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K M Flowers

Publications and source records attributed to K M Flowers.

11 recordsLinked to original sources
PubMed1996

eIF2B, the guanine nucleotide-exchange factor for eukaryotic initiation factor 2. Sequence conservation between the alpha, beta and delta subunits of eIF2B from mammals and yeast.

The guanine nucleotide-exchange factor eIF2B mediates the exchange of GDP bound to translation initiation factor eIF2 for GTP. This exchange process is a key regulatory step for the control of translation initiation in eukaryotic organisms. To improve our understanding of the structure, function and regulation of eIF2B, we have obtained and sequenced cDNA species encoding all of its five subunits. Here we report the sequences of eIF2B beta and delta from rat. This paper focuses on sequence similarities between the alpha, beta and delta subunits of mammalian eIF2B. Earlier work showed that the amino acid sequences of the corresponding subunits of eIF2B in the yeast Saccharomyces cerevisiae (GCN3, GCD7 and GCD2) exhibit considerable similarity. We demonstrate that this is also true for the mammalian subunits. Moreover, alignment of the eIF2B alpha, beta and delta sequences from mammals and yeast, along with the sequence of the putative eIF2B alpha subunit from Caenorhabditis elegans and eIF2B delta from Schizosaccharomyces pombe shows that a large number of residues are identical or conserved between the C-terminal regions of all these sequences. This strong sequence conservation points to the likely functional importance of these residues. The implications of this are discussed in the light of results concerning the functions of the subunits of eIF2B in yeast and mammals. Our results also indicate that the large apparent differences in mobility on SDS/PAGE between eIF2B beta and delta subunits from rat and rabbit are not due to differences in their lengths but reflect differences in amino acid composition. We have also examined the relative expression of mRNA species encoding the alpha, beta, delta and epsilon subunits of eIF2B in a range of rat tissues by Northern blot analysis. As might be expected for mRNA species encoding subunits of a heterotrimeric protein, the ratios of expression levels of these subunits to one another did not vary between the different rat tissues examined (with the possible exception of liver). This represents the first analysis of the levels of expression of mRNA species encoding the different subunits of eIF2B.

Amino Acid Sequence↗
PubMed1996

Cloning and characterization of complementary and genomic DNAs encoding the epsilon-subunit of rat translation initiation factor-2B.

Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide-exchange protein involved in the recycling of eIF-2 during peptide-chain initiation. Regulation of eIF-2B activity occurs under a wide range of conditions by diverse mechanisms. To better understand the regulation of eIF-2B activity as well as the coordinate expression of its five subunits, we have begun to clone and characterize the cDNAs and genes encoding these proteins. In the present study, complementary and genomic DNAs encoding the epsilon-subunit of rat eIF-2B were cloned and characterized. The cDNA is 2517 bp in length, including a 30 nt poly(A) tail, and recognizes both 2.7 and 3.5 kb mRNA species on Northern blots of rat RNA. The cDNA contains a 2151 bp open reading frame encoding 716 amino acids producing a protein with a predicted molecular mass of 80 kDa. The derived amino acid sequence contains regions identical to three peptides obtained from bovine liver eIF-2B epsilon and is 31% identical to Gcd6, the putative yeast eIF-2B epsilon. Examination of the derived amino acid sequence of rat eIF-2B epsilon reveals phosphorylation site motifs for several protein kinases which have been implicated in regulation of guanine nucleotide exchange activity. The mRNA for eIF-2B epsilon is expressed to a similar extent in most rat tissues examined with the exception of testis, where its expression is approx, three-fold greater. We have also isolated and sequenced the coding and 5'-flanking region of the rat eIF-2B epsilon gene. The 16 exons encoding rat eIF-2B epsilon are contained within 9.5 kb of genomic DNA. Examination of the promoter region of the gene reveals a consensus binding site for the alpha-Pal transcription factor as well as possible cytokine-response elements and binding sites for testis-specific transcription factors.

Amino Acid Sequence↗
PubMed1996

Altered expression of eukaryotic initiation factor 2B in skeletal muscle during sepsis.

Sepsis causes an inhibition of protein synthesis in skeletal muscles composed of fast-twitch fibers, in part, as a result of a decreased activity of the eukaryotic initiation factor 2B (eIF-2B). In the present study, we investigated the expression of two subunits of eIF-2B, i.e., the beta- and epsilon-subunits during sepsis. The expression of both beta- and epsilon-subunits of eIF-2B in gastrocnemius was decreased approximately 50% from control values during the first 5 days after induction of sepsis. The decreased expression of eIF-2B epsilon during sepsis correlated with similar reductions in eIF-2B epsilon mRNA. Restoration of protein synthesis (10 days postsurgery) was associated with a return of eIF-2B epsilon expression to values observed in control rats. Expression of eIF-2B epsilon was not altered in heart during sepsis or in gastrocnemius from nonseptic abscess animals. Amrinone, which ameliorated the inhibition of protein synthesis during sepsis, also prevented the fall in eIF-2B epsilon protein after 5 days of infection. The data provide evidence that expression of eIF-2B epsilon is markedly influenced in gastrocnemius during the course of the septic episode and support the concept that this change is a mechanism responsible for the inhibition of protein synthesis observed under this condition.

Amrinone↗
PubMed1995

Structure and sequence of the gene encoding the alpha-subunit of rat translation initiation factor-2B.

Eukaryotic initiation factor-2B (eIF-2B) is a heteropentameric guanine nucleotide exchange factor involved in the recycling of eIF-2 during the translation initiation phase of protein synthesis. The activity of eIF-2B is controlled by diverse mechanisms under a wide range of conditions. As part of our studies into the control of the expression of eIF-2B subunit mRNAs, we have isolated and characterized the rat gene encoding the alpha-subunit of eIF-2B. The gene is comprised of 9 exons which are contained within 8.5 kb of genomic DNA. A comparison of the 5'-flanking region of the gene with that of the previously isolated eIF-2B epsilon gene reveals that both possess a potential binding site for the alpha-Pal transcription factor as well as several other regions of homology.

Animals↗
PubMed1995

Molecular cloning and characterization of cDNA encoding the alpha subunit of the rat protein synthesis initiation factor eIF-2B.

Eukaryotic initiation factor 2B (eIF-2B) is an essential component of the pathway of peptide-chain initiation in mammalian cells, yet little is known about its molecular structure and regulation. To investigate the structure, regulation, and interactions of the individual subunits of eIF-2B, we have begun to clone, characterize, and express the corresponding cDNAs. We report here the cloning and characterization of a 1510-bp cDNA encoding the alpha subunit of eIF-2B from a rat brain cDNA library. The cDNA contains an open reading frame of 918 bp encoding a polypeptide of 305 aa with a predicted molecular mass of 33.7 kDa. This cDNA recognizes a single RNA species approximately 1.6 kb in length on Northern blots of RNA from rat liver. The predicted amino acid sequence contains regions identical to the sequences of peptides derived from bovine liver eIF-2B alpha subunit. Expression of this cDNA in vitro yields a peptide which comigrates with natural eIF-2B alpha in SDS/polyacrylamide gels. The predicted amino acid sequence exhibits 42% identity to that deduced for the Saccharomyces cerevisiae GCN3 protein, the smallest subunit of yeast eIF-2B. In addition, expression of the rat cDNA in yeast functionally complements a gcn3 deletion for the inability to induce histidine biosynthetic genes under the control of GCN4. These results strongly support the hypothesis that mammalian eIF-2 alpha and GCN3 are homologues. Southern blots indicate that the eIF-2B alpha cDNA also recognizes genomic DNA fragments from several other species, suggesting significant homology between the rat eIF-2B alpha gene and that from other species.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of a cDNA encoding rat PKR, the double-stranded RNA-dependent eukaryotic initiation factor-2 kinase.

We have isolated and sequenced a full-length cDNA encoding the double-stranded RNA-dependent protein kinase PKR from rat. The derived amino acid sequences of the protein kinase and RNA-binding domains are highly conserved between the rat, human and mouse enzymes. In addition, sequence elements in the 5'- and 3'-untranslated regions are also conserved between species.

Animals↗
PubMed1994

Cloning and characterization of cDNA encoding rat hemin-sensitive initiation factor-2 alpha (eIF-2 alpha) kinase. Evidence for multitissue expression.

Reticulocytes contain an eukaryotic initiation factor-2 alpha (eIF-2 alpha) kinase that is negatively regulated by hemin. This protein kinase, which has been termed heme-regulated inhibitor and heme-controlled repressor (HCR), has a strong inhibitory effect on the initiation of protein synthesis and plays an important role in translational control in these cells. Previous evidence has suggested that HCR may be an erythroid-specific enzyme. As reported herein, we have cloned and characterized the cDNA encoding an eIF-2 alpha kinase from rat brain. The predicted amino acid sequence of the kinase from rat brain shows 82% homology to rabbit reticulocyte HCR with the greatest variation concentrated in a central region of approximately 135 amino acids located between protein kinase subdomains IV and VI. We have expressed the rat brain cDNA in Escherichia coli and have demonstrated that the recombinant enzyme is a hemin-sensitive eIF-2 alpha kinase. We have also shown that mRNA recognized by the rat brain cDNA is expressed in a wide range of non-erythroid rat tissues at a lower but significant level compared with reticulocytes. These findings raise the possibility of additional, uncharacterized roles for HCR in non-erythroid cells.

Amino Acid Sequence↗
PubMed1994

Spironolactone administration and training-induced hypervolemia.

The purpose of this study was to determine the contribution of aldosterone to plasma volume expansion accompanying short-term exercise training. Twelve healthy males (age = 26.5 +/- 5.8) cycled for 120 min on three consecutive days at a relative exercise intensity of 65% VO2max. Half of the subjects were treated with 25 mg of spironolactone taken four times daily to suppress the effects of aldosterone. Resting plasma volume increased significantly (501 +/- 83 ml, p < 0.05) from pretraining in control subjects, but not for drug-treated subjects (163 +/- 94 ml). Spironolactone attenuated gains in resting plasma volume, serum sodium content, and serum osmolal content by 67%, 79% and 66%, respectively. Spironolactone-treated and non-treated subjects experienced similar increases in total serum protein content sufficient to increase the plasma volume by approximately 290 ml. It was not determined why drug-treated subjects attained less of a plasma volume expansion than that expected by the increase in oncotic pressure. In conclusion, two-fifths of the plasma volume expansion induced by 3 d of endurance cycling could be attributed to aldosterone activity, and the remaining three-fifths could be explained by the expansion of intravascular protein mass. Other neurohormonal influences may have contributed to the overall plasma volume expansion but this experiment did not allow for their exclusion.

Adolescent↗
PubMed1993

No evidence of oxidant stress during high-intensity rowing training.

In untrained subjects, strenuous exercise provokes the appearance of oxidant stress markers in blood and muscle. On the other hand, trained muscle is resistant to oxidant stress unless exercise challenges the muscle glycogen supply. It is not known whether chronic high-intensity exercise alters the susceptibility of skeletal muscle to oxidant stress, whether there are gender-related differences in markers of oxidant stress, or whether elevating muscle glycogen stores by increasing dietary carbohydrate can minimize any exercise-related oxidant stress. To address these issues, collegiate rowers (12 men, 11 women) were randomly assigned to a moderate-(MOD, 5 g/kg body wt) or high-carbohydrate (HI, 10 g/kg) diet in a double-blind design and underwent strenuous training for 4 wk. Training in the A.M. was 40 min at 70% maximal O2 consumption (VO2); in the P.M. it was either three 2,500-m time trials (to assess power output) or aerobic and lactate tolerance training. Total daily training time was 65 min at 70% maximal VO2 and 38 min at > or = 90% maximal VO2. Thrice-weekly morning blood samples were assayed for serum creatine kinase (CK), plasma thiobarbituric acid-reactive substances (TBARS), and serum beta-glucuronidase (beta-Gluc). Weekly muscle biopsies were obtained for analysis of glycogen and, when tissue sample quantity allowed, TBARS. HI rowers produced more power and improved power more (10.7 +/- 1.0 vs. 1.6 +/- 1.6%) over the 4 wk than did the MOD rowers. Preexercise muscle glycogen concentration was maintained at 119 mmol/kg in MOD but increased 65% in HI rowers (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Type II pneumocyte proliferation in vitro: problems and future directions.

In adult animals, the type II pneumocyte is progenitor of both the type I and type II alveolar epithelium. In primary culture, however, the fate of this cell is uncertain. Type II cells in culture lose their differentiated properties and eventually resemble type I cells, but a lack of specific markers has complicated the characterization of the phenotype acquired in vitro. Furthermore, limited proliferation of these cells in vitro has precluded definition of the relationship between type II cell proliferation and differentiation. Recent work in this laboratory has involved the correlation of flow cytometric cell cycle analysis with phenotype markers. Initial results indicate that isolation of type II cells induces cell cycle block similar to that sustained by other cell types in response to stress. In addition, preliminary evaluation of phenotype suggests that traditional markers become ambiguous beyond the 1st day of primary culture. These results raise concern related to the interpretation of experiments conducted in vitro. This report discusses the implications of these findings and directions for future work.

Animals↗