A modified partition affinity ligand assay (PALA) for direct reading of gamma-labels without further separation or washing.
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Antigen-Antibody Complex↗
Biomedical subjects
J Nilsson
Publications and source records attributed to J Nilsson.
Diagnosis of deep venous thrombosis by 99mTc-human serum albumin microcolloid.
99mTc-human serum albumin microcolloid (HSAC) was evaluated for diagnosis of deep venous thrombosis (DVT) in the legs of 38 consecutive patients. Five and thirty minutes after IV injection, the 99mTc-HSAC activity in both legs was measured by external counting using a collimated NaI (T1)-detector. The relative predominance of 99mTc-HSAC activity in the diseased leg was calculated. Phlebography was performed as a control after the 99mTc-HSAC test. In our hands the non-invasive 99mTc-HSAC test appeared to be rapid, easy to perform and convenient to the patient. The test showed a high sensitivity (11/13) and a low specificity (10/20) compared with phlebography. No adverse reactions were found. The results seem promising and further studies are in progress to establish the value of the 99mTc-HSAC test in screening for DVT.
Adult↗
Application of optical diffractometry in studies of cell fine structure. Comparison of arterial smooth muscle cells in contractile and synthetic state.
Arterial smooth muscle cells in contractile and synthetic state were analyzed by optical diffractometry. Cell sections (80-90 nm) were photographed in an electron microscope and diffraction patterns of the plates (negatives) were produced using a helium-neon laser. Radial and angular distributions of light intensity in the diffractograms were measured and digitized using an electronic detector plate consisting of ring- and wedge-shaped photosensitive elements; radial distributions provide information about size of structures and distances between them and angular distributions about spatial orientation of structures in the images. Micrographs of nuclei and cytoplasm were analyzed separately (40-50 plates in each group). Computerized statistical analysis of radial distributions of light intensity showed that the nuclear chromatin pattern differed between cells in contractile and synthetic state. The probability that the observed difference could have arisen purely by chance was less than 10(-5). Computer-aided classification to the a priori known cell group was correct in 96.5% of the cases. Analysis of radial distributions of light intensity similarly showed marked differences in cytoplasmic structure between cells in contractile state (dominated by bundles of myofilaments) and synthetic state (dominated by cisternae of rough endoplasmic reticulum). The probability that the observed difference could have arisen purely by chance was less than 10(-5). Computer-aided classification to the a priori known cell group was correct in 92.0% of the cases. In contrast, analysis of angular distributions of light intensity did not indicate any statistically significant differences between contractile and synthetic state cells. A likely reason is that both myofilaments and cisternae of rough endoplasmic reticulum were arranged in parallel. The results demonstrate that optical diffractometry is a useful method for image analysis in studies of cell fine structure. It provides information about size and orientation of structures with poorly defined shape and is particularly well suited for studies on cell differentiation and effects of pharmacological and other experimental treatments on cell fine structure. It represents an alternative and a complement to stereology for quantitative and objective evaluation of morphological data.
Animals↗
Radioimmunoassay of triiodothyronine (T3) and thyroxine (T4). An assay with both the bound and free fraction of the hormone present in the counting vessel.
A radioimmunoassay for triiodothyronine T3 and thyroxine T4 without any manual separation is described. By the use of an aqueous two-phase system the free hormone was separated from the immunologically bound fraction and lifted out of the counting area of the gamma counter. The coefficient of correlation between results obtained with this method and with a conventional RIA was 0.97 and 0.93 for T3 and T4, respectively.
Humans↗
Cell position regulates endodermal differentiation in embryonal carcinoma cell aggregates.
It has been suggested that cell position regulates endodermal differentiation in mouse embryo inner cell masses and in aggregates of embryonal carcinoma (EC) cells. This hypothesis states that cells at the interface between the cell mass and blastocoel fluid or culture medium differentiate into endoderm, whereas internally located cells follow alternative developmental pathways. To test the cell position hypothesis, pluripotent PSA-1 cells were aggregated with hypoxanthine phosphoribosyltransferase-deficient, parietal-like, endodermal cells. The resulting aggregates consisted of cores of PSA-1 cells surrounded by endodermal cells. Autoradiography was used to distinguish between endodermal cells that were the products of EC cell differentiation and the exogenous endoderm. Alkaline phosphatase staining was used to distinguish EC cells from endodermal cells. As predicted by the cell position hypothesis, the PSA-1 EC cells, all of which were internally located, did not differentiate into endodermal cells. Nonspecific inhibition of differentiation did not account for the lack of PSA-1-derived endoderm since the PSA-1 cells in such aggregates did differentiate into columnar ectodermal-like cells. Similar experiments were also conducted with F9 cells. In this case, aggregation cultures contained retinoic acid to induce F9 cells to differentiate into visceral endoderm. In cultures containing F9 cells surrounded by parietal-like endodermal cells, no F9-derived endoderm was detected either autoradiographically or by assaying for alpha-fetoprotein production, a visceral endoderm marker. Thus, retinoic acid-induced endodermal differentiation was also regulated by cell position. Collectively, the above results provide strong evidence for the hypothesis that cell position regulates endodermal differentiation in aggregates of EC cells.
Alkaline Phosphatase↗
Demonstration of stimulatory effects of platelet-derived growth factor on cultivated rat arterial smooth muscle cells. Differences between cells from young and adult animals.
The effects of platelet-derived growth factor (PDGF) on DNA synthesis and proliferation in cultures of arterial smooth muscle cells obtained from young and adult rats, respectively, were measured. Addition of 10-20 ng/ml of PDGF to medium MCDB 104 induced DNA synthesis in quiescent cultures of cells from young animals to a similar extent as 10-20% whole blood serum (WBS). PDGF further stimulated proliferation of the cells in medium MCDB 104, although less markedly than 10% WBS. Antibodies against PDGF partially inhibited the growth response after stimulation with serum. This shows that PDGF is a major growth factor in serum for these cells and that PDGF can promote entrance into and passage through S phase and mitosis independent o plasma factors. Cells from adult animals were also found to respond to PDGF, although a higher concentration (25 ng/ml) was required to obtain a maximum effect. These cells, however, responded better than cells from young animals to stimulation with serum. Further, antibodies against PDGF did not inhibit the growth-stimulatory effect of serum to any appreciable extent. Thus, serum contains growth factors other than PDGF that stimulate preferentiaLly the proliferation of smooth muscle cells from adult animals.
Aging↗
Endocytosis of cationic and anionic proteins in cultivated arterial smooth muscle cells.
Confluent secondary cultures of rat arterial smooth muscle cells were exposed to cationic and anionic derivatives of ferritin and horseradish peroxidase and studied electron microscopically in order to clarify the influence of molecular net charge on surface binding and endocytosis of proteins. The cationic markers bound uniformly to the plasma membrane. They were then ingested by membrane invagination and via small vesicles transported to lysosomes and the Golgi complex. These organelles were both labelled already after 30 min of incubation. With longer exposure times (2-4 h), an increasing accumulation within the lysosomes was observed, whereas the labelling of the Golgi complex decreased. In spite of continued interiorization of plasma membrane carrying the cationic markers, the cells retained their ability to bind the latter to the surface. The anionic markers did not bind to the cell surface, were taken up in the fluid phase, and later observed only in lysosomes. If assuming that the cationic and anionic proteins serve as markers for the plasma membrane and fluid phase, respectively, but do not affect the intracellular path of interiorized membrane, these results indicate that the endocytic vesicles fuse with and empty their content into lysosomes and that part of the incoming membrane subsequently is transferred to the Golgi complex for possible recirculation back to the cell surface. If, on the other hand, the net charge of the exogenous marker influences the path of the vesicles, there may exist more than one recovery route for membrane interiorized by endocytosis.
Animals↗
Effects of colchicine on DNA synthesis, endocytosis and fine structure of cultivated arterial smooth muscle cells.
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Animals↗
The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli.
The stability of different derivatives of plasmid vectors pBR322 and pACYC184 carrying the tryptophan operon of Escherichia coli was monitored in various media. It was found that in the absence of any special selective pressure, all plasmids were lost from the culture. The stability varied depending both on the orientation of the inserted tryptophan fragment and the growth media used. The pBR322::trp+ plasmids were lost at an average frequency of 0.3 to 0.8% per cell generation, while the pACYC184::trp+ plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost, indicating a high stability of the plasmid::cloned DNA as such. To increase the stability of the cloning vectors, the partition locus of plasmid pSC101 was added to both the pBR322::trp+ and pACYC184::trp+ plasmids. The addition of this gene increased the replicon stability at least 3- to 10-fold, with the pBR322::trp+-par+ plasmids being the most stable. Also in this case, the stability was dependent on the plasmid type and on the growth medium. In no case was there a discoordinate loss of the antibiotic-resistance and tryptophan genes from the vectors.
Cloning, Molecular↗
Surface binding and internalization of platelet-derived growth factor in human fibroblasts.
Surface binding and uptake of platelet-derived growth factor (PDGF) in human fibroblasts cultivated in vitro were studied by quantitative electron microscopic autoradiography using 125I-labeled PDGF and by indirect immunofluorescence using PDGF antibodies. After 120 min at 12 degrees C PDGF was found preferentially in coated regions of the plasma membrane. Warming the cells to 37 degrees C initiated rapid ingestion of the factor via small vesicles, usually lacking a membrane coat. After 10 min PDGF started to appear in lysosomes, and it showed maximal concentration within these organelles after 30 min. There were also signs of passage of PDGF through the Golgi complex, but only after 60 min. Treatment of the cells with chloroquine, a weak base that inhibits intralysosomal degradation, prevented disappearance of tracer from the lysosomes. The observations indicate that PDGF was internalized via coated regions of the plasma membrane and carried to lysosomes for degradation. Subsequent appearance of tracer within the Golgi complex could reflect receptor-ligand complexes that escaped degradation and were recirculated back to the cell surface.
Autoradiography↗
Phenotype modulation in primary cultures of arterial smooth muscle cells. On the role of platelet-derived growth factor.
Smooth muscle cells were isolated from adult rat aorta by collagenase digestion, grown in primary culture in the presence of 10% whole blood serum (WBS), and studied by quantitative electron microscopy and thymidine autoradiography in order to correlate cellular fine structure and proliferation. On day 2-4, the cells passed through a structural transition from contractile to synthetic state. In the former they were characterized by predominance of cytoplasmic microfilament bundles and in the latter by an extensive rough endoplasmic reticulum (RER) and a large Golgi complex. The disappearance of the microfilament bundles was accompanied by a transient increase in lysosomal volume density but no signs of bulk autophagy. This suggests that microfilaments were disassembled into subunit proteins and that lysosomes were engaged in adjusting the pool of free subunits into a new equilibrium. RER cisternae grew out from the nuclear envelope and successively spread throughout the cytoplasm. Stacks of Golgi cisternae were organized in a circumscribed juxtanuclear region. The structural modulation occurred also in medium containing 10% plasma-derived serum (PDS). Its onset was delayed by addition of antibodies (50 micrograms/ml) against platelet-derived growth factor (PDGF) to 10% WBS-medium and speeded up by addition of purified PDGF (25 ng/ml) to 10% PDS-medium. Otherwise, the kinetics of the structural modulation was the same in all experimental groups. The observations could not be explained by overgrowth of contaminating fibroblasts since (1) successive steps in the process were clearly evident, (2) the cells surrounded themselves by an incomplete basement membrane, a characteristic feature of smooth muscle, and (3) mitomycin C blocked cell growth but not conversion from contractile to synthetic state. After 3-4 days of culture in 10% WBS-medium, active DNA synthesis and cellular proliferation were initiated as determined autoradiographically and by cell counting. Electron microscopic autoradiography showed that all cells were morphologically in the synthetic state at the time of entrance into S-phase. Initially, the cells grew at a lower rate in the presence of PDGF antibodies but after 5-6 days of culture attained a rate similar to that in the controls. No distinct proliferation was obtained in 10% PDS-medium unless purified PDGF (10 ng/ml) was added during the first days of culture. The results suggest that the structural modulation of the smooth muscle is an absolute but not sufficient prerequisite for cellular proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Effects of nicotine on the fine structure of cultivated mouse peritoneal macrophages.
The effects of nicotine on the fine structure of thioglycollate-elicited mouse peritoneal macrophages cultivated in vitro was studied in order to elucidate further the mechanism behind its inhibitory effect on endocytosis and intracellular degradation of exogenous protein. In the concentrations (1.0-1,000 nM) and times (2-48 h) used here, nicotine lacked cytotoxic effect and did not appreciably affect the occurrence and distribution of different organelles. The most prominent change caused by the drug was the formation of large vacuoles believed to represent swollen lysosomes. This finding supports the idea that nicotine, like several other amines and weak bases, accumulates in the lysosomes by proton trapping. This could in turn impair the digestive capacity of the lysosomes by raising pH above the optimum of the acid hydrolases. It could also affect endocytosis and recycling of plasma membrane components. Additionally, the nicotine treatment led to an increased number of autophagic vacuoles consisting of lysosomes enclosing other lysosomes. A 'lysosomophagic' process of this type has previously been observed in other situations with decreased endocytosis. It is suggested to be a subtype of autophagy that serves the purpose of regulating the amount of lysosomes in connection with changes in the functional activity of this organelle.
Animals↗
Cell surface components and growth regulation in cultivated arterial smooth muscle cells.
The surface of rat arterial smooth muscle cells was characterized with respect to some of its chemical and functional properties. The effects of selective enzymic degradations (hyaluronidase, chondroitinases, heparitinase or neuraminidase) on [35S]sulphate-prelabelled cells and on binding sites for cationized ferritin (CF) were examined to assess the presence and relative importance of individual species of macromolecules on the cell surface. The results indicate that about half of the strongly anionic sites on the cell surface (binding CF at pH 2.0) could be ascribed to sulphate groups of glycosaminoglycans and about half to carboxyl groups of sialic acid residues in glycoproteins and/or glycolipids. Weaker anionic sites (binding CF at pH 7.0) largely originated from carboxyl groups of glycosaminoglycans. Chondroitin sulphate and heparan sulphate were the main glycosaminoglycans. The surface of cells from young animals showed a higher glycosaminoglycan and a lower sialic acid content than that of cells from adult animals. Continuous treatment of the cultures with neuraminidase stimulated serum-induced initiation of DNA synthesis, while treatment with hyaluronidase or heparitinase inhibited it. Addition of hyaluronic acid, heparin or heparan sulphate to the culture medium inhibited initiation of DNA synthesis as well as cell proliferation. The effect was more marked in cultures of cells from young animals than from adults, although the latter cells were found to grow at a higher rate and to higher densities. These results suggest a role for cell-surface and pericellular glycoconjugates in growth regulation. A possible mechanism of action is that these molecules, due to their anionic charge or by steric exclusion, interfere with the binding of platelet-derived growth factor, a highly cationic polypeptide, to its cell-surface receptor.
Animals↗
Fine structure of arterial smooth muscle cells cultured in the presence of whole blood serum or plasma-derived serum.
Normal diploid cells require serum to proliferate in culture. Platelet-derived growth factor has been identified as the main serum component responsible for this effect. Here, smooth muscle cells were isolated enzymatically from the aorta of 5-day-old rats and cultured in the presence of 10% whole blood serum (WBS) or plasma-derived serum (PDS), i.e. with or without platelet factor, and studied by transmission electron microscopy. The cells proliferated actively in WBS-medium but remained quiescent in PDS-medium. Fine structurally, cells from WBS-cultures demonstrated numerous mitochondria, an extensive rough endoplasmic reticulum (RER), a large Golgi complex, a few lysosomes, and microfilaments arranged in parallel bundles. After transfer to PDS-medium, the RER- and Golgi cisternae were markedly dilated and the number of membrane-associated ribosomes decreased. Segregation of fragments of cytoplasm within autophagosomes was frequently observed and the number of lysosomes increased. Lipid droplets were more abundant and often gathered in the Golgi area. Moreover, the cells had become more irregular in shape and showed many bleb-like processes at their surface. Microfilament bundles had also become more prominent and crossed each other in different directions. These observations show that the removal of platelet factor from the medium clearly modifies the fine structure of cultured smooth muscle cells. The findings are in good agreement with the concept that platelet factor not only supports the proliferation of cultured cells but also stimulates their secretory activity.
Adult↗
Effects of neuraminidase on DNA synthesis, proliferation and endocytosis of cultivated arterial smooth muscle cells.
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Animals↗
Diagnosis of deep venous thrombosis by phlebography and 99Tcm-plasmin.
One hundred and thirty-four patients admitted to the medical emergency ward due to suspect deep venous thrombosis (DVT) were examined. The uptake of intravenously injected porcine 99Tcm-plasmin was estimated in both legs. Thereafter, phlebography was performed using a high osmolar contrast medium. All phlebographies were evaluated independently. All patients with negative phlebography were examined clinically after 3-5 days. The plasmin test and phlebography were repeated when called for. The sensitivity of the plasmin test was 100% and the specificity 51% when compared to phlebography. The extension of the DVT as demonstrated by the plasmin test was similar to that determined by phlebography. Post-phlebographic thrombosis was very rare. It is concluded that 99Tcm- plasmin test is a rapid method, convenient to the patient and well suitable as a screening test. The results indicate that a negative plasmin test excludes DVT while a positive test necessitates additional examination by phlebography.
False Negative Reactions↗
Effects of nicotine on endocytosis and intracellular degradation of horseradish peroxidase in cultivated mouse peritoneal macrophages.
Thioglycollate-elicited mouse peritoneal macrophages were cultivated in vitro in control medium or medium containing nicotine (1 nM-1 microM). The drug caused a moderate lysosomal vacuolation and formation of vesicles with a dense core or ring. Furthermore, it partially inhibited uptake and intracellular degradation of horseradish peroxidase without affecting the specific activities of the lysosomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase in lysates of the cells. A slight inhibitory effect on the phagocytosis of latex beads was also noted. Since nicotine was used in concentrations of the same magnitude that may be obtained in the blood after smoking one cigarette, it seems worthwhile to further explore these findings in relation to the medical risks of smoking.
Animals↗
Lumbar back muscle activity in relation to trunk movements during locomotion in man.
The function of lumbar back muscles was studied by relating their activity patterns to trunk movements in 7 healthy adult males during normal walking (1.0-2.5 m/s) and running (2.0-7.0 m/s) on a treadmill. The movements of the trunk in the sagittal and frontal planes were recorded with a Selspot optoelectronic system using infrared light emitting diodes as markers. The electromyographic (EMG) activity from the two main portions of the lumbar erector spinae muscles (Multifidus and Longissimus) was recorded bilaterally with intramuscular wire electrodes. The angular displacements of the trunk showed regular oscillations, but their shape, magnitude and relation to the step cycle were different in the two planes (sagittal and frontal) and varied with speed and mode of progression. The EMG pattern in both muscles showed a bilateral cocontraction with two main bursts of activity per step cycle starting just before each foot was placed on the ground. Relating the EMG to the movements of the trunk indicated that the main function of the lumbar erector spinae muscles is to restrict excessive trunk movements. During walking this restricting action is most evident for movements in the frontal plane, whereas in running the lumbar back muscles mainly control the movements in the sagittal plane.
Adaptation, Physiological↗