A cellular protein binds to a conserved sequence in the adenovirus type 2 enhancer.

A sensitive gel retention assay has been utilized to detect proteins from uninfected Hela nuclei which interact with the adenovirus type 2 enhancer. This assay has been employed to monitor fractionation of nuclear extracts. Three enhancer binding factors were resolved by chromatography on DEAE-Sepharose and one of the factors was further purified by chromatography on heparin-Sepharose. DNase protection experiments have shown that the heparin-Sepharose fraction contains a factor which binds predominantly to the conserved sequence GTGGAAATTT present at position 160 in the adenovirus type 2 genome and found in many viral and cellular enhancers. Protection of this sequence from DNase I digestion was abolished by competition with a synthetic duplex oligonucleotide spanning bases 144-181. This region corresponds to the sequence defined by Hen et al. as possessing enhancer function. Competition experiments indicated that the enhancer binding factor also bound, albeit with reduced affinity, to multiple sites in the Ela upstream region located between positions 192 and 353. Within the sequences which compete are regions with homology to the high affinity site at position 160. The enhancer binding factor also binds with high affinity to sequences within the SV40 enhancer demonstrating that this factor interacts with sequences common to both the adenovirus and SV40 enhancers.     

The effects of a topical PGE2 analogue on global flap ischemia in rats

The present study evaluated the ability of DHV-PGE2ME, a topically effective 16-vinyl prostaglandin E2 analogue, to improve the tolerance of skin flaps to a period of ischemia. DHV-PGE2ME and placebo were applied to bilateral island flaps on 70 anesthetized rats; then the vascular pedicle of each flap was clamped for 10 hours. Treated flaps evidenced significantly better reperfusion, as documented by quantification of fluorescein dye delivery at 90 minutes after clamp release, and they had significantly greater ultimate viability (p less than 0.05, by ANOVA). While less than 3 percent of untreated flaps survived, those treated with 1.75 and 17.5 microgram/cm2 of drug evidenced 76 and 86 percent survival, respectively. Treatment of a given flap did not affect its contralateral mate, since there was no evidence of a systemic effect. Especially since its effect can be limited to the site of application, DHV-PGE2ME should be valuable for the treatment of compromised perfusion in a variety of settings.     

Proteolytic generation of constitutive tyrosine kinase activity of the human insulin receptor.

We measured rates of protein synthesis in vivo in subcellular fractions (soluble, myofibrillar and stromal fractions) of the heart and the gastrocnemius from rats after fasting or under hypoxic conditions (i.e. atmospheres containing 5% or 10% O2). Such interventions are known to inhibit protein synthesis under some circumstances. The recovery of tissue protein after fractionation was 80-100%. The proportions of protein present in the soluble and stromal fractions were different in the two muscles. The rates of protein synthesis in the myofibrillar and stromal fractions were less than those for total mixed tissue protein, whereas the rate for soluble protein was greater. Both fasting and moderate hypoxia (10% O2 for 24 h) inhibited protein synthesis in the gastrocnemius. In this tissue, the synthesis of the myofibrillar fraction was apparently the most sensitive to inhibition, and this resulted in some significant increases in the soluble-fraction/myofibrillar-fraction protein-synthesis rate ratios. In the heart, fasting inhibited protein synthesis, but moderate hypoxia (10% O2 for 24 h) did not. The rate of protein synthesis in the cardiac myofibrillar fraction was again more sensitive to fasting than were the rates in the other fractions, but it was not as sensitive as that in the gastrocnemius. Under severely hypoxic conditions (5% O2 for 1 or 2 h), protein synthesis was decreased in all fractions in both tissues. These results suggest that the rates of protein synthesis in these relatively crude subcellular fractions vary.     

Immunohistochemical localization of prostaglandin endoperoxide synthase in human fetal membranes and decidua

Prostaglandins play an important role during the maintenance of pregnancy and the initiation of parturition. Prostaglandin endoperoxide synthase activity has been demonstrated in human fetal membranes and decidua. Using immunohistochemical techniques, we identified in these tissues the cell types that contain prostaglandin endoperoxide synthase. A total of 33 specimens, ranging from 8 wk to 42 wk gestation, were studied. Decidualized stromal cells stained the most intensely and consistently of all cell types. Cytotrophoblast of the chorion and early placental villi and syncytotrophoblast of all gestational ages demonstrated a lighter, more variable staining pattern. Regardless of gestational age, amnion stained in a heterogeneous fashion, with some cells demonstrating an intense staining and other cells having no staining. There were no observable differences in laboring compared to nonlaboring term specimens. In summary, the specific cell types that contain immunoreactive prostaglandin endoperoxide synthase have been identified in fetal membranes and decidua.     

Urea hydrolysis by immobilized urease in a fixed-bed reactor: analysis and kinetic parameter estimation

Urea hydrolysis by urease immobilized onto ion exchange resins in a fixed-bed reactor has been studied. A modified Michaelis-Menten rate expression is used to describe the pH-dependent, substrate- and product-inhibited kinetics. Ionic equilibria of product and buffer species are included to account for pH changes generated by reaction. An isothermal, heterogeneous plug-flow reactor model has been developed. An effectiveness factor is used to describe the reaction-diffusion process within the particle phase. The procedure for covalent immobilization of urease onto macroporous cation exchangers is described. Urea conversion data are used to estimate kinetic parameters by a simplex optimization method. The best-fitted parameters are then used to predict the outlet conversions and pH values for systems with various inlet pH values, inlet urea and ammonia concentrations, buffers, particle sizes, and spacetimes. Very good agreement is obtained between experimental data and model predictions. This immobilized urease system exhibits quite different kinetic behavior from soluble urease because the pH near the enzyme active sites is different from that of the pore fluid. This effect results in a shift of the optimal pH value of the V(max) (pH) curve from 6.6 (soluble urease) to ca. 7.6 in dialysate solution, and ca. pH 8.0 in 20mM phosphate buffer. The reactor model is especially useful for estimating intrinsic kinetic parameters of immobilized enzymes and for designing urea removal columns.     

Activation of neutrophils by recombinant interleukin 6

The cytokine interleukin 6 (IL-6) has been shown to have multiple biological activities against many cellular targets. The present studies were designed to determine whether these activities extended to the neutrophil (PMN). Initially, we investigated the ability of IL-6 to modulate PMN-mediated antibody-dependent cellular cytotoxicity. The presence of IL-6 stimulated 51Cr release from labeled, opsonized targets by 67.1% (from 21.6 +/- 1.4% to 36.1 +/- 1.3% at 10 U of IL-6 (P less than 0.01)). IL-6 was not directly toxic to the target cells and stimulation of ADCC was shown to occur across a range of effector-to-target ratios. To investigate the basis of the capacity of IL-6 to stimulate PMN, we studied the effects of IL-6 on PMN chemotaxis, degranulation, and the respiratory burst. IL-6 was not chemotactic or chemokinetic for PMN. However, IL-6 stimulated lysozyme secretion from 14.1 +/- 2.5 to 23.7 +/- 3.6% at 100 U (P less than 0.01). IL-6 was a complete secretagogue, being able to induce the secretion of both the secretory granule marker lactoferrin (11.2 +/- 2.0 to 23.5 +/- 2.2%) and the primary granule marker beta-glucuronidase (5.0 +/- 1.0 to 18.2 +/- 4.0%). IL-6 was not able to directly stimulate the PMN respiratory burst. However, IL-6 did "prime" PMN, enhancing superoxide secretion by fMLP (10(-7) M)-treated PMN by 50.8% (5.9 +/- 1.0 to 8.9 +/- 1.5 nmol superoxide at 100 U of IL-6; P less than 0.01) and PMA (5.0 nM) by 54.3% (8.1 +/- 2.6 to 12.5 +/- 3.6 nmol; P less than 0.05). In conclusion, IL-6 is a PMN stimulant, enhancing the toxicity of PMN in an antibody-dependent cellular cytotoxicity assay. Enhanced cytotoxicity may have been mediated, at least in part, by the stimulation of secretion of toxic components from PMN targets and by the priming of stimulating respiratory burst activity.     

Structure-function relationships of elongation factor Tu. Isolation and activity of the guanine-nucleotide-binding domain

The guanine-nucleotide-binding domain (G domain) of elongation factor Tu(EF-Tu) consisting of 203 amino acid residues, corresponding to the N-terminal half of the molecule, has been recently engineered by deleting part of the tufA gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141-3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF-Tu towards thermal denaturation (t50% = 41.3 degrees C vs. 46 degrees C, respectively). Unlike EF-Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF-Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH+4, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF-Tu. K+ and Na+ are the most active followed by NH+4 and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3-5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7-8, similar to that of EF-Tu. The 70-S ribosome is the only EF-Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF-Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF-Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain.GTP complex and aa-tRNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular Analysis of Recombination Events in Drosophila

The locations of crossover junctions and gene conversion tracts, isolated in the rosy gene of Drosophila melanogaster, were determined using DNA sequencing and denaturing gradient gel electrophoresis. Frequent DNA sequence polymorphisms between the parental genes served as unselected genetic markers. All conversion tracts were continuous, and half of the reciprocal crossover events had conversion tracts at the crossover junction. These experiments have also identified the sequence polymorphisms responsible for altered gene expression in two naturally occurring rosy variants.     

Isolation and Characterization of Mutants of Clostridium acetobutylicum ATCC 824 Deficient in Acetoacetyl-Coenzyme A:Acetate/Butyrate:Coenzyme A-Transferase (EC 2.8.3.9) and in Other Solvent Pathway Enzymes

Mutants of Clostridium acetobutylicum ATCC 824 exhibiting resistance to 2-bromobutyrate or rifampin were isolated after nitrosoguanidine treatment. Mutants were screened for solvent production by using an automated alcohol test system. Isolates were analyzed for levels of butanol, ethanol, acetone, butyrate, acetate, and acetoin in stationary-phase batch cultures. The specific activities of NADH- and NADPH-dependent butanol dehydrogenase and butyraldehyde dehydrogenase as well as those of acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (butyrate-acetoacetate coenzyme A-transferase [EC 2.8.3.9]) (CoA-transferase), butyrate kinase, and phosphotransbutyrylase were measured at the onset of stationary phase. Rifampin-resistant strain D10 and 2-bromobutyrate mutant R were found to be deficient in only CoA-transferase, while several other mutants exhibited reduced butyraldehyde dehydrogenase and butanol dehydrogenase activities as well. The colony morphology of 2-bromobutyrate mutant R was similar to that of the parent on RCM medium; however, it had about 1/10 the level of CoA-transferase and increased levels of butanol dehydrogenase and butyraldehyde dehydrogenase. A nonsporulating, spontaneously derived degenerated strain exhibited reduced levels of butyraldehyde dehydrogenase, butanol, dehydrogenase, and CoA-transferase compared with those of the original strain. When C. acetobutylicum ATCC 824 was grown on medium containing low levels of 2-bromobutyrate, an altered colony morphology was observed. Not all strains resistant to 2-bromobutyrate (12 mM) were non-solvent-producing strains.