Cytogenetic stability of <Emphasis Type="Italic">Centaurea ragusina</Emphasis> long-term culture

Centaurea ragusina L. (Asteraceae) is an endemic Croatian plant species, which developed xeromorphic characteristics as a consequence of its natural environment – vertical limestone cliffs above the Adriatic sea. Cytogenetic status of C. ragusina long-term culture (94th subculture) and C. ragusina seedlings was analysed and compared after 4 weeks of growth. Cytogenetic stability was investigated in root meristem cells of C. ragusina cultured plants originated from Pen đa (cliffs near Dubrovnik) and seedlings originated from three different localities in south Adriatic (Penđa, Pasjača – cliffs near Dubrovnik and island of Komiža) using mitotic index and mitotic and chromosomal abnormalities as parameters. Mitotic indices of cultured plants and ‘Penđa’ seedlings were similar and showed significant increase compared to mitotic indices of ‘Komiža’ and ‘Pasjača’ seedlings. Although the highest number of mitotic abnormalities was recorded in root meristem cells of cultured plants, it was only a bit higher than in root tips of ‘Pasjača’ and ‘Penđa’ seedlings, while that of ‘Komiža’ was two times lower compared to cultured plants. Pattern of analysed mitotic abnormalities was very similar in root tips of cultured plants and ‘Pasjača’ and ‘Penđa’ seedlings, with exception of ‘Komiža’ seedlings. Presented results suggest that long-term cultivation of C. ragusina has almost no effect on culture ageing considering similar distribution of scored mitotic abnormalities as in ‘Penđa’ and ‘Pasjača’ seedlings.     

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.     

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.     

Heterologous Escherichia coli Expression, Purification and Characterization of the GrmA Aminoglycoside-Resistance Methyltransferase

The mechanism of resistance to aminoglycosides based on methylation of their target, 16S rRNA, was until recently described only in antibiotic producing microorganisms. However, equivalent methyltransferases have now also been identified among numerous clinical Gram-negative pathogenic isolates. We have cloned, expressed, and purified GrmA, the aminoglycoside-resistance methyltransferase from Micromonospora purpurea, producer of gentamicin complex. Two vectors were created that express protein with an N-terminal 6× histidine tag with and without an enterokinase recognition producing proteins His₆-EK-GrmA and His₆-GrmA, respectively. The activity of both recombinant proteins was demonstrated in vivo. After optimized expression and native purification both protein variants proved to be active in in vitro methylation assays. This work lays a foundation for future detailed biochemical, structural and pharmacological studies with this member of an important group of aminoglycoside-resistance enzymes.     

The role of the Vlax Roma in shaping the European Romani maternal genetic history

The Roma are comprised of many founder groups of common Indian origins but different socio-cultural characteristics. The Vlax Roma are one of the founder Roma populations characterized by a period of bondage in the historic Romanian principalities, and by the archaic Romanian language. Demographic history suggests different migration routes of Roma populations, especially after their arrival in Mesopotamia and the eastern boundary of the Byzantine Empire. Although various genetic studies of uniparental genetic markers showed a connection between Roma genetic legacy and their migration routes, precise sampling of Roma populations elucidates this relationship in more detail. In this study, we analyzed mitochondrial DNA of 384 Croatian Vlax Roma from two geographic locations in the context of 734 European Roma samples. Our results show that Roma migration routes are marked with two Near-Eastern haplogroups, X2 and U3, whose inverse proportional incidence clearly separates the Balkan and the Vlax Roma from other Roma populations that reached Europe as part of the first migration wave. Spatial and temporal characteristics of these haplogroups indicate a possibility of their admixture with Roma populations before arrival in Europe. Distribution of haplogroup M35 indicates that all Vlax Roma populations descend from one single founder population that might even reach back to the original ancestral Indian population. Founder effects followed by strict endogamy rules can be traced from India to contemporary small, local communities, as in the case of two Croatian Vlax Roma populations that show clear population differentiation despite similar origins and shared demographic history.     

The influence of antioxidants in the thiyl radical induced lipid peroxidation and geometrical isomerization in micelles of linoleic acid

Abstract

The biomimetic model of micelles of linoleic acid containing 2-mercaptoethanol and the antioxidant was examined under gamma irradiation up to 400?Gy in aerobic or deoxygenated conditions where thiyl radicals are the main reactive species. Lipid peroxidation was retarded by ascorbic acid and α-tocopherol, whereas this process was strongly inhibited by resveratrol as effectively as the ascorbic acid/α-tocopherol mixture. Furthermore, antioxidants have a much stronger inhibitory effect on the peroxidation in the presence of 2-mercaptoethanol, and at the same time show protective properties of the double bond, decreasing the cis–trans isomerization. Under anaerobic conditions, cis–trans isomerization occurred and antioxidants efficiency increased along the series: resveratrol < α-tocopherol?<?ascorbic acid. This result is explained taking into account the double bond localization in the hydrophobic core of the micelle and the need of co-localization of the antioxidant in order to get an anti-isomerizing activity and protection of the natural lipid geometry.     

Absence of integrin-mediated TGFbeta1 activation in vivo recapitulates the phenotype of TGFbeta1-null mice

The multifunctional cytokine transforming growth factor (TGF) beta1 is secreted in a latent complex with its processed propeptide (latency-associated peptide [LAP]). TGFbeta1 must be functionally released from this complex before it can engage TGFbeta receptors. One mechanism of latent TGFbeta1 activation involves interaction of the integrins alpha v beta6 and alpha v beta8 with an RGD sequence in LAP; other putative latent TGFbeta1 activators include thrombospondin-1, oxidants, and various proteases. To assess the contribution of RGD-binding integrins to TGFbeta1 activation in vivo, we created a mutation in Tgfb1 encoding a nonfunctional variant of the RGD sequence (RGE). Mice with this mutation (Tgfb1(RGE/RGE)) display the major features of Tgfb1(-/-) mice (vasculogenesis defects, multiorgan inflammation, and lack of Langerhans cells) despite production of normal levels of latent TGFbeta1. These findings indicate that RGD-binding integrins are requisite latent TGFbeta1 activators during development and in the immune system.