Does prothymosin alpha affect the phosphorylation of elongation factor 2?

Prothymosin alpha is a small, acidic, essential nuclear protein that plays a poorly defined role in the proliferation and survival of mammalian cells. Recently, Vega et al. proposed that exogenous prothymosin alpha can specifically increase the phosphorylation of eukaryotic elongation factor 2 (eEF-2) in extracts of NIH3T3 cells (Vega, F. V., Vidal, A., Hellman, U., Wernstedt, C., and Domínguez, F. (1998) J. Biol. Chem. 273, 10147-10152). Using similar lysates prepared by four methods (detergent lysis, Dounce homogenization, digitonin permeabilization, and sonication) and three preparations of prothymosin alpha, one of which was purified by gentle means (the native protein, and a histidine-tagged recombinant prothymosin alpha expressed either in bacteria or in COS cells), we failed to find a response. A reconstituted system composed of eEF-2, recombinant eEF-2 kinase, calmodulin, and calcium was also unaffected by prothymosin alpha. However, unlike our optimized buffer, Vega's system included a phosphatase inhibitor, 50 mM fluoride, which when evaluated in our laboratories severely reduced phosphorylation of all species. Under these conditions, any procedure that decreases the effective fluoride concentration will relieve the inhibition and appear to activate. Our data do not support a direct relationship between the function of prothymosin alpha and the phosphorylation of eEF-2.     

Models of Plant Disease Epidemics. II: Gradients of Bean Rust

The spread of bean rust from three types of inoculum sources (area, line, and point) was studied under field conditions in two seasons. Disease intensity (y) over time was quantified as incidence of diseased plants, incidence of diseased leaves, and disease severity at distances (d) of 0.3, 0.7, 1.5, 2.7, 4.3, 6.5, and 8.7 m from the inoculum sources. Seven curvilinear models were compared and the models that best explained the gradients of bean rust were y = ad^?b exp(cd), y = a exp(–bd^c) and y = a exp(–bd); where a was proportion of disease near the source, d was distance in metres, b was the slope, and c was a shape parameter. For general analysis to compare the slopes of the gradients, the gradient curves were linearized by ln(y) versus d and the parameters ln(a) (intercept) and b (slope) were estimated. The three measures of disease intensity and the three types of inoculum sources had estimated gradient parameters that were statistically different. The incidence of diseased plants rapidly approached y = 1.0 and the gradient slopes were near zero. Also with the ln(y) linearization, the gradients of the incidence of diseased leaves had slopes flatter than the gradients of disease severity. The gradient slopes from area sources of inoculum were flatter than from line sources, which in turn were flatter than from point sources. Late in the epidemics, the b-values became similar for all combinations of inoculum sources × assessment types. The interpretation of flattening of the gradients was confounded because aberrations in transformations occurred with most models. That is, when the intensity of disease (incidence or severity) approached the maximum, the increases in the intercepts were restricted and this restriction was responsible for most of the flattening. No flattening of gradients occurred over time when the gompit gradient model [–ln(–ln(y/y_max)) = –ln(–ln(a))?b ln(d)] was used for these same disease values. The fitting of models to the gradients served little purpose except to classify the curves in a general way. A new statistic, the area under the disease gradient curve (AUDGC), is proposed to compare epidemics.     

The functional intronless S-adenosylmethionine decarboxylase gene of the mouse (Amd-2) is linked to the ornithine decarboxylase gene (Odc) on Chromosome 12 and is present in distantly related species of the genus Mus

S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the biosynthesis of polyamines. We have previously identified a mouse AdoMetDC gene that exhibits the hallmarks of a retroposon; that is, it has no introns, is flanked by direct repeats, and has a poly(dA) tract at its 3′-end. This gene, termed Amd-2, is not a processed pseudogene; rather, it is transcribed in a variety of mouse tissues and encodes a functional enzyme. In the current report, we present the sequence of a 6.7-kb genomic segment of the Amd-2 locus. Several sequences of interest, including an intercisternal A particle (IAP) element, a transposon-related sequence, and several expressed sequence tags (ESTs), were found within or near Amd-2. We also show, through analysis of an interspecific backcross, that Amd-2 is located on Chr 12, tightly linked to the gene (Odc) that encodes ornithine decarboxylase, another key enzyme in polyamine synthesis. Finally, we show that Amd-2 is present among several divergent species of the genus Mus. Thus, the integration event that generated Amd-2 may have occurred early during Mus evolution. Received: 1 December 1998 / Accepted: 31 March 1999     

A dedicated flavin-dependent monooxygenase catalyzes the hydroxylation of demethoxyubiquinone into ubiquinone (coenzyme Q) in Arabidopsis

Ubiquinone (Coenzyme Q) is a vital respiratory cofactor and liposoluble antioxidant. In plants, it is not known how the C-6 hydroxylation of demethoxyubiquinone, the penultimate step in ubiquinone biosynthesis, is catalyzed. The combination of cross-species gene network modeling along with mining of embryo-defective mutant databases of Arabidopsis thaliana identified the embryo lethal locus EMB2421 (At1g24340) as a top candidate for the missing plant demethoxyubiquinone hydroxylase. In marked contrast with prototypical eukaryotic demethoxyubiquinone hydroxylases, the catalytic mechanism of which depends on a carboxylate-bridged di-iron domain, At1g24340 is homologous to FAD-dependent oxidoreductases that instead use NAD(P)H as an electron donor. Complementation assays in Saccharomyces cerevisiae and Escherichia coli demonstrated that At1g24340 encodes a functional demethoxyubiquinone hydroxylase and that the enzyme displays strict specificity for the C-6 position of the benzoquinone ring. Laser-scanning confocal microscopy also showed that GFP-tagged At1g24340 is targeted to mitochondria. Silencing of At1g24340 resulted in 40 to 74% decrease in ubiquinone content and de novo ubiquinone biosynthesis. Consistent with the role of At1g24340 as a benzenoid ring modification enzyme, this metabolic blockage could not be bypassed by supplementation with 4-hydroxybenzoate, the immediate precursor of ubiquinone''s ring. Unlike in yeast, in Arabidopsis overexpression of demethoxyubiquinone hydroxylase did not boost ubiquinone content. Phylogenetic reconstructions indicated that plant demethoxyubiquinone hydroxylase is most closely related to prokaryotic monooxygenases that act on halogenated aromatics and likely descends from an event of horizontal gene transfer between a green alga and a bacterium.     

Interference between strains in live virus vaccines, I: Combined vaccination with measles, mumps and rubella vaccine

One to four different lots of four commercially available trivalent measles-mumps-rubella vaccines were tested for their efficacy as measured by the induction of antibodies to the three vaccine viruses. All of the products were satisfactory although a 100% seroconversion rate was attained regularly only with rubella vaccine. The live virus in the different measles and mumps vaccine components and especially the relative amounts of measles to mumps virus varied widely. Obviously, the efficacy of a vaccine should not be judged only by the virus content. Of main importance is the further adjustment of the dosage of the interfering vaccine virus strains in relation to their attenuation.     

Pleurotus species convert monoterpenes to furanoterpenoids through 1,4-endoperoxides

Enzymatic synthesis of furanoterpenoids from β-myrcene and related monoterpenes was observed using a solubilised enzyme fraction of mycelium lyophilisates of several Pleurotus species. The initial enzymatic step, the incorporation of molecular oxygen into the conjugated 1,3-diene moiety, was similar to a 2 + 4 cycloaddition of 1,3-dienes with dienophilic ¹O₂, and was followed by a non-catalysed degradation sequence leading to the furans. The cyclic peroxides 3,6-dihydro-4-(2-(3,3-dimethyloxiran-2-yl)ethyl)-1,2-dioxine and 5-(3,6-dihydro-1,2-dioxin-4-yl)-2-methylpentan-2-ol were identified as key intermediates. Biotransformation of β-myrcene in ¹⁸O-labelled HEPES-buffer did not yield a detectable label in perillene, so a water addition to 3,10-epoxy-β-myrcenes as an alternative was ruled out. The pathway suggested presents a substantiated biogenetic scheme for the formation of monoterpenoid furans and opens biotechnological access to valuable natural flavour compounds, such as perillene and rosefurane. Only one metabolite, identified as the new natural compound 6-methyl-2-methylene-hept-5-enal, carried the ¹⁸O-label. The enzymatic formation of this compound through a 1,2-endoperoxide (3-(5-methyl-1-methylene-hex-4-enyl)-[1,2]-dioxetane) is suggested. The label may simply result from a chemical oxygen exchange between the carbonyl group and the ¹⁸O-labelled water.     

Decreased intestinal polyp multiplicity is related to exercise mode and gender in ApcMin/+ mice.

Moderate-intensity treadmill running can alter male Apc(Min/+) mouse polyp formation. This purpose of this study was to examine whether exercise mode differentially affects Apc(Min/+) mouse intestinal polyp development in male and female mice. Male and female Apc(Min/+) mice were randomly assigned to control, treadmill (18 m/min; 60 min/day; 6 days/wk), or voluntary wheel running (24-h access) groups. Nine weeks of training decreased total intestinal polyps by 29% in male treadmill runners (66 +/- 9; P = 0.038) compared with male controls (93 +/- 7). The number of large polyps (>/=1-mm diameter) were also reduced by 38% in male treadmill runners (49 +/- 6; P = 0.005) compared with male controls (79 +/- 6). Treadmill running in female Apc(Min/+) mice and wheel running in both genders did not affect polyp number or size. Spleen weight decreased in male treadmill runners (91 +/- 9 mg; P = 0.011) and wheel runners (75 +/- 6 mg; P = 0.004) compared with controls (141 +/- 13 mg). Plasma IL-6 was reduced by 96% in male treadmill runners (1.2 +/- 0.6 pg/ml) and 78% in male wheel runners (6.6 +/- 3.3 pg/ml) compared with control mice (27.9 +/- 2.8 pg/ml; P < 0.05). Female mice responded similarly with an 86% decrease in plasma IL-6 with treadmill running (3.2 +/- 1.2 pg/ml) and 90% decrease with wheel running (2.9 +/- 2.0 pg/ml) compared with control mice (21.1 +/- 5.3 pg/ml; P < 0.05). The crypt depth-to-villus height ratio in the intestine, an indirect marker of intestinal inflammation, decreased by 21 (P = 0.024) and 24% (P = 0.029), respectively, in male and female treadmill runners but not wheel runners. Physical activity-induced attenuation of intestinal polyp number and size is dependent on exercise mode and differs between genders. The modulation of systemic and intestinal inflammation may also depend on exercise mode.