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Papers by L. Jefferson

Biochimica Et Biophysica Acta-gene Structure and Expression, 1994

The American journal of physiology, 1984

The goal of these studies was to evaluate acute changes in protein metabolism in skeletal muscle ... more The goal of these studies was to evaluate acute changes in protein metabolism in skeletal muscle in response to contractile activity. Rates of protein synthesis were measured by following L-[U-14C]phenylalanine incorporation into protein in muscles of the perfused rat hindlimb at rest, during 10 min of maximal isometric muscle contractions, and during 10 min of recovery. Synthesis measurements were carried out under conditions that ensured that the specific radioactivity of the tRNA-bound precursor amino acid was equal to that of extracellular phenylalanine. Protein degradation was estimated by measuring the release of Nt-methylhistidine. Rates of synthesis were markedly inhibited in response to muscle contractions in tibialis anterior, gastrocnemius, and plantaris but were unaffected in soleus. Rates of synthesis returned toward those observed in the resting condition during the recovery period. Rates of degradation were also markedly inhibited in response to muscle contractions. D...

Oncogene, 2000

We have previously demonstrated that the protein encoded by the retinoblastoma susceptibility gen... more We have previously demonstrated that the protein encoded by the retinoblastoma susceptibility gene (Rb) functions as a regulator of transcription by RNA polymerase I (rDNA transcription) by inhibiting UBFmediated transcription. In the present study, we have examined the mechanism by which Rb represses UBFdependent rDNA transcription and determined if other Rb-like proteins have similar eects. We demonstrate that authentic or recombinant UBF and Rb interact directly and this requires a functional A/B pocket. DNase footprinting and band-shift assays demonstrated that the interaction between Rb and UBF does not inhibit the binding of UBF to DNA. However, the formation of an UBF/Rb complex does block the interaction of UBF with SL-1, as indicated by using the 48 kDa subunit as a marker for SL-1. Additional evidence is presented that another pocket protein, p130 but not p107, can be found in a complex with UBF. Interestingly, the cellular content of p130 inversely correlated with the rate of rDNA transcription in two physiological systems, and overexpression of p130 inhibited rDNA transcription. These results suggest that p130 may regulate rDNA transcription in a similar manner to Rb. Oncogene (2000) 19, 4988 ± 4999.

Molecular and Cellular Biology, 2001

Journal of Molecular and Cellular Cardiology, 1977

Journal of Immunological Methods, 1988

Various visualization methods were compared for quantitation of proteins by the dot-immunobinding... more Various visualization methods were compared for quantitation of proteins by the dot-immunobinding assay. Comparisons were carried out using a multi-subunit protein, eukaryotic initiation factor 2, and monospecific antibodies directed against two of the factor's subunits. The protein was spotted onto nitrocellulose and the membranes were incubated with primary antibody. The antigen-antibody complex was visualized by one of six methods using either alkaline phosphatase-, horseradish peroxidase-, or glucose oxidase-conjugated IgG, or colloidal gold-labelled IgG, colloidal gold-labelled IgG with silver enhancement, or 125I-labelled protein A. The amount of secondary antibody bound was quantitated by densitometric scanning of the nitrocellulose membrane after staining or autoradiography. The sensitivity of each of the methods was similar; each of the visualization methods could detect less than 1 ng of protein by the dot-immunobinding assay. Curves of protein concentration vs. densitometric absorbance were found to fit a parabolic relationship (r 2 = 0.99) over a wide range of concentrations.

FEBS Letters, 1994

Brefeldin A is a fungal metabolite which disrupts protein traffic through the Golgi apparatus and... more Brefeldin A is a fungal metabolite which disrupts protein traffic through the Golgi apparatus and thereby inhibits protein secretion. Recently, it has been shown that Brefeldin A also causes a marked decrease in the rate of protein synthesis in cells in culture [1992, FEBS Lett. 314, 371-3741. We show here that treatment of rat GH3 pituitary cells with Brefeldin A leads to an inhibition of protein synthesis at the level of peptide~hain initiation through a mechanism involving the phosphorylation of the a-subunit of eukaryotic initiation factor-2 (eIF-2a).

Experimental Cell Research, 1979

Isolated rat hepatocytes release an acidic glycoprotein(s) that can selectively promote the growt... more Isolated rat hepatocytes release an acidic glycoprotein(s) that can selectively promote the growth of transformed cells. This factor has a molecular weight of 60000-70000 D. Liver microsomal and cytosol fractions contain two species of stimulatory activity-t4000 and 3 500 D. Mitochondrial and nuclear fractions contain only the lower molecular weight factor. A factor present in perfused rat liver can METHODS promote the growth of cells transformed by viruses, chemicals, or by culture at high Liver cell suspensions density [l, 21. Indeed transformed fibro-Livers of normal male Sprague-Dawley rats are perblasts; epithelial cells, and lymphoblasts fused in situ as described nreviouslv I31 with the following modifications. The bicarbonate buffer concan be induced to divide by this factor. The tained only 10% bovine erythrocytes without calcium ions, bovine albumin, and amino acids. Mono-10% of the pellet. The pellet (approx. 2 ml of packed cells) is resuspended, except for the bottom 10% acteristics of TCGF will be presented in this which contains large clumps of cells, with 20 vol of bicarbonate buffer containing 3 % bovine albumin and report. 2.4 mM CaCl*. The washing procedure is repeated Em Cell RPS 124 (1979)

Experimental Cell Research, 1975

Perfused rat liver and rat hemicorpus preparations release a factor that promotes the growth of S... more Perfused rat liver and rat hemicorpus preparations release a factor that promotes the growth of SV3T3 cells. This material (TFGF) has a molecular weight of approx. 35 000 D and is sensitive to proteolytic enzymes. TFGF does not promote the multiplication of sparse 3T3 cells or initiate DNA synthesis in confluent 3T3 cells.

Biochimie, 1994

The initiation of peptide chains on ribosomes represents the first step in translation and is a d... more The initiation of peptide chains on ribosomes represents the first step in translation and is a dominant control point for regulation of protein synthesis in mammalian cells. An inhibition of peptide-chain initiation occurs in both rat liver treated with calcium-mobilizing hormones and in rat skeletal muscle in response to the insulin deprivation associated with diabetes mellitus or fasting. The inhibition of peptide-chain initiation in both tissues is accompanied by a shift in polysomal aggregation toward an accumulation of free ribosomal particles and a reduction in formation of 43S preinitiation complexes. Furthermore, in both tissues, the inhibition involves a reduction in the activity of a protein termed eukaryotic initiation factor (eIF)-2B. However, the mechanisms involved in the reduction in eIF-2B activity, and thus protein synthesis, in the two tissues under these conditions appear to be distinct. In liver treated with calcium-mobilizing hormones, the reduction in eIF-2B activity is the result of an increase in the phosphorylation state of the alpha-subunit of a second initiation factor, eIF-2. In contrast, the inhibition of initiation that occurs in skeletal muscle deprived of insulin is not due to a change in the phosphorylation state of eIF-2 alpha. Instead, the activity of eIF-2B may be directly modulated in response to insulin. Studies by others have shown that the epsilon-subunit of eIF-2B is a substrate for at least three different protein kinases and that phosphorylation by at least one of these kinases stimulates the activity of the factor in in vitro assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and Biophysical Research Communications, 1988

The present study examined the effects of diabetes and insulin treatment of diabetic rats on the ... more The present study examined the effects of diabetes and insulin treatment of diabetic rats on the activity of the protein synthesis initiation factor, the guanine nucleotide exchange factor. In extracts from gastrocnemius and psoas muscles from two-day diabetic rats, guanine nucleotide exchange factor activity was reduced to 80% and 67% of control values, respectively. Insulin treatment (2 h) restored guanine nucleotide exchange factor activity to control values in both muscles. In contrast, guanine nucleotide exchange factor activity was unchanged in extracts from either soleus muscle or heart from diabetic rats compared to controls. Also, insulin treatment did not increase guanine nucleotide exchange factor activity in extracts from soleus and heart. The results suggest that the diabetes-induced impairment in peptide-chain initiation in fast-twitch skeletal muscle (i.e. gastrocnemius and psoas) is related to an inhibition of guanine nucleotide exchange factor activity and that slow-twitch muscle is spared from the effect on initiation due to the preservation of guanine nucleotide exchange factor activity. 9 1988 Ac~demio P ..... I~c. Protein synthesis is impaired in skeletal muscle and heart in diabetic rats 0eviewed in 1). Whereas the rate of protein synthesis is depressed in all types of muscle during diabetes, the extent of inhibition following two days of diabetes is less in muscles composed of slow-twitch fibers than in muscles composed primarily of fast-twitch fibers (2,3). Also, the basis for the impairment is different between the two types of muscle. In slow-twitch muscle, the decreased rate of protein synthesis is due primarily to a fall in the content of RNA; i.e., a decrease in the capacity for protein synthesis (2-4). In contrast, the rate of protein synthesis in muscle composed primarily of fasttwitch fibers is limited by an impairment in peptide-chain initiation; i.e., a decreased efficiency (2-6). The impairment in peptide-chain initiation in fast-twitch muscle is rapidly reversed by insulin (2,6,7). In other types of cell an inhibiton of peptide-chain initiation with characteristics similar to those observed in fast-twitch skeletal muscle (5) has been shown to involve an inhibition of one of the initiation factors, the guanine nucleotide exchange factor (GEF) (8,9). In this paper, we show that GEF activity is decreased during diabetes only in extracts from those muscles that demonstrate an impairment in protein synthetic efficiency. In addition, the inhibition of GEF activity in extracts of fast-twitch skeletal muscle from diabetic rats is rapidly reversed by insulin treatment of the

Biochemical and Biophysical Research Communications, 1970

Biochemical and Biophysical Research Communications, 1981

Biochimica et Biophysica Acta (BBA) - General Subjects, 1994

Two polypeptide chain initiation factors, eukaryotic initiation factor 2 (eIF-2) and guanine nucl... more Two polypeptide chain initiation factors, eukaryotic initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF), were isolated from rat liver. Two forms of eIF-2 were identified, one contained three subunits (a, 8, and y), and the other contained only the a-and y-subunits. The three-subunit form was similar to eIF-2 from rabbit reticulocytes with respect to the sedimentation coefficient, Stokes radius, molecular weight of the a-and y-subunits, ability to restore protein synthesis in hemin-deficient reticulocyte lysate, and immunological cross-reactivity of the a-subunits using antibodies against liver eIF-2. In contrast, the 6subunits of the liver and reticulocyte factors were distinct; they had different molecular weights, and antibodies against rat liver eIF-28 did not recognize the @subunit of the reticulocyte factor. Furthermore, the GDP dissociation constant for reticulocyte eIF-2 was more than twice that of the liver factor. GEF from rat liver reversed GDP inhibition of the ternary complex assay and catalyzed the exchange of eIF-2-bound GDP for free GDP or GTP, characteristics ascribed to the corresponding protein from rabbit reticulocytes. However, its subunit composition and molecular weight were different from those reported for reticulocyte GEF. The Tab for GDP exchange mediated by GEF was about 5-fold slower with two-subunit than with threesubunit eIF-2. In addition, the Kn for GDP was lower for two-subunit than for three-subunit eIF-2 when GEF was present. Taken together, these data demonstrate species-associated variability in the @-subunit of eIF-2 and suggest a crucial role for the &subunit in the functional interaction of eIF-2 and GEF. The transfer of initiator Met-tRNA and GTP to the 40 S ribosomal subunit is mediated through eukaryotic initiation factor 2 (eIF-2)' (1). The 40 S preinitiation complex can then bind to mRNA, and a 60 S ribosomal subunit to form a

Biochimica et Biophysica Acta (BBA) - General Subjects, 1978

... 703712. View Record in Scopus | Cited By in Scopus (5). 2L.S. Jefferson, JW Robertson and EL... more ... 703712. View Record in Scopus | Cited By in Scopus (5). 2L.S. Jefferson, JW Robertson and EL Tolman In: A. Pecile and EE Miller, Editors, Growth and Growth Hormone, Excerpta Medica, Amsterdam (1972), pp. 106123. 3K.H. Woodisde and GE Mortimore, J. Biol. Chem. ...

AJP: Endocrinology and Metabolism, 2004

The studies described herein were designed to investigate the effects of 5-aminoimidazole-4-carbo... more The studies described herein were designed to investigate the effects of 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR), an activator of the AMP-activated protein kinase (AMPK), on the translational control of protein synthesis and signaling through the mammalian target of rapamycin (mTOR) in rat liver. Effects of AICAR observed in vivo were compared with those obtained in an in situ perfused liver preparation to investigate activation of AMPK in the absence of accompanying changes in hormones and nutrients. AMPK became hyperphosphorylated, as assessed by a gel-shift analysis, in response to AICAR both in vivo and in situ; however, increased relative phosphorylation at the Thr172 site on the kinase was observed only in perfused liver. Phosphorylation of AMPK either in vivo or in situ was associated with a repression of protein synthesis as well as decreased phosphorylation of a number of targets of mTOR signaling including ribosomal protein S6 kinase 1, eukaryotic initi...

AJP: Cell Physiology, 2010

The mammalian target of rapamycin (mTOR) assembles into two distinct multiprotein complexes known... more The mammalian target of rapamycin (mTOR) assembles into two distinct multiprotein complexes known as mTORC1 and mTORC2. Of the two complexes, mTORC1 acts to integrate a variety of positive and negative signals to downstream targets that regulate cell growth. The lipid second messenger, phosphatidic acid (PA), represents one positive input to mTORC1, and it is thought to act by binding directly to mTOR, thereby enhancing the protein kinase activity of mTORC1. Support for this model includes findings that PA binds directly to mTOR and addition of PA to the medium of cells in culture results in activation of mTORC1. In contrast, the results of the present study do not support a model in which PA activates mTORC1 through direct interaction with the protein kinase but, instead, show that the lipid promotes mTORC1 signaling through activation of the ERK pathway. Moreover, rather than acting directly on mTORC1, the results suggest that exogenous PA must be metabolized to lysophosphatidic a...

The Journal of …, 2005

The potential roles of insulin and dietary amino acids in the regulation of skeletal muscle prote... more The potential roles of insulin and dietary amino acids in the regulation of skeletal muscle protein synthesis were examined in adult and old rats. Animals were fed over 1 h with either a 25% or a 0% amino acid/protein meal. In each nutritional condition, postprandial insulin secretion was either maintained or blocked with diazoxide injections. Protein synthesis in gastrocnemius and soleus muscles was assessed in vivo using the flooding dose method. Insulin suppression decreased protein synthesis in both muscles irrespective of the nutritional condition and age of the rats. Moreover, reduced insulinaemia was associated with 4E-BP1 dephosphorylation, enhanced assembly of the 4E-BP1−eIF4E inactive complex and hypophosphorylation of eIF4E, p70 S6k and protein kinase B, key intermediates in the regulation of translation initiation and protein synthesis. Old rats did not differ from adult rats. The lack of amino acids in the meal of insulin-suppressed rats did not result in any additional decrease in protein synthesis. In the presence of insulin secretion, dietary amino acid suppression significantly decreased gastrocnemius protein synthesis in adult but not in old rats. Amino acid suppression was associated with reduced phosphorylation of 4E-BP1 and p70 S6k in adults. Along with protein synthesis, only the inhibition of p70 S6k phosphorylation was abolished in old rats. We concluded that insulin is required for the regulation of muscle protein synthesis irrespective of age and that the effect of dietary amino acids is blunted in old rats.