Metabolism,cellular actions,and cytotoxicity of selenomethionine in cultured cells

Selenomethionine metabolism and the biochemical basis for its cytotoxicity were analyzed in cultured human and murine lymphoid cells. The metabolic pathways were also addressed, using purified mammalian enzymes and crude tissue extracts. Selenomethionine was found to be effectively metabolized to S-adenosylmethionine analog, and that analog was further metabolized in transmethylation reactions and in polyamine synthesis, similarly to the corresponding sulphur metabolites of methionine. Selenomethionine did not block these pathways, nor was there a specific block on the synthesis of DNA, RNA, or proteins when added to the culture medium. Selenomethionine showed cytotoxicity at above 40 microM levels. Yet, low selenomethionine levels (10 microM) could replace methionine and support cell growth in the absence of methionine. Selenomethionine toxicity took place concomitantly with changes in S-adenosylmethionine pools. D-form was less cytotoxic than L-form. Methionine concentration modified the cytotoxicity. Together, this indicates that selenomethionine uptake and enzymic metabolism are involved in the cytotoxicity in a yet unknown way.     

Why do pied flycatcher females mate with already-mated males?

The contribution of the pied flycatcher Ficedula hypoleuca male in parental care was studied to examine why some females mate with already-mated males. No difference in feeding rate was found between older and yearling monogamous males, when comparing nests at the same time. Monogamous and primary females were helped significantly more in parental care by the male than were secondary females of polygynous males. Females could only partly compensate for the absence of a male and of nestlings were reduced in nests with low male assistance. Differences in mate and territory quality were far too slight to make it advantageous for females to choose already-mated males instead of mating with monogamous males. We suggest that males, by being polyterritorial, deceive females into accepting polygyny; and females can be deceived since they do not have time to find out the marital status of males.     

Differential proteome and cellular adhesion analyses of the probiotic bacterium Lactobacillus acidophilus NCFM grown on raffinose – an emerging prebiotic

Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT‐29 cells increased three‐fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D‐DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress‐related protein increasing in a range of +2.1 ? +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to ?2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α‐galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β‐galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host.     

Increase in CD30 ligand/CD153 and TNF-α expressing mast cells in basal cell carcinoma

Mast cells are a significant source of tumor necrosis factor (TNF) superfamily members, such as TNF-α, CD30 ligand/CD153 (CD30L) and CD40L/CD154. Furthermore, the expression of some of these proteins in mast cells has been associated with tumorigenesis, and mast cells have been found to be increased in number in the basal cell carcinoma (BCC) lesion. In this study, we have examined the expression of TNF-α, CD30L and CD40L immunoreactivity in mast cells in the healthy-looking skin and lesional skin of ten patients with superficial spreading BCC. Also, the counterparts of these molecules, TNF receptor (TNFR) I and II as well as CD30 and CD40, were analysed immunohistochemically. We found that numbers of mast cells and Kit-positive cells were significantly increased in the dermal BCC lesion. The percentage of CD30L-positive mast cells and the number of CD30-positive cells were significantly increased in the upper dermis of the BCC lesion as well. In addition, the numbers of TNF-α-positive mast cells and cells with TNFRI and TNFRII were markedly increased in the upper lesional dermis. In contrast, no mast cells positive for CD40L could be detected, even though the lesional dermis contained increased numbers of CD40 positive cells. The BCC epithelium was positive for TNFRI, TNFRII and CD40, but not for CD30, though the larger basal buds appeared to be less intensely stained for TNFRI and CD40. In conclusion, mast cells positive for CD30L and TNF-α, but not CD40L, are increased in number in the lesional dermis in BCC. These data suggest plausible pathways whereby mast cells can be activated and to interact with other cells and thereby contribute to the tumorigenesis in BCC.     

Mapping of structural determinants for the oligomerization of p58, a lectin-like protein of the intermediate compartment and cis-Golgi.

Shortly after synthesis, p58, the rat homologue of the mannose-binding lectin ERGIC-53/MR60, which localizes to pre-Golgi and cis-Golgi compartments, forms dimers and hexamers, after which an equilibrium of both forms is reached. Mature p58, a type I membrane protein, contains four cysteine residues in the lumenal domain which are capable of forming disulphide bonds. The membrane-proximal half of the lumenal domain consists of four predicted alpha-helical domains, one heavily charged and three amphipathic in nature, all candidates for electrostatic or coiled-coil interactions. Using single-stranded mutagenesis, the cysteines were individually changed to alanines and the contribution of each of the alpha-helical domains was probed by internal deletions. The N-terminal cysteine to alanine mutants, C198A and C238A and the double mutant, C198/238A, oligomerized like the wild-type protein. The two membrane-proximal cysteines were found to be necessary for the oligomerization of p58. Mutants lacking one of the membrane proximal cysteines, either C473A or C482A, were unable to form hexamers, while dimers were formed normally. The C473/482A double mutant formed only monomers. Deletion of any of the individual alpha-helical domains had no effect on oligomerization. The dimeric and hexameric forms bound equally well to D-mannose. The dimeric and monomeric mutants displayed a cellular distribution similar to the wild-type protein, indicating that the oligomerization status played a minimal role in maintaining the subcellular distribution of p58.     

Sexual selection in the wolf spider Hygrolycosa rubrofasciata: female preference for drum duration and pulse rate

The unusual form of sexual signaling, the drumming producedby the wolf spider Hygrolycosa rubrofasciata, allows exceptionallydetailed studies of female preference patterns against signalcharacteristics. It is easy to manipulate the signals and touse large numbers of females in playback experiments. Malesof H. rubrofasciata produce drums by striking their abdomenagainst dry leaves on the ground. Drums travel not only as substrate-borne vibrations, but also as airborne acoustic signals.Females respond sooner to drums transferred as substrate borne,but the mode of signal transfer has no effect on female preferencefor different types of drums. We investigated the effects oftwo key components of the drums, duration and pulse rate, onfemale choice. Previous studies have shown that the durationof the drum is both repeatable within males and variable betweenmales. In contrast, pulse rate shows high within-male repeatabilitybut only little variability between males. Using playbacksof manipulated drums, we found that females preferred drumsof longer duration but that pulse rate had no effect on femalepreference. Our results suggest that drum duration may functionas an indicator of male quality for choosy females. Pulse rate,on the other hand, is less likely to be an important traitin intersexual selection. Female preference for drum durationwas open-ended within the natural variation of the drum durations,but it leveled off outside the normal range. Thus supernomalstimuli would not pay for males using this energetically demanding acoustic signal.     

Can ultraviolet cues function as aposematic signals?

The fact that birds are sensitive to ultraviolet light (UV,320-400 nm) has been largely ignored by previous studies ofaposematism. Therefore, in the present article we investigatedwhether great tits preferred ultraviolet-reflecting colorscompared to colors without UV reflection and whether UV cuesalone could function as aposematic signals. We were able to manipulate prey visibility in UV light by changing the UV reflectanceof prey items as well as altering the lighting conditions.In order to perform a preference experiment we used three pairsof colors (green+UV vs. green, gray+UV vs. gray, yellow+UVvs. yellow) on a black background. The birds ate both UV typesequally for all three colors. Thus, there was no avoidance of the UV-reflecting prey. Next we tested the possibility thatUV reflection may affect avoidance learning. We used eithergreen or green+UV as a signal for unpalatability. In this set-upthe difference in UV did not allow avoidance learning to occur.Our experiment with great tits does not support the hypothesisthat UV cues alone might work effectively as aposematic signals.     

Phospholipases A2 in the reproductive system of the bull

1. Phospholipase A2 activities were studied in the reproductive organs, seminal plasma and spermatozoa of adult bulls. 2. Phosphatidylethanolamine and phosphatidylcholine with 14C-labelled linoleic (lino-PE, lino-PC) or arachidonic acid (ara-PE, ara-PC) at sn-2 position as well as a fluorescent derivative (4-pyrenylbutyric acid) of phosphatidylcholine (PPC) were used as substrates. 3. The radioactive substrates were hydrolysed most strongly by homogenates of the prostate and Cowper's gland, but also seminal vesicle and its secretory fluid, seminal plasma and ejaculated spermatozoa contained hydrolytic activity. The fluorescence substrate was most strongly hydrolysed by homogenates of ampulla and seminal vesicle as well as its secretory fluid, seminal plasma and ejaculated spermatozoa. 4. Seminal plasma and seminal vesicle fluid contained a Ca2(+)-independent enzyme (enzyme I), which hydrolysed only PPC, while another Ca2(+)-dependent enzyme (enzyme II) hydrolysed only the radioactive substrates. 5. Both enzymes were purified from the seminal vesicle fluid and their biochemical properties were analysed. In SDS-PAGE enzyme I preparation resulted in two major bands with molecular weights of 16,000 and 60,000 in equal quantities and minor band at 15,000. The binding of the enzyme I to Con A-Sepharose indicated that it is a glycoprotein and it had multiple pI-values from 3.75 to 5.0. Enzyme II gave in SDS-PAGE two closely located bands with molecular weights of about 15,000 and 16,000 (major band). Isoelectric focusing showed one band at pI 4.7. Both enzymes appear to bind to spermatozoa at ejaculation but their function remains to be shown.     

The effect of lignin model compound structure on the rate of oxidation catalyzed by two different fungal laccases

Laccases (EC 1.10.3.2) are multicopper oxidases able to oxidize various substrates, such as phenolic subunits of lignin. The substrate range can be widened to non-phenolic units by the use of mediators. Since discovery of the laccase-mediator system, direct reactions of lignin and laccase without mediated electron-transfer have gained much less attention. The objective of this study was to investigate lignin as a substrate for fungal laccases by using lignin model compounds. These model compounds contained guaiacylic and syringylic moieties and also compounds of guaiacylic origin at a higher oxidation level. Some of these compounds are commercially available, but most of them were synthesized. The oxidation reaction rates of the lignin model compounds were studied by monitoring consumption of the co-substrate oxygen, in reactions catalyzed by laccases from two different fungi; Melanocarpus albomyces and Trametes hirsuta, possessing different molecular and catalytic properties. These reaction rate studies were compared to physicochemical properties of the lignin model compounds: relative redox potentials determined using cyclic voltammetry and pK_a-values. Docking of syringylic and biphenylic compounds to the active sites of both laccases was performed and the resulting model complex structures were used to further interpret the reaction rate results. Reaction rates of laccases are mainly affected by the ability of a lignin model compound to be oxidized and the pK_a-value of the substrate seems to be less important. As a consequence, syringylic compounds are oxidized with the highest rates and compounds at a higher oxidation level and redox-potential, such as vanillin, are oxidized at a much lower rate. Both guaiacylic and syringylic type compounds fit well in the active sites of both laccases. Only for a biphenylic compound, steric clashes were observed, and they are likely to have an effect on the reaction rate. When the oxidation rates on the selected model compounds with the two different laccases were compared, the redox-potential difference between laccases T1 copper and the lignin model compound (ΔE) was not the only property that determined the oxidation rate. In the case of lignin model substrates, also the selectivity of a specific laccase, reflected in the k_cat/K_m value, plays an important role.     

Maternal antibodies in a wild altricial bird: effects on offspring immunity, growth and survival

1. In many animals immunity is not fully developed until adulthood but the young still need protection against various sets of pathogens. Thus, bird nestlings are highly dependent on antibodies received from their mother (in the eggs) during their rapid early growth period. The relationship between maternal immunity and the development of neonates' own immunity has been poorly studied. 2. It has been suggested that immune function plays an important part in mediating resource competition between different life-history traits, e.g. growth and reproduction. Maternal investment of antibodies has potentially permanent effects on offspring phenotype. Thus, the trade-offs between the immune function and other important life-history traits in the offspring will also affect the fitness of the mother. 3. Our supplemental feeding experiment in the magpie Pica pica indicates that the immunoglobulin levels of offspring at hatching are dependent on a mother's nutritional condition. In addition, the amount of maternal immunoglobulins transferred to offspring increases along the laying order within a nest. 4. We also found that at the age of 8-10 days the immunoglobulin production of the offspring has already begun. Furthermore, the maternal immunoglobulin levels of the offspring at hatching were positively related to their immunoglobulin levels on day 10. 5. Maternal immunoglobulins did not significantly affect offspring growth, but there was a negative relationship between self-produced immunoglobulins and growth over the first 10 days, indicating a trade-off between these traits. Nestlings' weight, however, had a positive relationship with immunoglobulin production suggesting that the observed trade-off between growth and immunoglobulin production is due to catch-up growth of nestlings with a low hatching weight. We found that within nests nestlings with higher maternal antibody levels had higher survival rate until day 20, but between nests there was an opposite relationship. 6. Evidently, there is a trade-off, in magpies, between maternal resources, immune function and growth, shaping the evolution of maternal investment in offspring immunity.