The use of [14C]eukaryotic initiation factor 2 to measure the endogenous pool size of eukaryotic initiation factor 2 in rabbit reticulocyte lysate.

[14C]Eukaryotic initiation factor 2 (eIF-2), obtained by reductive methylation of the purified initiation factor, was shown to be active in the unfractionated reticulocyte lysate. This allowed a direct measurement of the endogenous pool size of eIF-2 in rabbit reticulocyte lysate according to the principle of isotope dilution. A value of 20 to 30 pmol/ml of lysate was obtained. Although translational inhibition resulting from hemin deficiency appears to be characterized by a change from catalytic to stoichiometric utilization of eIF-2, the pool size of eIF-2 is too small to account for the normal period of protein synthesis before the onset of translation inhibition. This suggests, therefore, that additional events to eIF-2 alpha phosphorylation may be required for translational inhibition.     

Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein.

The adeno-associated virus (AAV) nonstructural protein Rep 68 is required for viral DNA replication. An in vitro assay has been developed in which addition of Rep 68 to an extract from uninfected HeLa cells supports AAV DNA replication. In this paper, we report characterization of the replication process when a fusion of the maltose binding protein and Rep 68, expressed in Escherichia coli, was used in the assay. Replication was observed when the template was either linear double-stranded AAV DNA or a plasmid construct containing intact AAV DNA. When the recombinant plasmid construct was used as the template, there was replication of pBR322 DNA as well as the AAV DNA; however, linear pBR322 DNA was not replicated. When the plasmid construct was the template, replication appeared to initiate on the intact plasmid and led to separation of the AAV sequences from those of the vector, a process which has been termed rescue. There was no evidence that replication could initiate on the products of rescue. Rep 68 can make a site-specific nick 124 nucleotides from the 3' end of AAV DNA; the site of the nick has been called the terminal resolution site. Our data are most consistent with initiation occurring at the terminal resolution site and proceeding toward the 3' terminus. When the template was the plasmid construct, either elongation continued past the junction into pBR322 sequences or the newly synthesized sequence hairpinned, switched template strands, and replicated the AAV DNA. Replication was linear for 4 h, during which time 70% of the maximal synthesis took place. An additional finding was that the Rep fusion could resolve AAV dimer length duplex intermediates into monomer duplexes without DNA synthesis.     

AMPK regulation of the growth of cultured human keratinocytes

AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). At concentrations of 10(-4) and 10(-3) M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10(-6) M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D3 (10(-7) and 10(-6) M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished as they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p < 0.05) as keratinocytes grown in control medium went from 25% to 100% confluence. In conclusion, the data are consistent with the hypothesis that activation of AMPK acts as a signal to diminish the proliferation of cultured keratinocytes as they approach confluence. They also suggest that AMPK activators, such as AICAR and troglitazone, inhibit keratinocyte growth and that the inhibition of cell growth by 1,25-dihydroxyvitamin D3 is AMPK-independent.     

Effects of hypoxia on the development of intestinal enzymes in neonatal and juvenile rats

Hypoxia in the neonate is known to alter the activity of hepatic and pancreatic enzymes involved in lipid and carbohydrate metabolism. The purpose of this study was to evaluate the effect of neonatal hypoxia on the activity of intestinal enzymes, and to determine whether the administration of glucocorticoids to neonates can mimic the effects of hypoxia. Hypoxia in neonatal rats (0-7 days) increased protein content, and lactase and maltase activity in the duodenal and the jejunal segments of the small intestine compared with normoxic controls. Hypoxia in juvenile rats (28-35 days) did not change these enzymes. Two weeks after returning hypoxic (0-7 days) pups to normoxia, their body weight remained lower than the age-matched controls. In the group recovering from hypoxia, sucrase, maltase, and leucine aminopeptidase activities were lower in the duodenal and the jejunal segment. Compared with controls, LDH activity was lower only in the jejunal intestine in the group recovering from hypoxia. All enzyme activities returned to control levels 3 weeks after recovery. Neonatal rats treated with dexamethasone had a decrease in body weight, but increases in sucrase and maltase activity in both the duodenal and the jejunal segment. Hypoxia in newborn rats caused a delayed maturation of small intestinal enzymes. Increases in serum glucocorticoids after hypoxic exposure probably do not play a major role in the delayed maturation of the disaccharidase activity in the small intestine.     

Dexamethasone treatment in the newborn rat: fatty acid profiling of lung, brain, and serum lipids.

Dexamethasone is used as treatment for a variety of neonatal syndromes, including respiratory distress. The present study utilized the power of comprehensive lipid profiling to characterize changes in lipid metabolism in the neonatal lung and brain associated with dexamethasone treatment and also determined the interaction of dexamethasone with hypoxia. A 4-day tapering-dose regimen of dexamethasone was administered at 0800 on postnatal days 3 (0.5 mg/kg), 4 (0.25 mg/kg), 5 (0.125 mg/kg), and 6 (0.05 mg/kg). A subgroup of rats was exposed to hypoxia from birth to 7 days of age. Dexamethasone treatment elicited numerous specific changes in the lipid profile of the normoxic lung, such as increased concentrations of saturated fatty acids in the phosphatidylcholine and cholesterol ester classes. These increases were more profound in the lungs of hypoxic pups. Additional increases in cardiolipin concentrations were also measured in lungs of hypoxic pups treated with dexamethasone. We measured widespread increases in serum lipids after dexamethasone treatment, but the effects were not equivalent between normoxic and hypoxic pups. Dexamethasone treatment in hypoxic pups increased 20:4n6 and 22:6n3 concentrations in the free fatty acid class of the brain. Our results suggest that dexamethasone treatment in neonates elicits specific changes in lung lipid metabolism associated with surfactant production, independent of changes in serum lipids. These findings illustrate the benefits of dexamethasone on lung function but also raise the potential for negative effects due to hyperlipidemia and subtle changes in brain lipid metabolism.     

Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner.

The human globin locus control region-binding protein, NF-E2, was purified by DNA affinity chromatography. Its tissue-specific component, p45 NF-E2, was cloned by use of a low-stringency library screen with murine p45 NF-E2 cDNA (N. C. Andrews, H. Erdjument-Bromage, M. B. Davidson, P. Tempst, and S. H. Orkin, Nature [London] 362:722-728, 1993). The human p45 NF-E2 gene was localized to chromosome 12q13 by fluorescent in situ hybridization. Human p45 NF-E2 and murine p45 NF-E2 are highly homologous basic region-leucine zipper (bZIP) proteins with identical DNA-binding domains. Immunoprecipitation experiments demonstrated that p45 NF-E2 is associated in vivo with an 18-kDa protein (p18). Because bZIP proteins bind DNA as dimers, we infer that native NF-E2 must be a heterodimer of 45- and 18-kDa subunits. Although AP-1 and CREB copurified with NF-E2, no evidence was found for heterodimer formation between p45 NF-E2 and proteins other than p18. Thus, p18 appears to be the sole specific partner of p45 NF-E2 in erythroid cells. Cloning of human p45 NF-E2 should permit studies of the role of NF-E2 in globin gene regulation and erythroid differentiation.     

Beta thymosins as actin binding peptides

The beta thymosins are a highly conserved family of strongly polar 5 kDa polypeptides that are widely distributed among vertebrate classes; most are now known to bind to monomeric actin and inhibit its polymerization. One beta thymosin, beta four, (Tβ⁴) is the predominant form in mammalian cells, present at up to 0.5 mM. Many species are known to produce at least two beta thymosin isoforms, in some cases in the same cell. Their expression can be separately regulated. When present outside the cell, the N-terminal tetrapeptide of beta four appears to affect cell cycle regulation; beta thymosins or smaller fragments derived from them may have additional regulatory functions. We suggest that many developmental changes in beta thymosin levels within cells and tissues may be related to changes in G-actin pool size.     

Thymosin-beta(4) changes the conformation and dynamics of actin monomers

Thymosin-beta(4) (Tbeta(4)) binds actin monomers stoichiometrically and maintains the bulk of the actin monomer pool in metazoan cells. Tbeta(4) binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) conjugated to Cys(374) of actin monomers. The K(d) of the actin-Tbeta(4) complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation. At 25 degrees C, the free energy of Tbeta(4) binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on subdomain 2 is >12 times faster when Tbeta(4) is bound. The C terminus of Tbeta(4) contacts actin near this cleavage site, at His(40). By tritium exchange, Tbeta(4) slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that Tbeta(4) changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of Tbeta(4) to sequester actin monomers and inhibit nucleotide exchange.     

Reduced activity of the interferon-induced double-stranded RNA-dependent protein kinase during a heat shock stress

Previous studies have shown that the antiviral response induced by interferon in murine cells could be degraded after a heat shock. Here we have confirmed that a similar effect occurs also in interferon-treated human HeLa cells subjected to a heat shock. In addition, we have investigated the fate of the interferon-induced, double-stranded RNA-dependent protein kinase in heat-shocked cells. This protein kinase is a Mr 68,000 protein (p68 kinase) which, when autophosphorylated, catalyzes phosphorylation of the protein synthesis eukaryotic initiation factor-2, thus mediating inhibition of protein synthesis. After heat shock of interferon-treated HeLa cells, the double-stranded RNA-dependent autophosphorylation of p68 kinase in cytoplasmic extracts is greatly reduced whereas the phosphorylation of other cellular proteins is not affected. In vivo, autophosphorylation of p68 kinase is also reduced in heat-shocked cells whereas there is no apparent effect on the phosphorylation state of other proteins. In such cells, the interferon-mediated antiviral response becomes modified according to the virus challenge, i.e. these cells remain resistant to vesicular stomatitis virus but become partially sensitive to encephalomyocarditis virus (EMCV) infection. The reduction in the activity of p68 kinase is due to its reduced nonionic detergent solubility occurring during the heat shock period. The resultant reduced detergent extractibility of p68 kinase is dependent on the intensity of the thermal stress. In contrast to the effect after a heat shock, arsenite treatment of interferon-treated HeLa cells induces heat shock proteins, but neither modifies the antiviral response nor affects the extractibility of p68 kinase. These results indicate that the degradation of the anti-EMCV response and reduced p68 kinase activity occur in response to heat treatment independently of the induction of heat shock proteins. The role of p68 kinase in the mechanism of the antiviral response against EMCV and vesicular stomatitis virus is discussed.