Entrapment of peptidoglycans and adamantyltripeptides into liposomes: an HPLC assay for determination of encapsulation efficiency

The encapsulation of different immunomodulating peptides, the peptidoglycan monomer, its semisynthetic derivatives (Adamant-1-yl)-acetyl-peptidoglycan monomer and Boc-Tyr-peptidoglycan monomer, respectively, and of two diastereoisomers of adamantyltripeptides into the large negatively charged multilamellar liposomes was investigated. The reproducible quantitative method using HPLC was established for the determination of the entrapped compounds. It was shown that the tested compounds could be efficiently incorporated into liposomes using either the film or modified film method. The results confirmed that the peptidoglycans with lipophilic substituents and particularly the adamantyltripeptides were incorporated into liposomes with higher efficiency than the peptidoglycan monomer using either of the described methods. Liposome preparations were stable at 4 degrees C up to seven days as shown by minimal leaking of the entrapped material.     

Dipeptidyl peptidase IV (DPPIV) enzyme activity on immature T-cell line R1.1 is down-regulated by dynorphin-A(1-17) as a non-substrate inhibitor

In this study we examined surface expression of CD26 and the corresponding enzyme activity of dipeptidyl peptidase IV (DPPIV) on the cells of immature murine T-cell line, R1.1. The data obtained have shown that R1.1 cells express high density of surface CD26 as compared to normal thymus cells. This was associated with strong enzyme activity, which, based on substrates and inhibitor specificity, corresponded to DPPIV. The DPPIV enzyme activity of R1.1 cells was 10 times stronger than that found on normal murine thymus cells (V(max) = 39 micromol/min/10(6) cells, vs 3.7 micromol/min/10(6) cells, respectively). Upon activation with anti-CD3, up-regulation of both membrane CD26, as well as of DPPIV enzyme activity on R1.1 cells were observed. The finding of strong DPPIV on R1.1 cells makes them suitable model for testing putative substrates/inhibitors of the enzyme in its natural microenvironment. Since in addition to strong DPPIV, R1.1 cells also express kappa opioid receptors (KOR) [European Journal of Pharmacology 227 (1992) 257], we tested the effect of dynorphin-A(1-17), an endogenous opioid peptide with KOR selectivity, on DPPIV of R1.1 cells. Dynorphin-A(1-17) down-regulated DPPIV in a dose-dependent manner, with the potency similar to that of substance P, a known natural DPPIV substrate [Journal of Pharmacology and Experimental Therapeutics 260 (1992) 1257]. DPPIV down-regulation was resistant to bestatin and thiorphan, the inhibitors of two cell surface peptidases (APN and NEP, respectively) with potential of dynorphin-A(1-17) degradation, suggesting that the mechanism underlying the observed effect does not involve degradative products of dynorphin-A(1-17). DPPIV down-regulation was also resistent to KOR antagonist, NBI, suggesting that the mechanism underlying the observed phenomenon involves neither cointernalization of KOR and DPPIV. Collectively, cells of immature T cell line, R1.1 exert strong DPPIV enzyme activity, which could be down-regulated in the presence of dynorphin-A(1-17) by mechanism that presumably includes non-substrate inhibition. By down-regulating DPPIV, dynorphin-A(1-17) may indirectly affect activity and/or specificity of natural substrates of DPPIV, such as substance P, RANTES, and endomorphins.     

Self-image and refugee status in adolescents from Bosnia and Herzegovina and Croatia

The first aim is focused in refugees comparing them with a comparison group in clinical characteristics. In addition, the authors compared scores on the self-image (S-I) scale of refugees and comparison subjects, after they had stratified them into four groups by gender and age. Subjects were 133 refugees from Bosnia and Herzegovina and comparison subjects were from Zagreb, Croatia (n = 189). The subjects were assessed by psychotherapeutic interview, Offer Self-Image Questionnaire (OSIQ), questionnaires of depressive symptoms, war stressors, posttraumatic reactions (PTS-reactions) and general data. Refugees had significantly higher scores on the questionnaires of war stressors and PTS-reactions (p < 0.001). Refugees didn't differ in the majority of 12 scales of OSIQ from comparison subjects, in all four groups. Younger male refugees had the most scales with significantly higher raw scores in S-I, four in total. S-I can give us useful insight into different dimensions of personality of refugees.     

Ultrasound measurement of the volume of musculus quadriceps after knee joint injury

The monitoring of the recovery of femoral muscles, after the knee-joint injury, is possible by the method of ultrasound measurement of the muscular volume. In a clearly defined longitudinal study, our object was to standardize the method of ultrasound measurement of muscular volume and to evaluate its adequacy in practical application in quadriceps muscle rehabilitation. The ultrasound measurements of m. rectus femoris and m. vastus intermedius were conducted in three intervals: in the first 24 hours after the injury; after 1 week, when immobilization was removed; and after 6 weeks, when rehabilitation was finished. The study comprised 30 patients with knee-joint injury, and 30 asymptomatic subjects, who formed the control group. The results showed significant decrease of muscular volume (mm3) after joint immobilization on injured leg and a significant increase of volume after rehabilitation. The same differences were observed on healthy legs, but without significance. Within the same intervals, there were no changes in the muscular mass in the control group. M. rectus femoris was completely recovered in greater number of patients (54.1%), comparing to m. vastus intermedius (25.4%). We conclude that the ultrasound is an appropriate method for monitoring the process of muscular atrophy during immobilization, as well as the course of muscular restitution during the physical therapy.     

19F NMR study of the leucine-specific binding protein of Escherichia coli: mutagenesis and assignment of the 5-fluorotryptophan-labeled residues

The Escherichia coli L-leucine receptor is an aqueous protein and the first component in the distinct transport pathway for hydrophobic amino acids. L-leucine binding induces a conformational change, which enables the receptor to dock to the membrane components. To investigate the ligand-induced conformational change and binding properties of this protein, we used (19)F NMR to probe the four tryptophan residues located in the two lobes of the protein. The four tryptophan residues were labeled with 5-fluorotryptophan and assigned by site-directed mutagenesis. The (19)F NMR spectra of the partially ligand free proteins show broadened peaks which sharpen when L-leucine is bound, showing that the labeled wild-type protein and mutants are functional. The titration of L-phenylalanine into the 5-fluorotryptophan labeled wild-type protein shows the presence of closed and open conformers. Urea-induced denaturation studies support the NMR results that the wild-type protein binds L-phenylalanine in a different manner to L-leucine. Our studies showed that the tryptophan to phenylalanine mutations on structural units linked to the binding pocket produce subtle changes in the environment of Trp18 located directly in the binding cleft.     

Testing the adaptive plasticity of Iris pumila leaf traits to natural light conditions using phenotypic selection analysis

A multivariate selection analysis has been used to test the adaptiveness of several Iris pumila leaf traits that display plasticity to natural light conditions. Siblings of a synthetic population comprising 31 families of two populations from contrasting light habitats were grown at an open dune site and in the understory of a Pinus nigra stand in order to score variation in phenotypic expression of six leaf traits: number of senescent leaves, number of live leaves, leaf length, leaf width, leaf angle, and specific leaf area. The ambient light conditions affected the values of all traits studied except for specific leaf area. In accordance to ecophysiological expectations for an adaptive response to light, both leaf length and width were significantly greater while the angle between sequential leaves was significantly smaller in the woodland understory than at the exposed dune site. The relationship between leaf traits and vegetative fitness (total leaf area) differed across light habitats as predicted by functional hypotheses. The standardized linear selection gradient (β′) for leaf length and width were positive in sign in both environments, but their magnitude for leaf length was higher in the shade than under full sunlight. Since plasticity of leaf length in the woodland shade has been recognized as adaptive, fitness cost of producing plastic change in leaf length was assessed. In both of the available methods used, the two-step and the multivariate regression procedures, a rather high negative association between the fitness value and the plasticity of leaf length was obtained, indicating a cost of plasticity. The selection gradient for leaf angle was weak and significant only in the woodland understory. Genetic correlations between trait expressions in contrasting light environments were negative in sign and low in magnitude, implying a significant genetic variation for plasticity in these leaf traits. Furthermore, leaf length and leaf width were found to be genetically positively coupled, which indicates that there is a potential for these two traits to evolve toward their optimal phenotypic values even faster than would be expected if they were genetically independent.     

Cytokinins in the perianth, carpels, and developing fruit of Helleborus niger L

Reproductive development in the Christmas rose (Helleborus niger L.) differs from that in commonly investigated model plants in two important aspects: (i) the perianth develops a photosynthetic system, after fertilization, and persists until seed ripening; and (ii) the ripe seed contains an immature embryo which continues to mature off the mother plant. The possible roles of cytokinins in these processes are investigated here by analysing extracts of the perianth and the carpels/maturing fruit prepared during anthesis and four stages of post-floral development. trans-Zeatin, dihydrozeatin, N6-(Delta2-isopentenyl)adenine, and their ribosides were identified by tandem mass spectrometry. Single ion monitoring in the presence of deuterated internal standards demonstrated the additional presence of the corresponding riboside-5'-monophosphates, O-glucosides, and 9-glucosides, and afforded quantitative data on the whole set of endogenous cytokinins. Fruit cytokinins were mostly localized in the seeds. Their overall concentrations increased dramatically during early seed development and remained high for 6-8 weeks, until shortly before seed ripening (the last time point covered in this work). Overall cytokinin levels in the perianth did not change markedly in the period covered, but the level of N6-(Delta2-isopentenyl)adenine-type cytokinins appeared to increase slightly and transiently during the greening phase. The perianths of unpollinated or depistillated flowers, which survived, but did not pass through the complete greening process, contained significantly less cytokinins than observed in fruit-bearing flowers. This suggests that perianth greening requires defined cytokinin levels and supports the role of the developing fruit in their maintenance.     

Production of phenolic compounds,antioxidant and antimicrobial activities in hairy root and shoot cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L.

Three hairy root clones of Hypericum perforatum (HR 2, HR 15 and HR 27) transformed with Agrobacterium rhizogenes A4M70GUS and their corresponding regenerated shoot culture clones (HRRS) were compared for differences in growth, production of phenolic compounds, antioxidant and antimicrobial activities. Transgenic clones were selected on the basis of morphological evaluation, genetic and molecular analyses. The clone HR 2 had the highest biomass accumulation, while HR 27 showed the highest shoot regeneration potential. The total phenolics and flavan-3-ols were enhanced in all tested transgenic cultures, while total flavonoids and hypericins were augmented in HRRS clones compared to non-transformed shoots. The HRRS clones produced substantial amounts of chlorogenic acid and 3-p-coumaroylquinic acid. Regarding the flavonoids, they produced significant contents of luteolin hexoside (HRRS 2), quercitrin and quercetin (HRRS 15) and isoquercetin (HRRS 27), while HR 2 and 15 accumulated 4-O-methylkaempferol-O-hexoside and quercetin 6-C-glucoside, respectively. The HR 15 was promising for the production of catechin and procyanidin derivatives and together with its HRRS clone exhibited a high potential for hyperforin and adhyperforin production. All identified naphtodianthrones were confirmed in HRRS 2 and 15 clones. Among xanthones, mangiferin was found as the major compound in HRRS, while trihydroxy-1-metoxy-C-prenyl xanthone was dominant in HR clones. Antimicrobial activity of transgenic cultures revealed that HRRS 15 strongly inhibited the growth of Bacillus cereus, Micrococcus flavus, Pseudomonas aeruginosa and Escherichia coli. Altogether, H. perforatum HR and HRRS cultures could be proposed as promising experimental systems for enhanced production of phenolic compounds with antioxidant and antibacterial properties.     

Phenotypic plasticity in response to environmental heterogeneity contributes to fluctuating asymmetry in plants: first empirical evidence

Fluctuating asymmetry (FA) is widely used to quantify developmental instability (DI) in ecological and evolutionary studies. It has long been recognized that FA may not exclusively originate from DI for sessile organisms such as plants, because phenotypic plasticity in response to heterogeneities in the environment might also produce FA. This study provides the first empirical evidence for this hypothesis. We reasoned that solar irradiance, which is greater on the southern side than on the northern side of plants growing in the temperate zone of the Northern Hemisphere, would cause systematic morphological differences and asymmetry associated with the orientation of plant parts. We used geometric morphometrics to characterize the size and shape of flower parts in Iris pumila grown in a common garden. The size of floral organs was not significantly affected by orientation. Shape and particularly its asymmetric component differed significantly according to orientation for three different floral parts. Orientation accounted for 10.4% of the total shape asymmetry within flowers in the falls, for 11.4% in the standards and for 2.2% in the style branches. This indicates that phenotypic plasticity in response to a directed environmental factor, most likely solar irradiance, contributes to FA of flowers under natural conditions. That FA partly results from phenotypic plasticity and not just from DI needs to be considered by studies of FA in plants and other sessile organisms.