- Professor emeritus (biochemistry/cell biology)edit
The number of Krebs II tumour cells recovered from the ascitic fluid of mice fed for 8 days on a lactalbumin (La) control diet was about three times higher than that in animals fed a phytohaemagglutinin-containing (PHA) diet. Feeding a... more
The number of Krebs II tumour cells recovered from the ascitic fluid of mice fed for 8 days on a lactalbumin (La) control diet was about three times higher than that in animals fed a phytohaemagglutinin-containing (PHA) diet. Feeding a PHA diet for less than 8 days after tumour cell injection also led to a reduction in tumour cell growth. There was an apparent inverse relationship between the total tumour cell count and the intracellular content of putrescine, spermidine and spermine. Hyperplasia of the small intestine occurred in the mice during the development of the ascites. A series of other organs were not affected in the same manner. The results indicate that the polyamine content of Krebs II ascites cells must increase by more than threefold in order to achieve the intracellular concentration necessary to be able to enter the S-phase. A partial synchronization of the tumour cell population is suggested. Hyperplastic growth of the small intestine would appear to compete with tumour cells for polyamines from a common body pool.
Research Interests: Cancer and Plant Lectins
The exposure of L-cells to a period of 15 min incubation in ice followed by a return to normal culture conditions caused distinct alterations in the distribution pattern of 3H-choline incorporation in phospholipids in subcellular... more
The exposure of L-cells to a period of 15 min incubation in ice followed by a return to normal culture conditions caused distinct alterations in the distribution pattern of 3H-choline incorporation in phospholipids in subcellular fractions. The amount of radioactivity appearing in the nuclei and nuclear-associated endoplasmic reticulum decreased while that in the mitochondrial and endoplasmic reticulum membrane fractions increased, suggesting a precursor-product relationship. These changes appeared to occur in a linear fashion. Furthermore, the increase in radioactivity in individual endoplasmic reticulum subfractions differed in that label increased fivefold in light rough membranes but only about twofold in the HR and S subfractions.
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When MPC-11 cells are disrupted by nitrogen cavitation in the presence of buffer containing 25-40 mM KCl then endoplasmic reticulum membranes can be separated into three subfractions by sucrose density gradient centrifugation: heavy rough... more
When MPC-11 cells are disrupted by nitrogen cavitation in the presence of buffer containing 25-40 mM KCl then endoplasmic reticulum membranes can be separated into three subfractions by sucrose density gradient centrifugation: heavy rough (HR), light rough (LR) and smooth (S) membranes. An increase in the salt concentration of the buffer to 50 mM or above results in the occurrence of only the LR and S membranes in gradients. However, when cells equilibrated at high pressure in the bomb in 100 mM KCl buffer were expelled into a diluting buffer such that the final buffer concentration was reduced to 25 mM KCl upon cell disruption, then appreciable amounts of HR membranes are observed in sucrose gradients. The results would suggest that salt concentrations above 25-40 mM KCl stabilize the interaction between HR membranes and the cytoskeleton to such a degree that these membranes are pelleted at low speed together with the nuclei. The yields of LR and S membranes are apparently not affected to any significant degree by altered salt concentration.
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The incorporation of 3H-glucosamine, 3H-choline and 14C-fucose into subcellular fractions of MPC-11 cells was studied. After a 20 min period of labelling with both 3H-glucosamine and 3H-choline, greatest incorporation was observed in... more
The incorporation of 3H-glucosamine, 3H-choline and 14C-fucose into subcellular fractions of MPC-11 cells was studied. After a 20 min period of labelling with both 3H-glucosamine and 3H-choline, greatest incorporation was observed in nuclear-associated endoplasmic reticulum (NER). 14C-fucose, however, was incorporated to a greater extent in endoplasmic reticulum (ER) membranes. Pulse-chase experiments with 3H-glucosamine showed a loss of radioactivity from NER and a simultaneous increase in the ER fraction. In comparison to NER, ER membranes were poorly labeled with 3H-glucosamine after a 20 min pulse. Following a 2 h incubation there was a 12 fold increase in radioactivity in ER membranes in comparison to a 1.2 fold increase in NER. There were no individual differences between subfractions of ER membranes with respect to 3H-glucosamine content after the pulse, or following the 2 h incubation. The results indicate that the NER is a major, early site of the synthesis of 3H-glucosamine labeled membrane glycoproteins, and that these proteins migrate into other ER membranes early after their synthesis.
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Plant lectins have the common property of being capable of binding specifically and reversibly to carbohydrate residues. They are present in common plant food such as fruits, vegetables, legumes and nuts. Among important features of plant... more
Plant lectins have the common property of being capable of binding specifically and reversibly to carbohydrate residues. They are present in common plant food such as fruits, vegetables, legumes and nuts. Among important features of plant lectins is their ability to survive digestion in the gastrointestinal tract, and some also show resistance to denaturation by food processing. Plant lectins have physiological effects, such as binding to glycoproteins on the epithelial surface of the small intestine, where they may elicit local and/or systemic reactions, e.g. modulation of the immune system and the micro flora in the gut. Although much progress has been made in the study of lectins, particularly in relation to their molecular structure, questions regarding their true properties and functions in the human diet remain virtually unanswered. We seek here to summarize the potential beneficial effects of plant lectins on human health.
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When microsomes, isolated from MPC-11 cells after nitrogen cavitation of cells in buffer containing 100 mM KCl, were separated into light rough (LR) and smooth (S) fractions by discontinuous gradient centrifugation it was observed that... more
When microsomes, isolated from MPC-11 cells after nitrogen cavitation of cells in buffer containing 100 mM KCl, were separated into light rough (LR) and smooth (S) fractions by discontinuous gradient centrifugation it was observed that [3H]-choline label and A260 nm absorption did not coincide in the LR region of the gradient. This was in contrast to the situation when microsomes were isolated from cells disrupted by nitrogen cavitation at 25 mM KCl. The A260 nm absorbing material that appeared in gradient fractions (1-5) below the position of LR membranes was found to consist of polysomal material. This material gave a "richer" polysome profile than that released from the LR membranes by addition of detergent. Labeling experiments with [3H]-leucine showed that nascent polypeptides associated with monosomes and polysomes in fractions 1-5 were of shorter length than the corresponding ones in the LR fraction. A mere contamination of LR microsomes by free polysomes appeared most unlikely. The results are consistent with an effect of "shearing" on the membrane-bound polysomes of the LR microsomes under specific experimental conditions. This effect results in the production of a 5' mRNA fragment (short polypeptide chains) and a 3' mRNA fragment (long polypeptide chains), the former fragment migrating further down the gradient tube free of LR membranes, whilst the latter remained attached to the LR membranes.
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The incorporation of [3H]-glucosamine into polypeptides of three fractions of polysomes in MPC-11 cells was studied. After short term incubation greatest incorporation was observed in a fraction of membrane-bound polysomes, which after... more
The incorporation of [3H]-glucosamine into polypeptides of three fractions of polysomes in MPC-11 cells was studied. After short term incubation greatest incorporation was observed in a fraction of membrane-bound polysomes, which after nitrogen cavitation of cells, remained bound to the endoplasmic reticulum (ER) associated with the nucleus (fraction 2). Polypeptide chains on membrane-bound polysomes in the microsomal fraction (fraction 1) and free polysomes contained much less radioactivity. Since nascent polypeptide chains contained within membrane-bound polysomes of fraction 2 are glycosylated at an earlier stage than those in fraction 1 it is likely that this represents a difference in type of proteins synthesized in the respective fractions of ER.
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The signal peptide of the luciferase secreted by the marine copepod Gaussia princeps has been shown to promote high-level protein synthesis/secretion of recombinant proteins, being far superior to mammalian counterparts. The main aim of... more
The signal peptide of the luciferase secreted by the marine copepod Gaussia princeps has been shown to promote high-level protein synthesis/secretion of recombinant proteins, being far superior to mammalian counterparts. The main aim of the present study was to investigate the effects of seven selected signal peptides derived from oikosins, house proteins of the marine organism Oikopleura dioica, on synthesis/secretion of recombinant proteins. Vector constructs were made in which the coding regions of two naturally secreted proteins, Gaussia luciferase and human endostatin (hEndostatin), were “seamlessly” fused to the signal peptide coding sequences of interest. CHO cells were transfected with the plasmids and populations of stably transfected cells established. The amounts of reporter proteins in cell extract and medium samples were determined and the results compared to those obtained from cells stably transfected with a reference vector construct. In addition, the amounts of luciferase or hEndostatin encoding mRNAs in the cells were determined and related to the protein levels obtained. The levels of reporter protein produced varied greatly among the seven oikosin signal peptides tested. Whereas the oikosin 1 signal peptide resulted in about 40% production of Gaussia luciferase compared to the reference construct, oikosins 2–7 were extremely ineffective (<1%). mRNA levels were not dramatically affected such that inadequate availability of transcript for translation was not the underlying reason for the observations. The oikosin 1 signal peptide was also the most effective regarding synthesis/secretion of hEndostatin. No secreted product was observed using the oikosin 3 signal peptide. Interestingly, the molecular weight of hEndostatin in cell extracts prepared from cells transfected with oikosin 2 and 3 constructs was higher than that using the oikosin 1 signal peptide. The overall findings indicate that the signal peptide affects the efficiency of protein synthesis and secretion through a mechanism operating at the post-transcriptional level. The results described here provide substantial support to our previous observations which suggested that the choice of the signal peptide is imperative when aiming to achieve optimal synthesis and secretion of a recombinant protein using transfected mammalian cells.
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Cultures of mouse plasmacytoma cells (MPC-11) grown within the range 6-23 x 10(5) cells/ml showed considerable variation in cell cycle distribution profiles and also differences with regard to relative amounts of microsomal subfractions.... more
Cultures of mouse plasmacytoma cells (MPC-11) grown within the range 6-23 x 10(5) cells/ml showed considerable variation in cell cycle distribution profiles and also differences with regard to relative amounts of microsomal subfractions. The variability of appearance of heavy rough (HR) and light rough (LR) microsomal subfractions was not merely due to differences in nutritional state of the culture. Cultures containing a high S/G2 + M cell cycld distribution ratio showed a high content of HR microsomal membranes; as the S/G2 + M ratio decreased, so too decreased the amount of HR material whilst the amount of LR microsomal membranes increased. The results indicate that there is a direct correlation between phase of cell cycle and both amount and relative distribution of rough microsomal membranes, the smooth fraction (S), however, remains relatively unchanged.
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To compare the effects of chlorpromazine (CPZ), prochlorperazine (PCP), trifluoperazine (TFP), clozapine (CLO), haloperidol (HPD), quetiapine (QTP), pimozide (PMZ), and olanzapine (OLP) as well as the tricyclic antidepressants... more
To compare the effects of chlorpromazine (CPZ), prochlorperazine (PCP), trifluoperazine (TFP), clozapine (CLO), haloperidol (HPD), quetiapine (QTP), pimozide (PMZ), and olanzapine (OLP) as well as the tricyclic antidepressants amitriptyline AMI, imipramine IMI, and nortriptyline NTP on thrombin-induced liberation of arachidonic acid AA in platelets. This work was carried out at the Department of Biomedicine, University of Bergen, Norway in 2006-2007. Human platelets pre labelled with [3H] arachidonate were incubated with thrombin in the absence and presence of the drugs, and the amount of free [3H] arachidonate liberated was determined. Myosin light chain (MLC) phosphorylation was determined in [32P] phosphate-labelled platelets after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The effects of the drugs on the molecular area and surface pressure of phospholipid monolayers were determined in the Langmuir apparatus. All drugs reduced arachidonate liberation with the ranking order of increasing potency: OLP&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;QTP&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;HPD&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;AMI&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;IMI&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;PMZ&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;NTP&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;CPZ&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;TFP&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;PCP. Since phenothiazines are calmodulin antagonists, this property of the drugs was tested as reduction of thrombin-induced phosphorylation of MLC in [32P] Pi-labelled platelets. Only PCP, CPZ, TFP, and NTP reduced MLC phosphorylation. All 11 drugs studied markedly increased the mean molecular area of dipalmitoyl phosphatidylserine monolayers at 37 degrees. The mechanisms for reduction of arachidonate liberation is thought to interfere with activation of cytosolic phospholipase A2 (cPLA2) by alteration of the PLA2 phospholipid substrate structure in platelet membranes.
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NMRI mice injected subcutaneously with Krebs II lymphosarcoma cells and fed on a diet containing the kidney bean lectin phytohemagglutinin (PHA) within the range 0.45-7.0 mg/g diet, developed tumors during a 10 day period which on average... more
NMRI mice injected subcutaneously with Krebs II lymphosarcoma cells and fed on a diet containing the kidney bean lectin phytohemagglutinin (PHA) within the range 0.45-7.0 mg/g diet, developed tumors during a 10 day period which on average were only 35% of the dry weight of tumors in lactalbumin (La) fed mice (control). The reduction in growth occurred in a dose-dependent manner in the range 0.45-3.5 mg/g diet. The degree of hyperplasia of the small intestine in response to feeding the PHA diets was higher in non-injected compared to injected mice. A lipolytic effect of PHA was observed above 1.75 mg/g diet in control mice and the highest concentration had a major effect on body weight. Since the index of hyperplasia at the lowest PHA concentration tested did not correlate with the reduction in tumor size, it is suggested that other factors in addition to the initial lectin-induced gut hyperplasia are involved in slowing down the progression of tumor growth.
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In biological systems, there is an equilibrium state between pro-oxidant processes and antioxidant defence systems. If this state is disturbed in favour of pro-oxidant processes, oxidative stress will occur, the consequence of which is... more
In biological systems, there is an equilibrium state between pro-oxidant processes and antioxidant defence systems. If this state is disturbed in favour of pro-oxidant processes, oxidative stress will occur, the consequence of which is increased production of free radicals. Oxidative stress, no matter what its cause, can ultimately result in cell toxicity. Free radicals attack cellular components indiscriminately including lipids, carbohydrates and macromolecules such as proteins and DNA and thus affect their functionality. Lipids, by virtue of their location in cell membranes, are particularly vulnerable to free radical attack and lipid peroxides are produced as a consequence. Membrane stabilization (integrity) is a mandatory issue for survival of any biological system. Vitamin E, the lipid soluble essential micronutrient, embedded within membranes, has a high degree of scavenging activity. It is a highly efficient lipid-soluble chain-breaking antioxidant that has the function of m...
A variety of studies have shown that incubation of different tumour cell lines with mistletoe lectins (MLs) in vitro has a marked cytotoxic effect. In the concentration range of low cytotoxicity cell death induced by ML-I is... more
A variety of studies have shown that incubation of different tumour cell lines with mistletoe lectins (MLs) in vitro has a marked cytotoxic effect. In the concentration range of low cytotoxicity cell death induced by ML-I is quantitatively due to apoptotic processes. The first events observed being membrane perforation and protusions. Simultaneous treatment of certain tumour cells with MLs rendered them more sensitive to induction of apoptosis by TNFalpha. The immunomodulatory activity of ML-I was investigated by measuring cytokine release and the results confirmed that cytokine induction by the lectin is regulated at the transcriptional level. ML-I has been shown to potentiate the effect of chemotherapeutic drugs. In addition to an in vitro effect a number of workers have demonstrated that MLs suppress tumour growth in vivo. Mistletoe lectins have been administered to animals locally to the tumour, systemic, subcutaneously or by the oral route via the diet. In many cases apoptosis ...
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... J. 310, 193-196 4. Vedeler, A,, Pryme, I., & Hesketh, J. (1991) Mol. Cell Biochem. 100, 183-193 5. Johannessen, A,, Pryme, I. & Vedeler, A. (1995) Mol. Cell Biochem. ... Bird, R. & Sells, B. (1986) Biochim. Biophys. Acta... more
... J. 310, 193-196 4. Vedeler, A,, Pryme, I., & Hesketh, J. (1991) Mol. Cell Biochem. 100, 183-193 5. Johannessen, A,, Pryme, I. & Vedeler, A. (1995) Mol. Cell Biochem. ... Bird, R. & Sells, B. (1986) Biochim. Biophys. Acta 868, 215-225 11. ...
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The present study concerns the importance of the timing of feeding mice a PHA-containing diet (7 mg g-1 diet) on tumor formation. The major decrease in tumor weight occurred in mice fed on the PHA diet for 11 days. A marked reduction was... more
The present study concerns the importance of the timing of feeding mice a PHA-containing diet (7 mg g-1 diet) on tumor formation. The major decrease in tumor weight occurred in mice fed on the PHA diet for 11 days. A marked reduction was also observed in animals pre-fed for 3 days with PHA before tumor cells were injected and the diet then changed to lactalbumin, La. A large decrease in tumor weight was also evident when a change of diet from La to PHA was made on the day of tumor cell inoculation. Despite the presence of the developing tumor PHA was able to induce hyperplasia of the small intestine in all groups of animals fed PHA during a part or the whole of the experiment. The dry weights of tumors attained in each of the experimental groups plotted as a function of duration of PHA feeding, and the percentage lipid content of the tumors, mirrored almost exactly one another, suggesting that the availability of essential lipid material is severely reduced by the lectin. This would...
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ABSTRACT Normal cell growth and activity require not only the correct proteins synthesized in the correct amounts, but also that they are delivered to their site of function. Thus, protein targeting is crucial for cell organization.... more
ABSTRACT Normal cell growth and activity require not only the correct proteins synthesized in the correct amounts, but also that they are delivered to their site of function. Thus, protein targeting is crucial for cell organization. Interactions between the cytoskeleton and protein synthetic apparatus have the potential to provide a mechanism for the transport and compartmentalization of mRNAs and polysomes. In addition to being associated with endoplasmic reticulum (ER) membranes, ribosomes were also detected in close proximity to fine filamentous elements now recognized as components of the cytoskeleton. Aggregates of ribosomes, judged to be polysomal structures, were also detected in pockets at junctions where filaments were arranged in a cross-like network. These electron microscopical observations provided the first evidence for the existence of a class of polysomes found in association with cytoskeletal filaments. The presence of remnants of ER has been demonstrated in the cytomatrix indicates that this non-ionic detergent residue would also contain ribosomes associated with the rough ER (RER). Studies on mRNA-cytoskeletal interaction were made possible by the introduction of the combination of electron microscopy with in situ hybridization. This allowed the simultaneous study of individual components of the cytoskeletal filament system and the location of specific mRNA species.
The growth of a murine non-Hodgkin lymphoma (NHL) tumour, either as an intraperitoneal ascites tumour or as a solid subcutaneous tumour, has been shown to be greatly reduced by including phytohaemagglutinin (PHA), a lectin present in raw... more
The growth of a murine non-Hodgkin lymphoma (NHL) tumour, either as an intraperitoneal ascites tumour or as a solid subcutaneous tumour, has been shown to be greatly reduced by including phytohaemagglutinin (PHA), a lectin present in raw kidney bean (Phaseolus vulgaris) in the diet. The reduced rate of growth occurred in a dose-dependent manner. Based on the experimental observations it has been suggested that a competition occurs between the gut tissue undergoing hyperplasia and the developing tumour for nutrients (including polyamines) from a common body pool. This may be an important factor with regard to the observed initial low level of tumour growth following the feeding of a PHA-containing diet. Results showing that the level of hyperplasia of the small intestine in response to feeding the PHA diets was higher in non-injected mice compared to those which had been injected with tumour cells substantiated the concept of competition between gut and tumour for nutrients etc. required for growth. The observations suggest that lectins, which exhibit growth-promoting effects on the gut, may have interesting applications in the formulation of new approaches with respect to cancer treatment.
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Neoplastic proliferation requires the availability of polyamines. Phytohaemagglutinin (PHA) is a potent growth factor which induces polyamine-dependent growth of the gut. In the hope of directing polyamines and other nutrients away from... more
Neoplastic proliferation requires the availability of polyamines. Phytohaemagglutinin (PHA) is a potent growth factor which induces polyamine-dependent growth of the gut. In the hope of directing polyamines and other nutrients away from the tumour to the growing gut, mice fed PHA-containing or lactalbumin diets were injected intraperitoneally with Krebs II ascites cells and the number of tumour cells and the weights of internal organs were followed. PHA-treatment significantly slowed down the proliferation of tumour cells. Changes in the weight and polyamine content of tissues indicated that inter-organ competition between the tumour and vital organs can be used to manipulate the metabolism of tumour-bearing mice.
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This synopsis reviews published in vivo studies on possible health consequences of genetically modified food and feed where the ingredients in question have consisted of genetically modified plant materials. The following, however, have... more
This synopsis reviews published in vivo studies on possible health consequences of genetically modified food and feed where the ingredients in question have consisted of genetically modified plant materials. The following, however, have not been taken into consideration:--ingredients consisting of genetically modified microorganisms or parts of animals/fish--ingredients produced by/from genetically modified organisms but without any DNA present--studies on consequences for the environment or biodiversity--in vitro studies or computer simulations. According to a Norwegian report &amp;amp;amp;amp;quot;Gen-mat&amp;amp;amp;amp;quot; (NOU 2000:29), and a more recent search in Medline and Citations Index, to our knowledge a total of ten studies have been published on the health effects of GM-foods and feeds. In this minireview the data made available in these published studies is discussed.
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Cytosolic, cytoskeleton and membrane fractions were extracted from HepG2 cells by a sequential detergent/salt extraction procedure. The cytosolic fraction contained 93% of the lactic dehydrogenase activity while the cytoskeleton fraction... more
Cytosolic, cytoskeleton and membrane fractions were extracted from HepG2 cells by a sequential detergent/salt extraction procedure. The cytosolic fraction contained 93% of the lactic dehydrogenase activity while the cytoskeleton fraction was enriched in actin and vimentin. The distribution of mRNAs for c-myc, glucose transporter 1, ribosomal proteins L4 and S6 and cyclin A were investigated by Northern hybridization of total RNA extracted from polysomes isolated from cytosolic, cytoskeleton and membrane fractions. The membrane-bound polysomes were enriched in the glucose transporter 1 mRNA and the cytoskeleton-bound polysomes were enriched in the mRNAs for the two ribosomal proteins, c-myc and cyclin A. The results suggest that the mRNAs for nuclear proteins are one class of mRNAs which are translated on polysomes associated with the cytoskeleton; this may be related to the requirement to transport the newly synthesized protein to the nucleus.
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Insulin induced Krebs II ascites cells to attach to the substratum and to adopt a flattened morphology associated with normal adhesion and movement. The changes were associated with a reorganization of cellular actin. The results show... more
Insulin induced Krebs II ascites cells to attach to the substratum and to adopt a flattened morphology associated with normal adhesion and movement. The changes were associated with a reorganization of cellular actin. The results show that insulin has important effects on cell structure and morphology. Insulin may thus be involved in the nutritional control of normal and malignant growth.
Research Interests: Cell Adhesion, Adhesion, Cytoskeleton, Cell Culture, Insulin, and 3 moreMice, Animals, and Cell Motility
From 30 min to 1h of step-up conditions there was a redistribution of polysomes between free, cytoskeletal-bound and membrane-bound fractions such that more polysomes were recovered bound to the cytoskeleton and less in the free fraction.... more
From 30 min to 1h of step-up conditions there was a redistribution of polysomes between free, cytoskeletal-bound and membrane-bound fractions such that more polysomes were recovered bound to the cytoskeleton and less in the free fraction. After 1h incubation with insulin there was a higher proportion of polysomes in the cytoskeletal fraction with a decrease occurring in the membrane-bound fraction. At 2h little change was observed in the presence of insulin while a large increase occurred in the cytoskeletal-bound fraction and a decrease in membrane-bound polysomes was seen in cells incubated in the absence of insulin. The results indicate that the proportions of polysomes in the three different fractions can be modulated by physiological stimuli, such as media replenishment and insulin.
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Research Interests: Biochemistry, Radiation, RNA, Metabolism, Cell line, and 16 moreTissue culture, Carbon Isotopes, Mice, Animals, Multiple Myeloma, Cell Fractionation, Nitrogen, Cell nucleus, Detergents, Culture Media, Cell free System, Plasmacytoma, Biochemistry and cell biology, Adenosine Triphosphate, Polyribosomes, and Cell Membrane
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ABSTRACT
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When microsomes, isolated from MPC-11 cells after nitrogen cavitation of cells in buffer containing 100 mM KCl, were separated into light rough (LR) and smooth (S) fractions by discontinuous gradient centrifugation it was observed that... more
When microsomes, isolated from MPC-11 cells after nitrogen cavitation of cells in buffer containing 100 mM KCl, were separated into light rough (LR) and smooth (S) fractions by discontinuous gradient centrifugation it was observed that [3H]-choline label and A260 nm absorption did not coincide in the LR region of the gradient. This was in contrast to the situation when microsomes were isolated from cells disrupted by nitrogen cavitation at 25 mM KCl. The A260 nm absorbing material that appeared in gradient fractions (1-5) below the position of LR membranes was found to consist of polysomal material. This material gave a &quot;richer&quot; polysome profile than that released from the LR membranes by addition of detergent. Labeling experiments with [3H]-leucine showed that nascent polypeptides associated with monosomes and polysomes in fractions 1-5 were of shorter length than the corresponding ones in the LR fraction. A mere contamination of LR microsomes by free polysomes appeared most unlikely. The results are consistent with an effect of &quot;shearing&quot; on the membrane-bound polysomes of the LR microsomes under specific experimental conditions. This effect results in the production of a 5&#39; mRNA fragment (short polypeptide chains) and a 3&#39; mRNA fragment (long polypeptide chains), the former fragment migrating further down the gradient tube free of LR membranes, whilst the latter remained attached to the LR membranes.
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The incorporation of [3H]-glucosamine into polypeptides of three fractions of polysomes in MPC-11 cells was studied. After short term incubation greatest incorporation was observed in a fraction of membrane-bound polysomes, which after... more
The incorporation of [3H]-glucosamine into polypeptides of three fractions of polysomes in MPC-11 cells was studied. After short term incubation greatest incorporation was observed in a fraction of membrane-bound polysomes, which after nitrogen cavitation of cells, remained bound to the endoplasmic reticulum (ER) associated with the nucleus (fraction 2). Polypeptide chains on membrane-bound polysomes in the microsomal fraction (fraction 1) and free polysomes contained much less radioactivity. Since nascent polypeptide chains contained within membrane-bound polysomes of fraction 2 are glycosylated at an earlier stage than those in fraction 1 it is likely that this represents a difference in type of proteins synthesized in the respective fractions of ER.
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After transfer of Krebs II ascites cells from the mouse peritoneum to suspension culture addition of the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) causes an early stimulation of 3H-choline incorporation into... more
After transfer of Krebs II ascites cells from the mouse peritoneum to suspension culture addition of the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) causes an early stimulation of 3H-choline incorporation into phosphatidylcholine (PC). Choline transport into the treated cells, however, was unaffected. Within 30 min of TPA treatment 3H-choline incorporation was almost 300% above the control level. During a 5 hr period of suspension culture the overall patterns of 3H-choline incorporation were similar in TPA-treated and control cultures though the rate was greatly accentuated by the presence of the phorbol ester. Incubation of cells with cycloheximide prior to incubation with TPA did not result in an inhibition of the TPA-directed 3H-choline incorporation. After 3 hr incubation with TPA there were large increases in radioactivity in all subcellular fractions. At 20 hr, however, the values were not far from those of the control. During the first 3 hr of incubation with TPA the incorporation of 3H-choline into light rough (LR) and smooth (S) membranes was stimulated to levels of 400% and 320% respectively above control values. At later times the profiles of radioactivity in membrane subfractions in TPA-treated and control cultures were similar. The results illustrate an early effect of TPA on PC biosynthesis in Krebs II ascites cells while at later times of incubation the stimulatory effect was virtually abolished.