Glycogen supercompensation in rat soleus muscle during recovery from nonweight bearing

The time course of glycogen changes in soleus muscle recovering from 3 days of nonweight bearing by hindlimb suspension was investigated. Within 15 min and up to 2 h, muscle glycogen decreased. Coincidentally, muscle glucose 6-phosphate and the fractional activity of glycogen phosphorylase, measured at the fresh muscle concentrations of AMP, increased. Increased fractional activity of glycogen synthase during this time was likely the result of greater glucose 6-phosphate and decreased glycogen. From 2 to 4 h, when the synthase activity remained elevated and the phosphorylase activity declined, glycogen levels increased (glycogen supercompensation). A further increase of glycogen up to 24 h did not correlate with the enzyme activities. Between 24 and 72 h, glycogen decreased to control values, possibly initiated by high phosphorylase activity at 24 h. At 12 and 24 h, the inverse relationship between glycogen concentration and the synthase activity ratio was lost, indicating that reloading transiently uncoupled glycogen control of this enzyme. These data suggest that the activities of glycogen synthase and phosphorylase, when measured at physiological effector levels, likely provide the closest approximation to the actual enzyme activities in vivo. Measurements made in this way effectively explained the majority of the changes in the soleus glycogen content during recovery from nonweight bearing.     

Metabolism of amino acids,protein and glucose in fat pads of traumatized rats

Soft tissue injury to one hindlimb of rats was used to test the response to trauma of metabolism in epididymal fat pads. Degradation of [1?¹⁴C] leucine was lower on day 2 after injury, but not on days 1 or 3, whether or not glucose or insulin were provided. Although trauma did not affect the basal rate of release of ¹⁴CO₂, lactate or pyruvate from fat pads incubated with [U?¹⁴C] glucose, the stimulation by insulin of these processes was smaller in fat pads of 2 day traumatized than of normal animals. These results suggest that trauma due to injury may decrease the capacity for utilization of leucine and glucose by adipose tissue. Release of alanine, glutamine and glutamate by gat pads incubated with leucine was also lower on day 2. This decreased efflux could not be accounted for by changes in net protein breakdown or in pyruvate availability and probably reflected their reduced $\text{de novo}$ synthesis due to the diminished release of nitrogen from leucine.     

In vitro inhibition activity of different bacteriocin-producing Escherichia coli against Salmonella strains isolated from clinical cases

Aims:  To compare in vitro the inhibitory activity of four bacteriocin-producing Escherichia coli to a well-characterized panel of Salmonella strains, recently isolated from clinical cases in Switzerland.
Methods and Results:  A panel of 68 nontyphoidal Salmonella strains was characterized by pulsed-field gel electrophoresis analysis and susceptibility to antibiotics. The majority of tested strains were genetically different, with 40% resistant to at least one antibiotic. E. coli Mcc24 showed highest in vitro activity against Salmonella (100%, microcin 24), followed by E. coli L1000 (94%, microcin B17), E. coli 53 (49%, colicin H) and E. coli 52 (21%, colicin G) as revealed using a cross-streak activity assay.
Conclusions:  Escherichia coli Mcc24, a genetically modified organism producing microcin 24, and E. coli L1000, a natural strain isolated from human faeces carrying the mcb -operon for microcin B17-production, were the most effective strains in inhibiting in vitro both antibiotic resistant and sensitive Salmonella isolates.
Significance and Impact of the Study:  Due to an increasing prevalence of antibiotic resistant Salmonella strains, alternative strategies to fight these foodborne pathogens are needed. E. coli L1000 appears to be a promising candidate in view of developing biotechnological alternatives to antibiotics against Salmonella infections.     

Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron‐reducing enrichment culture

Anaerobic benzene degradation was studied with a highly enriched iron‐reducing culture (BF) composed of mainly Peptococcaceae‐related Gram‐positive microorganisms. The proteomes of benzene‐, phenol‐ and benzoate‐grown cells of culture BF were compared by SDS‐PAGE. A specific benzene‐expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N‐terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI‐MS/MS‐based shotgun proteomic analysis revealed other specifically benzene‐expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate‐CoA ligase (BamY) of Geobacter metallireducens and 67% to 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (～17 kb) composed of carboxylase‐related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX).     

A protein kinase inhibitor, staurosporine, mimics nerve growth factor induction of neurotensin/neuromedin N gene expression.

Nerve growth factor (NGF) cooperates with glucocorticoids, activators of adenylate cyclase, and lithium to induce the expression of teh gene encoding the neuropeptides neurotensin and neuromedin N (NT/N gene) in PC 12 pheochromocytoma cells. High level expression requires simultaneous treatment with three or all four inducers. To examine the mechanism underlying this complex synergism, we have examined the effects of protein kinase inhibitors and other agents which influence intracellular signal transduction on NT/N gene expression. Two structurally similar bacterial alkaloids, staurosporine and K-252a, inhibit several protein kinases in vitro, including protein kinase C and cyclic nucleotide-dependent kinases. K-252a has been reported to specifically inhibit the effects of NGF on PC12 pheochromocytoma cells. Surprisingly, staurosporine in combination with other inducers markedly potentiated NT/N gene expression. In contrast, K-252a had no effect on NT/N gene expression when added simultaneously with other inducers. Expression of the NT/N gene was also potentiated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which directly activates protein kinase C, and by bradykinin, which stimulates phosphatidylinositol turnover in PC12 cells, and these effects were not blocked by staurosporine. Staurosporine was generally more effective in stimulating NT/N gene expression when used in inducer combinations that did not include NGF. These results, taken together with recent evidence that staurosporine is also able to induce neurite outgrowth from PC12 cells, suggest that the effects of staurosporine and NGF may converge, in part, on a common intracellular target.     

Provision of nestboxes raises the breeding density of Great Tits Parus major equally in coniferous and deciduous woodland

Nestbox provision is a technique used to increase nest-site availability for secondary cavity-nesting birds. However, little is known about the demographic consequences of nestbox provision in different habitat types. To assess how nestbox provision affects the density of hole-nesting birds simultaneously in two contrasting habitats, we compared the breeding density of Great Tits along transects without nestboxes with that in transects where nestboxes were provided. Although the initial density of breeders was considerably higher in the deciduous habitat than in the coniferous habitat, provision of nestboxes increased density by a similar number of additional pairs in each habitat type. Thus, the provision of nestboxes in managed coniferous forests may be as effective in increasing the breeding opportunities of cavity nesters as in deciduous stands. Moreover, previous research showed that pairs in deciduous habitat with nestboxes have consistently lower breeding success than those in coniferous habitat with nestboxes. It is possible that the addition of nestboxes in the preferred habitat increased density to such an extent that density-dependent effects became apparent.     

Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP

Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His₁₀-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.The incorporation of one atom of oxygen during hydroxylation, epoxidation, sulfoxidation, or Baeyer-Villiger oxidation is a common initial step of the aerobic degradation of aromatic compounds by microorganisms. In bacteria, these reactions are most frequently catalyzed by inducible flavoprotein monooxygenases (EC 1.14.13 [57]). The majority of these enzymes (so-called single-component flavoprotein monooxygenases) utilize electrons from NAD(P)H, which are transferred to a non-covalently bound flavin adenine dinucleotide (FAD) in order to activate molecular oxygen as a flavin (hydro)peroxide. Depending on the protonation of this intermediate and the type of substrate, an oxygen atom is then incorporated by nucleophilic or electrophilic attack. More recently, different two-component flavoprotein monooxygenases have been characterized (57). These systems cover an NAD(P)H-dependent flavin reductase in order to generate reduced flavin and an oxygenase that utilizes this cofactor for the activation of oxygen.The exquisite regio- and stereoselectivities of oxygen insertion by flavoprotein monooxygenases favor these enzymes for biocatalytic applications (23, 24, 33). This is especially true because chemical synthesis approaches by hetero- or homogenic catalysis often do not yield a sufficiently high enantiomeric excess for the production of pharmaceuticals and their chiral building blocks. The use of oxygen as an inexpensive nontoxic oxidant and mild reaction conditions are additional advantages with the potential for increasing the environmental sustainability of oxygenase-catalyzed biotransformations.The necessity for expensive cofactors is perhaps the most striking drawback limiting the industrial application of flavoprotein monooxygenases. Different electrochemical and enzymatic procedures for in vitro cofactor regeneration are available (20, 21, 32, 52, 56), but these systems are currently lacking long-term stability. As a consequence, the practical application of flavoprotein monooxygenases is virtually restricted to in vivo systems in which cofactor regeneration is mediated by the metabolism of the expression host (45, 49). The limitations of whole-cell-mediated biotransformations by substrate and/or product toxicity can be overcome by means of two-phase systems, as was recently shown for the two-component styrene monooxygenase (SMO) from Pseudomonas sp. strain VLB120 (45).Two-component flavoprotein monooxygenases present additional challenges for biocatalytic applications. The need for two separate protein components may hamper attempts at recombinant enzyme expression, the application of immobilized enzymes in cell-free systems, and the detection of novel oxygenases during activity-based metagenome-screening approaches. Moreover, and as was already shown for the two-component SMO from Pseudomonas sp. strain VLB120 (44), the interprotein transfer of reduced FAD is accompanied to a certain extent by the auto-oxidative formation of reactive oxygen species such as hydrogen peroxide (Fig. ​(Fig.1).1). Auto-oxidation of reduced FAD not only decreases the efficiency of the oxygenation process but also negatively interferes with the physiological conditions of the cell. The extent of oxidative stress is considerably increased when FAD oxidoreductase activity exceeds oxygenase activity and uncoupling becomes dominant.Open in a separate windowFIG. 1.Catalytic mechanism of two-component SMOs and the formation of oxidative stress by uncoupling between FAD oxidoreductase (StyB) and oxygenase (StyA) (adapted from reference 36). FAD_OX and FAD_RED, oxidized and reduced forms of FAD, respectively.Presently the details of reduced-FAD transfer between the oxygenase and FAD oxidoreductase components of SMOs are not known. Recent kinetic studies have indicated that reduced FAD is transferred by a mixed mechanism in which direct contact of both proteins and free diffusion of the reduced cofactor play a role (25). This hypothesis is supported by the work of Otto and coworkers in which the formation of hydrogen peroxide was shown to be reciprocally proportional to the concentration of active oxygenase StyA (44). The high level of efficiency of self-sufficient cytochrome P450 enzymes compared to that of multicomponent types is attributed to the closer location between the heme-containing P450 domain (oxygenase) and a reductase domain (FAD/flavin mononucleotide [FMN] and NADH binding site), which should also promote the efficiency of diffusive transfer (38). These self-sufficient P450 systems are of high biocatalytic interest (8, 34, 39), and it is likely that other types of self-sufficient monooxygenases (e.g., flavoenzymes) behave in a similar way.Members of the gram-positive genus Rhodococcus are characterized by their exceptionally high level of metabolic versatility toward a broad range of organic substrates (31). Large genome sizes, the presence of megaplasmids, and a distinct gene redundancy (multiple enzyme homologs) favor these organisms as a promising source for novel enzymes (59). Moreover, several studies have provided evidence of functionally convergent evolution of the catabolic activities of rhodococci and proteobacteria (12, 37). Since most research on bacterial catabolic activities has so far been performed on the latter group, novel enzymes and mechanisms are likely to be identified in gram-positive bacteria.The nocardioform actinobacterium Rhodococcus opacus 1CP was originally isolated from contaminated soil by enrichment with 4-chloro- and 2,4-dichlorophenol as the sole carbon sources (18). To solve questions related to the enzymes involved in chlorophenol mineralization, an R. opacus 1CP clone library was generated, leading to the identification of a 9-kb genomic fragment harboring genes with a presumed function in styrene metabolism. Sequence analysis indicated the presence of an open reading frame (ORF) encoding a fusion protein composed of an oxygenase and a reductase subunit with a high level of similarity to the corresponding subunits of two-component SMOs from pseudomonads. Recombinant expression and biochemical characterization confirmed that it has enantioselective styrene epoxidation ability and showed that StyA2B is the first representative of a new class of NADH- and flavin-dependent single-component flavoprotein monooxygenases.