Energy metabolism of Inuit sled dogs

We explored how seasonal changes in temperature, exercise and food supply affected energy metabolism and heart rate of Inuit dogs in Greenland. Using open flow respirometry, doubly labeled water, and heart rate recording, we measured metabolic rates of the same dogs at two different locations: at one location the dogs were fed with high energy food throughout the year while at the other location they were fed with low energy food during summer. Our key questions were: is resting metabolic rate (RMR) increased during the winter season when dogs are working? Does feeding regime affect RMR during summer? What is the proportion of metabolic rate (MR) devoted to specific dynamic action (SDA), and what is the metabolic scope of working Inuit sled dogs? The Inuit dogs had an extremely wide thermoneutral zone extending down to ?25°C. Temperature changes between summer and winter did not affect RMR, thus summer fasting periods were defined as baseline RMR. Relative to this baseline, summer MR was upregulated in the group of dogs receiving low energy food, whereas heart rate was downregulated. However, during food digestion, both MR and HR were twice their respective baseline values. A continuously elevated MR was observed during winter. Because temperature effects were excluded and because there were also no effects of training, we attribute winter elevated MR to SDA because of the continuous food supply. Working MR during winter was 7.9 times the MR of resting dogs in winter, or 12.2 times baseline MR.     

Bacterial and nonbacterial expression of wild-type and mutant human phospholipid hydroperoxide glutathione peroxidase and purification of the mutant enzyme in the milligram scale

15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes is essential for normal cell function. Glutathione peroxidases contain selenocysteine as catalytically active amino acid and this selenocysteine is encoded by a TGA stop codon. Detailed protein chemical investigations on phospholipid hydroperoxide glutathione peroxidases and crystal trials have been hampered in the past by limited protein supply. There is no efficient natural source for large-scale enzyme preparation and overexpression of the functional protein in recombinant systems has not been reported so far. To avoid problems with recognition of the selenocysteine stop codon we mutated the selenocysteine to a cysteine and expressed the Sec46Cys mutant in milligram amounts in the baculovirus/insect cell system and as His-tag fusion protein in Escherichia coli. The recombinant enzyme species were purified by conventional fast protein liquid chromatography (nonfusion protein) or by affinity chromatography on a nickel matrix (His-tag protein). Surprisingly, we found that both protein variants were functional although their specific activities were reduced when compared with the wild-type enzyme. Basic protein chemical and enzymatic properties of the purified enzyme species were determined and monoclonal antibodies which recognize the native phospholipid hydroperoxide glutathione peroxidases were raised using our enzyme preparations as antigen. The described strategies for overexpression of mutant phospholipid hydroperoxide glutathione peroxidase species and their purification from recombinant sources provide sufficient amounts of enzyme for future protein chemical investigations and detailed crystal trials.     

Free radical scavenging activity and secondary metabolites from in vitro cultures of Sanicula graveolens

An in vitro propagation system was developed to obtain shoot and root cultures from the Andean spice Sanicula graveolens (Apiaceae). Propagation of shoots, roots and plantlets was achieved by the temporary immersion system. The free radical scavenging effect of the methanol/water (7:3 v/v) extracts was determined by the discoloration of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). Total phenolic, flavonoid, chlorogenic acid (CA) and quercetin 3-O-glucoside content in the samples was assessed by spectrophotometry and DAD-HPLC analysis, respectively. On a dry weight basis, the crude extracts showed total phenolic values ranging from 3.57 to 6.93%, with highest content for the root culture sample. Total flavonoid content ranged from 1.23 to 2.23% and was lower for the root culture. Chlorogenic acid and neochlorogenic acid were identified by TLC in all samples. Highest free radical scavenging effect was observed for the root culture which also presented the highest CA content. Two of the shoot culture samples, with similar IC50 values in the DPPH discoloration assay, also presented close quercetin-3-O-glucoside content.     

Identification of ςB-Dependent Genes in Bacillus subtilis Using a Promoter Consensus-Directed Search and Oligonucleotide Hybridization

A consensus-directed search for sigma(B) promoters was used to locate potential candidates for new sigma(B)-dependent genes in Bacillus subtilis. Screening of those candidates by oligonucleotide hybridizations with total RNA from exponentially growing or ethanol-stressed cells of the wild type as well as a sigB mutant revealed 22 genes that required sigma(B) for induction by ethanol. Although almost 50% of the proteins encoded by the newly discovered sigma(B)-dependent stress genes seem to be membrane localized, biochemical functions have so far not been defined for any of the gene products. Allocation of the genes to the sigma(B)-dependent stress regulon may indicate a potential function in the establishment of a multiple stress resistance. AldY and YhdF show similarities to NAD(P)-dependent dehydrogenases and YdbP to thioredoxins, supporting our suggestion that sigma(B)-dependent proteins may be involved in the maintenance of the intracellular redox balance after stress.     

Two soluble glycosyltransferases glycosylate less efficiently in vivo than their membrane bound counterparts

Many Golgi glycosyltransferases are type II membrane proteins which are cleaved to produce soluble forms that are released from cells. Cho and Cummings recently reported that a soluble form of alpha1, 3- galactosyltransferase was comparable to its membrane bound counterpart in its ability to galactosylate newly synthesized glycoproteins (Cho,S.K. and Cummings,R.D. (1997) J. Biol. Chem., 272, 13622-13628). To test the generality of their findings, we compared the activities of the full length and soluble forms of two such glycosyltransferases, ss1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/ GA2 synthase; GalNAcT) and beta galactoside alpha2,6 sialyltransferase (alpha2,6-ST; ST6Gal I), for production of their glycoconjugate products in vivo . Unlike the full length form of GalNAcT which produced ganglioside GM2 in transfected cells, soluble GalNAcT did not produce detectable GM2 in vivo even though it possessed in vitro GalNAcT activity comparable to that of full length GalNAcT. When compared with cells expressing full length alpha2,6-ST, cells expressing a soluble form of alpha2,6-ST contained 3-fold higher alpha2,6-ST mRNA levels and secreted 7-fold greater alpha2,6-ST activity as measured in vitro , but in striking contrast contained 2- to 4-fold less of the alpha2,6-linked sialic acid moiety in cellular glycoproteins in vivo . In summary these results suggest that unlike alpha1,3-galactosyltransferase the soluble forms of these two glycosyltransferases are less efficient at glycosylation of membrane proteins and lipids in vivo than their membrane bound counterparts.      

Host genotype and genetic diversity shape the evolution of a novel bacterial infection

Pathogens continue to emerge from increased contact with novel host species. Whilst these hosts can represent distinct environments for pathogens, the impacts of host genetic background on how a pathogen evolves post-emergence are unclear. In a novel interaction, we experimentally evolved a pathogen (Staphylococcus aureus) in populations of wild nematodes (Caenorhabditis elegans) to test whether host genotype and genetic diversity affect pathogen evolution. After ten rounds of selection, we found that pathogen virulence evolved to vary across host genotypes, with differences in host metal ion acquisition detected as a possible driver of increased host exploitation. Diverse host populations selected for the highest levels of pathogen virulence, but infectivity was constrained, unlike in host monocultures. We hypothesise that population heterogeneity might pool together individuals that contribute disproportionately to the spread of infection or to enhanced virulence. The genomes of evolved populations were sequenced, and it was revealed that pathogens selected in distantly-related host genotypes diverged more than those in closely-related host genotypes. S. aureus nevertheless maintained a broad host range. Our study provides unique empirical insight into the evolutionary dynamics that could occur in other novel infections of wildlife and humans.Subject terms: Molecular evolution, Bacterial evolution, Bacterial genetics