Epidermal growth factor or okadaic acid stimulates phosphorylation of eukaryotic initiation factor 4F

Eukaryotic initiation factor 4F, a multi-protein mRNA cap binding complex, was isolated by m7GTP-Sepharose affinity chromatography from human mammary epithelial cells (184A1N4) incubated with [32P] orthophosphate. Treatment of cells with epidermal growth factor resulted in enhanced phosphorylation of both p28 (eIF-4E) and p220 subunits. The identities of the p28 and p220 subunits were confirmed by immunoprecipitation. The phosphorylation was both rapid and sustained in duration; p28 attained maximal levels (2-3-fold) within 30 min of treatment and remained elevated for at least 2 h, while p220 reached one-half maximal levels by 30 min, and maximal levels (3-4-fold) by 2 h of treatment. Two phosphorylated isoforms of p28 and multiple phosphorylated forms of p220 were detected by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphoamino acid analysis of 6 N HCl hydrolyzates of p28 and p220 isolated from epidermal growth factor-treated and control cells indicated that serine is the predominant phosphorylated amino acid in both instances. In no case was phosphotyrosine observed. Pretreatment of cells with 1 microM okadaic acid resulted in the hyperphosphorylation of both p28 and p220 subunits. These results suggest that mitogenic growth factors and cellular serine/threonine phosphatases (pp1 and/or pp2A) serve essential roles in regulating phosphorylation levels of eukaryotic initiation factor 4F and support the concept that translational control is a component of the signal transduction mechanisms involved in growth regulation.     

Elevated cholesterol and decreased sterol carrier protein-2 in peroxisomes from AS-30D hepatoma compared to normal rat liver

Peroxisomes were isolated from AS-30D hepatoma and compared to normal rat liver cells for the purpose of investigating the cholesterol accumulation in the hepatoma cells. Cholesterol was found to be approximately 10-fold higher relative to protein in AS-30D peroxisomes as compared to peroxisomes from normal liver. The peroxisomes from the hepatoma cells were found to be more stable; catalase was not released from these peroxisomes during isolation or osmotic shock of the peroxisomal fraction. The elevated cholesterol level may stabilize the peroxisomal membrane. Sterol carrier protein-2 (SCP-2) levels were measured using a radioimmunoassay (RIA), which indicated the highest concentration of SCP-2 to be in peroxisomes. Hepatoma peroxisomes had a lower concentration of SCP-2 (2.5 micrograms/mg) than normal liver peroxisomes (8 micrograms/mg). Approximately half of all SCP-2 detected was found to be soluble in both hepatoma and normal rat liver cells. Immunoblots from both rat liver and AS-30D fractions demonstrated the presence of the 14-kDa form of SCP-2. The liver fractions also had a 57-kDa immunoreactive protein, which was barely detectable in the AS-30D fractions. The low abundance of the high molecular weight form of SCP-2 from hepatoma peroxisomes and the lower amounts of SCP-2 detected in the AS-30D peroxisomes may be related to the accumulation of cholesterol in the cells.     

Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase.

The spontaneous refolding of chemically denatured dihydrofolate reductase (DHFR) is completely arrested by chaperonin 60 (GroEL). This inhibition presumably results from the formation of a stable complex between chaperonin 60 and one or more intermediates in the folding pathway. While sequestered on chaperonin 60, DHFR is considerably more sensitive to proteolysis, suggesting a nonnative structure. Bound DHFR can be released from chaperonin 60 with ATP, and although chaperonin 10 (GroES) is not obligatory, it does potentiate the maximum effect of ATP. Hydrolysis of ATP is also not required for DHFR release since certain nonhydrolyzable analogues are capable of partial discharge. "Native" DHFR can also form a stable complex with chaperonin 60. However, in this case, complex formation is not instantaneous and can be prevented by the presence of DHFR substrates. This suggests that native DHFR exists in equilibrium with at least one conformer which is recognizable by chaperonin 60. Binding studies with 35S-labeled DHFR support these conclusions and further demonstrate that DHFR competes for a common saturable site with another protein (ribulose-1,5-bisphosphate carboxylase) known to interact with chaperonin 60.     

Acquired resistance of sheep to larvae of Lucilia cuprina, assessed in vivo and in vitro

The effect on subsequent larval survival of infesting sheep repeatedly with larvae of Lucilia cuprina was assayed in vivo and in vitro. One in vivo assay technique, in which implanted larvae were grown to third instar, indicated a significant reduction in larval survival; another in vivo technique, in which larvae were allowed to develop to second instar in small aluminium rings attached to the sheep, indicated no reduction in larval growth or survival. Larvae of Lucilia cuprina grown in vitro on media containing sera from previously infested sheep were significantly retarded in growth after 20 h compared with controls; no difference was detected when larvae were allowed to develop to pupation on two changes of the same media. No significant differences in survival of larvae either to 20 h or to pupation were obtained between the two treatments. ELISA antibody levels against crude soluble larval material were significantly higher for sera from infested sheep than for control sera, and the regression of antibody level on mean larval weight obtained after 20 h growth in vitro was significant. The immunoglobulin fraction isolated from sera of infested sheep significantly retarded larval growth when incorporated with normal serum in growth media. These results are consistent with an effect of specific anti-larval antibody produced by sheep in response to infestation.     

Biotic and abiotic factors affecting stability of Beauveria bassiana conidia in soil

Beauveria bassiana conidia were stored in sterile and nonsterile soil under various temperature, relative humidity, soil water content, and pH regimes. Survival of the conidia was primarily dependent on temperature and soil water content. Conidia half-lives ranged from 14 days at 25°C and 75% water saturation to 276 days at 10°C and 25% water saturation. Conidia held at ?15°C exhibited little or no loss in viability regardless of water content, relative humidity, or pH. Conidia were not recoverable after 10 days from soils held at 55°C. Conidia survival in nonsterile soil that was amended with carbon sources, nitrogen sources, or combinations of carbon and nitrogen was greatly decreased and loss was often complete in less than 22 days whereas sterile soil treated in the same manner showed dramatic increases in number, demonstrating that B. bassiana is capable of growth in sterile soil. The obvious fungistatic effect in amended nonsterile soils was possibly related to Penicillium urticae which was routinely isolated from the soils and is shown to produce a water-soluble inhibitor of B. bassiana. The fungistatic effect was shown to be an active inhibition rather than due to competition.     

Organelle Membranes from Germinating Castor Bean Endosperm: II. ENZYMES, CYTOCHROMES, AND PERMEABILITY OF THE GLYOXYSOME MEMBRANE

Glyoxysome ghosts were isolated from germinating castor bean endosperms using established methods. Electron microscopic examination showed that some matrix material was retained within the glyoxysomal membrane. Two cytochrome reductases and phosphorylcholine glyceride transferase co-sedimented with the alkaline lipase, a known component of the glyoxysome membrane, in sucrose gradient centrifugation of osmotically shocked glyoxysomes. The activities of these enzymes in the glyoxysome membranes were compared to those in the endoplasmic reticulum relative to phospholipid content. On this basis, the phosphorylcholine glyceride transferase was 10-fold more active in the endoplasmic reticulum, whereas the lipase was 50-fold more active in the glyoxysome membrane. The cytochrome reductases were only 2-fold more active in the endoplasmic reticulum, indicating that they are components of the two membranes. Difference spectroscopy of the glyoxysome membrane suspension revealed the presence of a b5-type cytochrome similar to that found in the endoplasmic reticulum. Since the glyoxysome membrane is apparently derived from the endoplasmic reticulum, components of the endoplasmic reticulum such as these are likely to be incorporated into the glyoxysome membrane during biogenesis.     

Teratogenic effects of diazepam in the hamster.

Pregnant hamsters were treated with different doses of oral and intravenous diazepam during the period of organogenesis. Teratogenic effects of diazepam were observed following oral treatment on days 8 and 10 and following intravenous treatment on day 11 of gestation. Types of malformations included cleft palate, exencephaly, limb anomalies, and hemorrhage. A dose-effect relationship was not observed. Comparison with reported literature seems to indicate that diazepam may be a mild teratogen in some species.     

Regulation of fatty acid synthesis.

Acetyl-CoA carboxylase and fatty acid synthetase are the two major enzymes involved in the synthesis of fatty acids in animals. The activities of both enzymes are affected by nutritional manipulations. Although acetyl-CoA carboxylase is considered generally to be the rate-limiting step in lipogenesis, there is evidence that suggests that fatty acid synthetase may become rate limiting under certain conditions. The principal support for the view that acetyl-CoA carboxylase is the rate-limiting enzyme for lipogenesis is that the activity of the enzyme is controlled by allosteric effectors that change the catalytic efficiency of the enzyme. Until recently, the only known control of fatty acid synthetase was through changes in rate of enzyme synthesis. Data are reviewed that show that fatty acid synthetase can exist in forms possessing different catalytic activities. Thus fatty acid synthetase appears to be subject to the type of control necessary for an enzyme to serve as a regulator of the rate of a biological process over a short term.     

Hematoxylin substitutes: fluorone black and methyl fluorone black (9-phenyl- and 9-methyl-2,3,7-trihydroxy-6-fluorone) as metachrome iron alum mordant dyes.

The phenyl and methyl trihydroxyfluorones, hitherto used histologically only in the rather difficult and unreliable Turchini technics for discriminating deoxyribonucleic from ribonucleic acid, find a new use as iron mordant metachrome dyes which act as nuclear stains. Nuclear staining is unaffected by acid extraction of nucleic acids, as with hematoxylin lakes. The two dyes, named by Liebermann and Lindenbaum 9-phenyl-2, 3, 7-trihydroxy-6-fluorone, have also acquired (illustrating with the phenyl homolog) longer chemical names of the form 2,6,7-trihydroxy-9-phenylisoxanthene-3-one (Eastman). Aldrich and Pfalz-Bauer adhere to the Liebermann-Lindenbaum nomenclature. The trivial name fluorone black is proposed for the phenyl homolog and methyl fluorone black for the methyl homolog. The iron lake of fluorone black appears to be a useful substitute for iron hematoxylin, methyl flurone black less useful. Neither dye has the diverse capability of hematoxylin.     

An assessment of the AFLP method for investigating population structure in the red alga Chondrus crispus Stackhouse (Gigartinales, Florideophyceae)

The appropriateness of the Amplified Fragment Length Polymorphism (AFLP) technique for investigating Chondrus crispus Stackhouse populations in the Maritime Provinces of Canada was assessed. The AFLP procedure was first subjected to reproducibility testing and three shortcomings were noted: 1) failure to reproduce band intensity between replicate runs for the same individual and primer pair; 2) failure of some bands to replicate; 3) lack of reproducibility for complete replicate runs for some individuals and primer pairs. In the last-mentioned case, the lack of reproducibility resulted in characteristic electropherograms indicative of weak reactions. These weak runs can be attributed to poor restriction digest/ligation reactions and/or substandard PCR, these failures ultimately resulting from low and inconsistent DNA quality. We recommend that reproducibility testing should be completed routinely in studies using the AFLP technique. In the current work, only fragments and individuals that gave reproducible results were used in subsequent analyses. The AFLP method resulted in highly variable markers within and between the populations of C. crispus included in this investigation, which prevented successful resolution of population structure. This situation could result from a lack of suitability for AFLP markers in population genetic studies, and/or too extensive genetic variation for C. crispus populations to be discerned by the AFLP technique. These two possible explanations are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.