Expression and characterization of recombinant human UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than other UGTs and was purified as an active dimeric enzyme

Eight human liver UDP-glucuronosyltransferases (UGTs) were expressed in baculovirus-infected insect cells as fusion proteins carrying a short C-terminal extension that ends with 6 histidine residues (His tag). The activity of recombinant UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B15 was almost fully inhibited by 0.2% Triton X-100. In the case of UGT1A9, however, glucuronidation of alpha-naphthol and scopoletin was resistant to such inhibition, whereas glucuronidation of entacapone and several other aglycones was sensitive. His-tagged UGT1A9 was purified by immobilized metal-chelating chromatography (IMAC). Purified UGT1A9 glucuronidated scopoletin at a high rate, whereas its glucuronidation activity toward entacapone was low and largely dependent on phospholipid addition. Recombinant UGT1A9 in which the His tag was replaced by hemagglutinin antigenic peptide (HA tag) was also prepared. Insect cells were co-infected with baculoviruses encoding both HA-tagged and His-tagged UGT1A9. Membranes from the co-infected cells, or a mixture of membranes from separately infected cells, were subjected to detergent extraction and IMAC, and the resulting fractions were analyzed for the presence of each type of UGT1A9 using tag-specific antibodies. In the case of separate infection, the HA-tagged UGT1A9 did not bind to the column. When co-infected with His-tagged UGT1A9, however, part of the HA-tagged enzyme was bound to the column and was eluted by imidazole concentration gradient together with the His-tagged UGT1A9, suggesting the formation of stable dimers that contain one His-tagged and one HA-tagged UGT1A9 monomers.     

H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl

H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.     

Evaluation of quantitative PCR and culture methods for detection of house dust fungi and streptomycetes in relation to moisture damage of the house

Aims: Microbial concentrations in vacuumed house dust samples (n = 71) were analysed by culture and quantitative polymerase chain reaction (qPCR) methods and their association with extent of moisture damage in the house was studied. Methods and Results: Microbial concentrations measured by qPCR correlated with concentrations obtained by culture method, but were orders of magnitude higher. qPCR also had better sensitivity. Concentrations of several microbes in house dust, determined with qPCR, were associated with the extent of moisture damage in the house. This association was strongest for Penicillium brevicompactum, one of the fungi detected in highest concentrations by qPCR. Furthermore, house dust concentrations of Wallemia sebi, Trichoderma viride, Cladosporium sphaerospermum, Eurotium amstelodami and the combined assay group for Penicillium spp., Aspergillus spp. and Paecilomyces variotii were significantly associated with the extent of the moisture damage. Conclusion: These species or assay groups could probably be used as indicators of moisture damage in the house. Significance and Impact of the Study: This finding indicates the benefits of the qPCR method, which is sensitive enough to reveal the differences in microbial concentrations of house dust between moisture‐damaged and undamaged houses.     

Phylogeography of the ant Myrmica rubra and its inquiline social parasite

Widely distributed Palearctic insects are ideal to study phylogeographic patterns owing to their high potential to survive in many Pleistocene refugia and-after the glaciation-to recolonize vast, continuous areas. Nevertheless, such species have received little phylogeographic attention. Here, we investigated the Pleistocene refugia and subsequent postglacial colonization of the common, abundant, and widely distributed ant Myrmica rubra over most of its Palearctic area, using mitochondrial DNA (mtDNA). The western and eastern populations of M. rubra belonged predominantly to separate haplogroups, which formed a broad secondary contact zone in Central Europe. The distribution of genetic diversity and haplogroups implied that M. rubra survived the last glaciation in multiple refugia located over an extensive area from Iberia in the west to Siberia in the east, and colonized its present areas of distribution along several routes. The matrilineal genetic structure of M. rubra was probably formed during the last glaciation and subsequent postglacial expansion. Additionally, because M. rubra has two queen morphs, the obligately socially parasitic microgyne and its macrogyne host, we tested the suggested speciation of the parasite. Locally, the parasite and host usually belonged to the same haplogroup but differed in haplotype frequencies. This indicates that genetic differentiation between the morphs is a universal pattern and thus incipient, sympatric speciation of the parasite from its host is possible. If speciation is taking place, however, it is not yet visible as lineage sorting of the mtDNA between the morphs.     

Polyamine flux analysis by determination of heavy isotope incorporation from 13C, 15N-enriched amino acids into polyamines by LC-MS/MS

The study of polyamine flux, i.e. the circulating flow of polyamines through the interconnected biosynthetic and catabolic pathways, is of considerable interest because of the established links between the polyamine metabolism and many diseases, such as cancer and diabetes. To study polyamine flux in detail, a novel method based on following the label incorporation from the (13)C, (15)N-labeled polyamine precursors, arginine, methionine and ornithine, into polyamines by LC-MS/MS was implemented. This methodology was tested on three distinct cell lines with different spermidine/spermine-N (1)-acetyltransferase (SSAT) expression levels, i.e. non-transgenic, transgenic and knockout. These trials allowed the identification of the critical conditions for the successful polyamine flux measurement, such as the functional time frame of label incorporation, until plateau phase with the selected precursor is reached. The novel LC-MS/MS-based method for polyamine flux overcame the limitations of previous existing methodologies, with baseline separation of the different polyamine species and the exact quantification of the incorporated label. Moreover, the obtained results clearly show that the increased SSAT expression is associated with accelerated polyamine flux.     

How do food quality and larval crowding affect performance of the autumnal moth,Epirrita autumnata?

Outbreak densities of autumnal moth, Epirrita autumnata (Borkhausen) (Lepidoptera: Geometridae), lead to high larval crowding. Phenotypic responses of E. autumnata to larval crowding and to food quality were studied by measuring growth and consumption as well as pupal weight and fecundity. Crowding may trigger increased consumption and faster development to avoid impending food shortage on good quality food. This is suggested by the result that on a good‐quality diet, the growth of crowded larvae was better than that of solitary larvae, though they did not consume more food than solitary larvae. Crowded larvae also completed the last instar earlier than solitary larvae. The fecundity of crowded autumnal moths was not lower than the fecundity of solitarily grown autumnal moths. This may provide conditions for extra rapid population build‐up of E. autumnata. During the population increase phase the crowding effect may facilitate larval performance; however, at peak density the crowding starts to have negative effects on the performance of larvae. On a poor‐quality diet, the performance of crowded and solitary larvae did not differ. The growth of larvae was better on a good‐quality diet than on a poor‐quality diet, due to higher efficiency in food utilization. Larvae feeding on low‐quality diet did not prolong their development time, but pupated at smaller size; this resulted in lower fecundity. A decrease in food quality can be seen as a cue of oncoming food shortage and resource depletion; it may be advantageous to pupate at a smaller size and ensure survival till reproduction, rather than risk prolonging development to achieve larger size and higher fecundity.     

Axillary shoot proliferation of blue honeysuckle

Callus cultures of Ajuga reptans flowers produced a complex mixture of cyanidin- and delphinidin-based pigments, of which more than 90% were acylated. The anthocyanin composition varied little during one growth period. During a time span of 5 years no new anthocyanin classes appeared. Quantitative differences in anthocyanin composition between the callus lines and during a 5 year time span were more pronounced. In general, the accumulation of delphinidin-based anthocyanins decreased. The percentage of acylated anthocyanins was stable in time. The accumulation of metabolically evolved anthocyanins (5′-substituted and acylated) decreased during passage from solid culture to liquid culture. The accumulation of acylated anthocyanins was influenced by the type of aeration in liquid cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Activity and stability of human kallikrein-2-specific linear and cyclic peptide inhibitors.

Human glandular kallikrein (KLK2) is a highly prostate-specific serine protease, which is mainly excreted into the seminal fluid, but part of which is also secreted into circulation from prostatic tumors. Since the expression level of KLK2 is elevated in aggressive tumors and it has been suggested to mediate the metastasis of prostate cancer, inhibition of the proteolytic activity of KLK2 is of potential therapeutic value. We have previously identified several KLK2-specific linear peptides by phage display technology. Two of its synthetic analogs, A R R P A P A P G (KLK2a) and G A A R F K V W W A A G (KLK2b), show specific inhibition of KLK2 but their sensitivity to proteolysis in vivo may restrict their potential use as therapeutic agents. In order to improve the stability of the linear peptides for in vivo use, we have prepared cyclic analogs and compared their biological activity and their structural stability. A series of cyclic variants with cysteine bridges were synthesized. Cyclization inactivated one peptide (KLK2a) and its derivatives, while the other peptide (KLK2b) and its derivatives remained active. Furthermore, backbone cyclization of KLK2b improved significantly the resistance against proteolysis by trypsin and human plasma. Nuclear magnetic resonance studies showed that cyclization of the KLK2b peptides does not make the structures more rigid. In conclusion, we have shown that backbone cyclization of KLK2 inhibitory peptides can be used to increase stability without losing biological activity. This should render the peptides more useful for in vivo applications, such as tumor imaging and prostate cancer targeting.     

Genetic differentiation between the ant Myrmica rubra and its microgynous social parasite

Hymenopteran inquiline species have been proposed to originate by sympatric speciation through intraspecific social parasitism. One such parasite, Myrmica microrubra, was recently synonymized with its Myrmica rubra host, because comparisons across Europe indicated insufficient genetic differentiation. Here, we use microsatellite markers to study genetic differentiation more precisely in a sample of Finnish M. rubra and its inquilines collected at two localities, supplemented with mitochondrial DNA sequences. The parasite had much lower genetic variation than the host at three of the four loci studied. Genetic differentiation between the host populations was moderate (F _ST = 0.089), whereas the parasite populations were more strongly subdivided (F _ST = 0.440). The host and parasite were highly genetically differentiated both across populations (F _ST = 0.346) and in strict sympatry (0.327, 0.364), a result that remained robust both in a haplotype network and in PCA ordination. Individual assignments of genotypes indicated that gene flow between sympatric host and inquiline populations is reduced by about an order of magnitude relative to the gene flow within the morphs. Our results suggest that the parasitic morph of M. rubra may be an incipient species, but it remains unclear to what extent the observed genetic differentiation between host and inquiline is due to possible assortative mating and selection against hybrids or to recurrent bottlenecking and genetic drift. We conclude that an explicitly functional species concept would be unambiguous in treating this inquiline as a full species, as it begets its own kind and maintains its integrity in spite of occasional interbreeding with the host.     

Breeding success and lutein availability in great tit (Parus major)

The relationship among temporal variation in the availability of carotenoid-rich food, tissue carotenoid levels and breeding success are poorly known. We studied how diet quality and quantity affect the carotenoid profile and fledging success of great tit (Parus major) nestlings along a pollution gradient. We found declining seasonal trend in lutein concentration of caterpillars, which may be the explanation for the declining trend in nestlings' lutein concentration of plasma with season, despite the increase in caterpillar biomass. This may be because the biomass of most lutein-rich caterpillars (autumnal moths) decreased and less lutein-rich caterpillars (sawflies) increased during the breeding season. The temporal difference in occurrence of different caterpillar species means that peak lutein availability does not coincide with peak caterpillar abundance. However the positive association between total larval biomass and the number of great tit fledglings may suggest that fledging success depends more on total caterpillar availability than on lutein concentration of caterpillars.