Cloning of the glycerol kinase gene of Bacillus subtilis

A 3.5 kb fragment of Bacillus subtilis DNA which contains wild type alleles of mutations in glpK (glycerol kinase) and glpD (glycerol-3-phosphate [G3P] dehydrogenase) was cloned in plasmid pHV32 in Escherichia coli. The cloned fragment expresses glycerol kinase in B. subtilis mutants carrying the mutations glpK11 and recE4 after induction with glycerol or G3P whereas it does not express G3P dehydrogenase. The cloned fragment thus contains the complete glpK but probably only part of glpD.     

Acquired temperature-sensitive paralysis as a biomarker of declining neuronal function in aging Drosophila

General locomotor activity decreases with normal aging in animals and could be partially explained by decreases in neuronal function. Voltage-gated Na⁺ channels are essential in initiating and propagating rapid electrical impulses underlying normal locomotor activity and behavior in animals. Isolation of mutations conferring temperature-sensitive (ts) paralysis has been an extremely powerful paradigm for identifying genes involved in neuronal functions, such as membrane excitability and synaptic transmission. For instance, decreased expression of wild-type Na⁺ channels in flies harboring the no-action-potential ( nap ) mutant allele ( mle^napts ) confers rapid and reversible ts paralysis, because of failure of action potential propagation. Here, we report that aging wild-type Drosophila gradually develops an acquired susceptibility to ts paralysis that is indistinguishable from that seen in young ts paralytic mle^napts mutants. Moreover, we show that this general age-dependent susceptibility is also present in mle^napts flies, although the effects are shifted to lower temperature regimes. The mle^napts flies also exhibit decreased lifespan and increased frailty. Paralysis and decreased lifespan of mle^napts flies were partially rescued by increasing the dosage of para , the structural gene for the major action potential Na⁺ channel in central nervous system of Drosophila . Lastly, we show a dramatic scaling of ts paralysis susceptibility with chronological age in short-lived and long-lived mutant flies, further demonstrating that this age-dependent risk is independent of genetic background. Thus, decreased neural transmission, a hallmark of which is ts paralysis, is a biomarker of aging.     

Decomposition of spruce litter needles of different quality by Setulipes androsaceus and Thysanophora penicillioides

Different components of functional biodiversity, such as functional type richness and composition, have been reported to affect the decomposition of litter mixtures. In spite of the numerous reports of these effects, mechanisms underlying patterns of decomposition in litter mixtures are still unclear. We analyzed whether mixture decomposition was affected by: (a) the number of species in the mixture (mixture richness); and (b) the mixture’s functional composition (% of fast- vs. slow-decomposing species included in the mixture). We then tested if variation between observed and expected values of decomposition in mixtures was associated to: (c) the initial litter characteristics of the component species (initial nitrogen, lignin, cellulose and hemicellulose content of litters); and (d) the chemical heterogeneity of the mixtures (variation in the same chemical compounds between the components in each mixture). When up to 5 species representing different functional types were included, both species richness and functional composition showed statistically significant non-additive, and in general positive, effects on litter mixture decomposition. The positive effect of mixture richness on decomposition did not disappear, but was much less marked, when considering mixture with slow-decomposing species only. Although the main driver of decomposition in a mixture is still the average decomposability of the component species (itself largely determined by litter quality), the species interactions in a mixture add a consistent source of variability that is worth considering when predicting the decomposability of a given mixture. We showed that a greater positive difference between observed decomposition rates and that expected from its component species alone was found in mixtures with higher mean nitrogen content and a higher heterogeneity in non-labile compounds. Our results offer quantitative proof that litter chemical heterogeneity, as well as the mean quality of the mixture, can affect the decomposability in litter mixtures.     

Age-dependent changes in structure and function of the male labial gland in Bombus terrestris

The cephalic region of the labial gland in the buff-tailed bumblebee, Bombus terrestris, consists of numerous acini (formed by associated secretory cells and a central lumen) and connecting ducts. Age-dependent changes in secretion production (both qualitative and quantitative) are associated with changes in the amount of rough endoplasmic reticulum (RER), Golgi apparatus, and smooth endoplasmic reticulum (SER). The main secretory organelle is RER in the youngest individuals (pharate, and less-than-a-day old males), Golgi apparatus in 1-day-old males, and SER in males older than 2 days. Secretory cell death starts at 5 days of age, with maximal longevity to 10 days. Pheromone production starts immediately after eclosion, with pheromone quantities increasing until day 7. 2,3-dihydrofarnesol, the main component of the male-marking pheromone, appears in 1-day-old male glands, and reaches a maximum at 7 days of age, when its presence in the gland starts to decrease gradually. Older glands contain compounds not present in young ones. Variation in pheromone quantity and composition are reflected sensitively in the response of the queen antennae. Though queen antennae responded to gland extracts of all ages examined, maximum sensitivity was observed in response to extracts of glands 2-10 days old, while extracts of older glands gradually lose their effectiveness. Both major and minor components of the labial gland secretion extract elicited queen antennal responses suggesting that the pheromone is a multicomponent blend. Age-dependent changes in pheromone production, accumulation and tuning of pheromone activity are all synchronized approximately with male flight from the hive.     

The comparison of biophysical properties of DB-67 and its ester DB-67-4ABTFA determined by fluorescence spectroscopy methods

Fluorescence spectroscopy methods are applied to the study of camptothecin analogue DB-67 and its ester DB-67-4ABTFA (trifluoroacetic acid salt of 20(S)-aminobutyrate substituted DB-67). Camptothecin and many of its analogues exhibit anticancer properties. They are fluorescent compounds, so using the method of fluorescence anisotropy measurements and fluorescence spectra recording many biophysical properties can be determined including affinity to proteins and membranes. One can also observe the process of conversion of the ester into DB-67. Active lactone form of camptothecin in fluids at pH 7.4 hydrolyses and converts into inactive carboxylate. Process of camptothecin deactivation is accelerated in plasma and after about 2h the total conversion to carboxylate form occurs. It is caused by fast and irreversible binding of carboxylate form to the human serum albumin (HSA). Camptothecin carboxylate bound to HSA does not lactonise. On the other hand, camptothecin lactone binding to membranes is reversible, but as long as lactone form bound to membranes does not hydrolyse. Knowledge of binding properties to proteins and membranes permits to select among many camptothecin analogues the ones exhibiting desirable behavior in physiological conditions: high affinity of lactone form to membranes and low affinity of carboxylate form to albumin. The studied DB-67 and DB-67-4ABTFA fulfill these requirements.     

The HSA affinity of warfarin and flurbiprofen determined by fluorescence anisotropy measurements of camptothecin

The determination of affinity of warfarin and flurbiprofen to human serum albumin (HSA) by fluorescence anisotropy measurements of carboxylate form of camptothecin (CPT-C) is the subject of this paper. A simple method based on measurements of fluorescence anisotropy of CPT-C allows to determine the affinity constant of CPT-C to HSA by computation of the fraction of bound CPT-C molecules with HSA It was observed, that adding of competing drug to plasma significant reduces the rate of increase of CPT-C fluorescence anisotropy with increase of albumin concentration and, the affinity constant of CPT-C to HSA decreases. The hypothesis of interactions between competing drug and CPT-C is presented. The results of these studies suggest that CPT-C displaces other drug from protein binding site and the degree of this displacement depends on concentration of drug and drug-HSA binding affinity. The presented in this paper biosystems research allows to estimate the affinity constant of warfarin and flurbiprofen. It was also confirmed that despite that most of drugs bind predominantly to Site I or Site II of HSA (only one of these sites is high-affinity site), at elevated concentrations, part of drug molecules can be bound to low-affinity site of HSA.     

A phylogenetic delimitation of the "Sphagnum subsecundum complex" (Sphagnaceae, Bryophyta)

A seemingly obvious but sometimes overlooked premise of any evolutionary analysis is delineating the group of taxa under study. This is especially problematic in some bryophyte groups because of morphological simplicity and convergence. This research applies information from nucleotide sequences for eight plastid and nuclear loci to delineate a group of northern hemisphere peat moss species, the so-called Sphagnum subsecundum complex, which includes species known to be gametophytically haploid or diploid (i.e., sporophytically diploid-tetraploid). Despite the fact that S. subsecundum and several species in the complex have been attributed disjunct ranges that include all major continents, phylogenetic analyses suggest that the group is actually restricted to Europe and eastern North America. Plants from western North America, from California to Alaska, which are morphologically similar to species of the S. subsecundum complex in eastern N. America and Europe, actually belong to a different deep clade within Sphagnum section Subsecunda. One species often considered part of the S. subsecundum complex, S. contortum, likely has a reticulate history involving species in the two deepest clades within section Subsecunda. Nucleotide sequences have a strong geographic structure across the section Subsecunda, but shallow tip clades suggest repeated long-distance dispersal in the section as well.     

Impact of specific environmental characteristics of the site of origin (shady,sunny) on anthocyanin and flavonol contents of replanted plants at common cyclamen (<Emphasis Type="Italic">Cyclamen purpurascens</Emphasis> Mill.)

The value of plant provenance (plant origin) is well-known phenomena in woody plants, but less is known in herbaceous plants (perennials). This study with common cyclamen (Cyclamen purpurascens Mill.) was conducted to reveal the importance of specific environmental site properties of plant origin for plant growth and plant quality in the next years. The plants were observed in years 2013 and 2014, more than 10 years after removing and replanting them from the original sites. Morphological characteristics of plants were evaluated by measuring the length and the width of plant rosettes, whereby plants originated from different sites did not show any significant differences. Additionally, the pigment composition, flavonol and anthocyanin content of plant leaves were evaluated. Plants removed from sunny sites showed significantly lower chlorophyll values (total chlorophyll, chlorophyll a) in the both observed years; lower carotenoid and total pigment values were measured only in year 2013. The prevailing anthocyanin in cyclamen leaves was malvidin-3,5-diglucoside with 57.28 µg l^?1 FW in the year 2013 and with 103.68 µg l^?1 FW in the year 2014. Plants originated from the sunny sites accumulated in 2013 significantly more malvidin-3,5-diglucoside in comparison with plants from shady sites of origin. The major substances from the flavonol group were quercetin-3-O-rutinoside and quercetin-dirhamnosyl-glucoside in both analysed years. The cyclamen leaves originated from sunny sites contained in 2013 significant more quercetin-dirhamnosyl-glucoside than cyclamen leaves from shady sites. The results of the study show that different stress parameters (irradiation and water supply in specific year) have a significant impact on the morphological and also internal parameters of cyclamen leaves.     

Use of immobilized cytochrome c as a ligand for affinity chromatography of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans

Three matrices were used for immobilizing the cytochrome c: Sepharose CL-4B, Silasorb SPH amine and a laboratory-prepared new matrix based on crosslinked triazine (2,4,6-tris(aminoethylamine)-1,3,5-triazine) (TAT). Cytochrome c was immobilized on the matrices by several procedures and the amount of incorporated cytochrome c was determined. Cytochrome c immobilized on Sepharose CL-4B with periodate activation, cytochrome c immobilized on Silasorb-amine with carbodiimide activation and cytochrome c immobilized on crosslinked triazine were suitable for purification of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans. The yield with all matrices was about 90%. The purification factor of the above matrices was about 15. A new matrix based on TAT with cytochrome c represented a suitable way for thiosulfate dehydrogenase purification.     

The impact of plasma folate levels of mothers and newborns on intrauterine growth retardation and birth weight

Folate plays an important role in the genomic stability of human cells. In our studies of the impact of environmental pollution on human health, we have found that air pollution can affect pregnancy outcome. As it may be also affected by nutrition, we examined the effect of plasma folate levels of mothers and newborns on intrauterine growth retardation (IUGR) and birth weight (BW) in cohorts from Prague (N=319) and Teplice (N=444). The lower plasma levels (<6.1 nmol/L) were observed in 7.1% of mothers in Prague, and in 9.6% of mothers in Teplice. The higher plasma levels (>36.5 nmol/L) were observed in 28.4% of mothers in Prague, and in 15.7% of mothers in Teplice. The higher plasma levels were observed in 75.4% of newborns in Prague, and 73.2% of newborns in Teplice. When comparing maternal high versus low folate plasma levels and IUGR by logistic regression, the risk of IUGR was significantly decreased for European cohort (according to ethnicity) with gestation age >36 weeks [N=536, OR=0.44, P=0.026], and even more pronounced in the group of European smokers [N=157, OR=0.14, P=0.015]. Using multiple regression analysis, plasma folate levels of mothers and newborns significantly affected the birth weight of newborns of smoking mothers (P<0.05). The obtained results seem to indicate a positive effect of folate on pregnancy outcome, especially its potential to decrease the risk of IUGR in European population and lower birth weight in smoking European mothers. It would be warranted to study the effect of folate levels on pregnancy outcomes in the relationship to different environmental pollution and life styles of mothers.