cell types (RPE, photoreceptor, and Müller) is consistent with its proposed role in transport of retinoids among cells. Localization of CRALBP in RPE corroborates previous biochemical studies; its presence in the Müller cell suggests that this glial cell may play a hitherto unsuspected role in vitamin A metabolism in retina.
Three-dimensional ultrastructure of a unicellular cyanobacterium.
Authors: Nierzwicki-Bauer S A , Balkwill D L , Stevens S E
Published: May 2004

The first complete three-dimensional ultrastructural reconstruction of a cyanobacterium was accomplished with high-voltage electron microscopy and computer-aided assembly of serial sections. The precise arrangement of subcellular features within the cell body was very consistent from one cell to another. Specialized inclusion bodies always occupied specific intracellular locations. The photosynthetic thylakoid membranes entirely surrounded the central portion of the cytoplasm, thereby compartmentalizing it from the rest of the cell. The thylakoid membranes formed an interconnecting network of concentric shells, merging only at the inner surface of the cytoplasmic membrane. The thylakoids were in contact with the cytoplasmic membrane at several locations, apparently to maintain the overall configuration of the thylakoid system. These results clarified several unresolved issues regarding structure-function relationships in cyanobacteria.
Effect of colchicine on the Golgi complex of rat pancreatic acinar cells.
Authors: Pavelka M , Ellinger A
Published: May 2004

Colchicine administered to adult rats at a dosage of 0.5 mg/100 g of body weight effected a disorganization of the Golgi apparatus in pancreatic acinar cells. The results obtained after various periods of treatment (10 min to 6 h) showed (a) changes in all components of the Golgi complex, and (b) occurrence of large vacuoles that predominated in cytoplasmic areas outside the Golgi region. The alterations in Golgi stacks concerned elements of the proximal and distal side: (a) accumulation of transport vesicles, (b) formation of small, polymorphic secretion granules, and (c) alterations in the cytochemical localization of enzymes and reaction product after osmification. Transport vesicles accumulated and accompanied short, dilated cisternae, which lack mostly the reaction products of thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase, and osmium deposits after prolonged osmification. After 4 to 6 h of treatment, accumulated transport vesicles occupied extensive cellular areas; stacked cisternae were not demonstrable in these regions. The changes on the distal Golgi side included GERL elements: condensing vacuoles were diminished; they were substituted by small, polymorphic zymogen granules, which appeared to be formed by distal Golgi cisternae and by rigid lamellae. Unusually extended coated regions covered condensing vacuoles, rigid lamellae, and polymorphic secretion granules. A cytochemical distinction between Golgi components and GERL was possible neither in controls nor after colchicine treatment. The cytochemical alterations in Golgi components were demonstrable 20-30 min following administration of colchicine; at 45 min, initial morphological changes--augmentation of transport vesicles and formation of polymorphic zymogen granules--became apparent. 20 min after administration of colchicine, conspicuous groups of large vacuoles occurred. They were located mostly in distinct fields between cisternae of the endoplasmic reticulum, and were accompanied by small osmium--reactive vesicles. Stacked cisternae were not demonstrable in these fields. Vacuoles and vesicles were devoid of reaction products of thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase. The results provide evidence that formation of stacked Golgi cisternae is impaired after colchicine treatment. The colchicine--induced disintegration of the Golgi complex suggests a regulatory function of microtubules in the organization of the Golgi apparatus.
Cell surface changes and enzyme release during hypoxia and reoxygenation in the isolated, perfused rat liver.
Authors: Lemasters J J , Stemkowski C J , Ji S , Thurman R G
Published: May 2004

We examined the effects of hypoxia and reoxygenation in isolated, perfused rat livers. Hypoxia induced by a low rate of perfusion led to near anoxia confined to centrilobular regions of the liver lobule. Periportal regions remained normoxic. Within 15 min, anoxic centrilobular hepatocytes developed surface blebs that projected into sinusoids through endothelial fenestrations. Periportal hepatocytes were unaffected. Both scanning and transmission electron microscopy suggested that blebs developed by transformation of preexisting microvilli. Upon reoxygenation by restoration of a high rate of perfusion, blebs disappeared. Other changes included marked shrinkage of hepatocytes, enlargement of sinusoids, and dilation of sinusoidal fenestrations. There was also an abrupt increase in the release of lactate dehydrogenase and protein after reoxygenation, and cytoplasmic fragments corresponding in size and shape to blebs were recovered by filtration of the effluent perfusate. We also studied phalloidin and cytochalasin D, agents that disrupt the cytoskeleton. Both substances at micromolar concentrations caused rapid and profound alterations of cell surface topography. We conclude that hepatic tissue is quite vulnerable to hypoxic injury. The morphological expression of hypoxic injury seems mediated by changes in the cortical cytoskeleton. Reoxygenation causes disappearance of blebs and paradoxically causes disruption of cellular volume control and release of blebs as cytoplasmic fragments. Such cytoplasmic shedding provides a mechanism for selective release of hepatic enzymes by injured liver tissue.
Scanning electron microscope-analysis of the protrusions (knobs) present on the surface of Plasmodium falciparum-infected erythrocytes.
Authors: Gruenberg J , Allred D R , Sherman I W
Published: May 2004

The nature of the surface deformations of erythrocytes infected with the human malaria parasite Plasmodium falciparum was analyzed using scanning electron microscopy at two stages of the 48-h parasite maturation cycle. Infected cells bearing trophozoite-stage parasites (24-36 h) had small protrusions (knobs), with diameters varying from 160 to 110 nm, and a density ranging from 10 to 35 knobs X micron-2. When parasites were fully mature (schizont stage, 40-44 h), knob size decreased (100-70 nm), whereas density increased (45-70 knobs X micron-2). Size and density of the knobs varied inversely, suggesting that knob production (a) occurred throughout intraerythrocytic parasite development from trophozoite to schizont and (b) was related to dynamic changes of the erythrocyte membrane. Variation in the distribution of the knobs over the red cell surface was observed during parasite maturation. At the early trophozoite stage of parasite development, knobs appeared to be formed in particular domains of the cell surface. As the density of knobs increased and they covered the entire cell surface, their lateral distribution was dispersive (more-than-random); this was particularly evident at the schizont stage. Regional surface patterns of knobs (rows, circles) were seen throughout parasite development. The nature of the dynamic changes that occurred at the red cell surface during knob formation, as well as the nonrandom distribution of knobs, suggested that the red cell cytoskeleton may have played a key role in knob formation and patterning.
Modulation of collagen synthesis by a growth factor and by the extracellular matrix: comparison of cellular response to two different stimuli.
Authors: Tseng S C , Savion N , Gospodarowicz D , Stern R
Published: May 2004

Cultured bovine corneal endothelial cells can be grown in three ways: on plastic, on plastic with fibroblast growth factor present in the media, and on their own preformed extracellular matrix. On plastic alone, cells grow in a disorderly fashion and secrete matrix on all cell surfaces. Cells grown on plastic with growth factor or on a matrix, at confluence, have matrix deposition only on the basal surface of the cells and an orderly contact-inhibited pattern of growth. This correlates with the polarity they demonstrate histologically. This cell-matrix pattern resembles the pattern observed in vivo. Both the soluble growth factor and the extracellular matrix are able to modulate the pattern of collagen synthesis and deposition by cells, but they do so in two entirely different ways. In cells grown on the extracellular matrix, total collagen synthesis is lower but more efficient. Collagen is deposited primarily into the cell layer even at the early sparse stage of culture. In cells grown on plastic with growth factor in the media, collagen is initially secreted into the media and does not become incorporated into the matrix. The deposition of collagen on the basal surface of cell occurs only late in the culture, and is achieved by increments in a stepwise manner. The in vivo-like pattern is not manifest until confluence has been reached. Thus, the extracellular matrix functions not only as a structural support, but is also instructional to the cells plated on it. In this case, the matrix regulates the level of collagen synthesis in the cells and modulates the pattern of collagen deposition. Soluble growth factors may act in part by enhancing a cell's ability to elaborate an appropriate matrix pattern necessary for the cell's own growth and accurate function.
Cell configuration-related control of vimentin biosynthesis and phosphorylation in cultured mammalian cells.
Authors: Ben-Ze'ev A
Published: May 2004

The cell configuration-related control of a cytoskeletal protein (vimentin) expression was examined by varying cell shape between flat and spherical. Cultivation of cells in monolayer or in a spherical configuration on poly-2-hydroxyethylmethacrylate-coated plates revealed a preferential down regulation of vimentin synthesis during suspension culture. The mechanism(s) regulating the decrease in the expression of vimentin in spherical cells appears to be at the level of translation, because mRNAs extracted from monolayer and suspension-cultured cells were equally active in directing vimentin synthesis in the rabbit reticulocyte cell-free system. When after prolonged suspension culture, the cells were allowed to reattach and spread, vimentin synthesis recovered rapidly to the control monolayer rate. The phosphorylation of vimentin was also reduced dramatically during suspension culture. However, unlike the rapid recovery of vimentin biosynthesis upon reattachment (less than 6 h), the recovery in the rate of vimentin phosphorylation was much slower (greater than 20 h) and paralleled the recovery to the monolayer growth rate. Although the control of vimentin biosynthesis in suspension culture is a cell configuration-related process, the decrease in the rate of vimentin phosphorylation in suspension culture appears to be the result of the slower growth rate and may reflect the reported correlation between the rate of vimentin phosphorylation and the accumulation of cells in mitosis.
Synthesis and regulation of acute phase plasma proteins in primary cultures of mouse hepatocytes.
Authors: Baumann H , Jahreis G P , Gaines K C
Published: May 2004

Adult mouse hepatocytes respond in vivo to experimentally induced acute inflammation by an increased synthesis and secretion of alpha 1-acid glycoprotein, haptoglobin, hemopexin, and serum amyloid A. Concurrently, the production of albumin and apolipoprotein A-1 is reduced. To define possible mediators of this response and to study their action in tissue culture, we established primary cultures of hepatocytes. Various hormones and factors that have been proposed to regulate the hepatic acute phase reaction were tested for their ability to modulate the expression of plasma proteins in these cells. Acute phase plasma and conditioned medium from activated monocytes influenced the production of most acute phase plasma proteins, and the regulation appears to occur at the level of functional mRNA. Purified hormones produced a significant anabolic response in only a few cases: dexamethasone was found to be effective in maintaining differentiated expression of the cells; and glucagon produced a specific inhibition of haptoglobin synthesis. When cells were treated with a combination of conditioned monocyte medium and dexamethasone, secretion of proteins was markedly reduced. The carbohydrate moieties of all plasma glycoproteins were incompletely modified, apparently as a result of decreased intracellular transport of newly synthesized plasma proteins. Although primary hepatocytes were not phenotypically stable in tissue culture, the cells nevertheless retained a broad response spectrum to exogenous signals. We propose this as a useful system to study the production of plasma proteins and thereby pinpoint the nature and activity of effectors mediating the hepatic acute phase reaction.
Outer doublet heterogeneity reveals structural polarity related to beat direction in Chlamydomonas flagella.
Authors: Hoops H J , Witman G B
Published: May 2004

Analysis of serial cross-sections of the Chlamydomonas flagellum reveals several structural asymmetries in the axoneme. One doublet lacks the outer dynein arm, has a beak-like projection in its B-tubule, and bears a two-part bridge that extends from the A-tubule of this doublet to the B-tubule of the adjacent doublet. The two doublets directly opposite the doublet lacking the arm have beak-like projections in their B-tubules. These asymmetries always occur in the same doublets from section to section, indicating that certain doublets have consistent morphological specializations. These unique doublets give the axoneme an inherent structural polarity. All three specializations are present in the proximal portion of the axoneme; based on their frequency in random cross-sections of isolated axonemes, the two-part bridge and the beak-like projections are present in the proximal one quarter and one half of the axoneme, respectively, and the outer arm is absent from the one doublet greater than 90% of the axoneme's length. The outer arm-less doublet of each flagellum faces the other flagellum, indicating that each axoneme has the same rotational orientation relative to the direction of its effective stroke. This strongly suggests that the direction of the effective stroke is controlled by a structural component within the axoneme. The striated fibers are associated with specific triplets in a manner suggesting that they play a role in setting up or maintaining the 180 degrees rotational symmetry of the two flagella.
Cell contact-mediated regulation of tyrosine hydroxylase synthesis in cultured bovine adrenal chromaffin cells.
Authors: Acheson A L , Thoenen H
Published: May 2004

The specific activity of tyrosine hydroxylase (TH) in bovine adrenal chromaffin cells can be controlled by changing cell density. Chromaffin cells initially plated at low density (2-3 X 10(4) cells/cm2), and subsequently replated at a 10-fold higher density showed a sixfold increase in specific TH activity within 48 h, resulting from enhanced synthesis (increased number of TH molecules as demonstrated by immunotitration and blockade by cycloheximide) rather than activation. The density-mediated TH induction was blocked by inhibitors of both messenger RNA synthesis (alpha-amanitin) and processing (9-beta-arabinofuranosyladenine), indicating a transcriptional level of regulation. Medium conditioned by high density replated cells could not mimic the effect of high density plating itself, thus direct cell contact, rather than a diffusible factor, is responsible for the density-mediated TH induction. Since neither acetylcholinesterase nor lactate dehydrogenase specific activities were increased by high cell density, it can be concluded that the contact-mediated induction of TH is rather specific, and not the result of a general process of enzyme induction.
Acidification of endocytic vesicles by an ATP-dependent proton pump.
Authors: Yamashiro D J , Fluss S R , Maxfield F R
Published: May 2004

One of the early events in the pathway of receptor-mediated endocytosis is the acidification of the newly formed endocytic vesicle. To examine the mechanism of acidification, we used fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) as a probe for endocytic vesicle pH. Changes in pH were determined from the change in fluorescein fluorescence at 490-nm excitation as measured with a microscope spectrofluorometer. After endocytosis of F-alpha 2M, mouse fibroblast cells were permeabilized by brief exposure to the detergent digitonin. Treatment with the ionophore monensin or the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused a rapid increase in the pH of the endocytic vesicle. Upon removal of the ionophore, the endocytic vesicle rapidly acidified only when MgATP or MgGTP was added. Neither ADP nor the nonhydrolyzable analog, adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) could support acidification. The ATP-dependent acidification did not require a specific cation or anion in the external media. Acidification was insensitive to vanadate and amiloride but was inhibited by Zn2+ and the anion transport inhibitor diisothiocyanostilbene disulfonic acid (DIDS). We also examined the acidification of lysosomes with the permeabilized cell system, using fluorescein isothiocyanate dextran as probe. DIDS inhibited the ATP-dependent reacidification of lysosomes, although at a lower concentration than that for inhibition of endocytic vesicle reacidification. These results demonstrate that endocytic vesicles contain an ATP-dependent acidification mechanism that shares similar characteristics with the previously described lysosomal proton pump.
Identification of tubulin in Dictyostelium discoideum: characterization of some unique properties.
Authors: White E , Tolbert E M , Katz E R
Published: May 2004

We used three antitubulin antibodies to localize Dictyostelium tubulin subunits on two-dimensional polyacrylamide gels by Western blotting. All three antibodies, a polyclonal antibody against sea urchin alpha- and beta-tubulin and two monoclonal antibodies against yeast alpha-tubulin, recognize the same set of polypeptides with a molecular weight of 55,000 while focusing at a pH far more basic than all other tubulins. Each antibody specifically stains the microtubule system of slime mold amoebae by indirect immunofluorescence. The microtubule system can be isolated as a major component of the amoeba cytoskeleton, and these preparations are greatly enriched for the presumptive tubulin subunits. The microtubules of these cytoskeletons are resistant to being depolymerized by millimolar concentrations of calcium, while they retain their cold sensitivity. Comparison of peptide maps of slime mold and brain alpha-tubulins indicates that the proteins are related but not identical. Possible explanations for these unusual characteristics are discussed.