Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species

Shoots of micropropagated Gentiana acaulis, G. cruciata, G. lutea, and G. purpurea were inoculated with suspensions of Agrobacterium rhizogenes cells, strains ATCC 15834 or A4M70GUS. Adventitious roots appeared at the sites of inoculation in all 4 species. Root tips were excised and cultured on growth regulator-free media for 2-6 years. They exhibited very high branching and plagiotropism. Spontaneous bud initiation occurred in roots of G. cruciata. Roots of G. lutea, G. acaulis and G. purpurea were cultured on media with high kinetin concentration, which induced the formation of friable callus tissues. Only in G. purpurea were these calluses organogenic. Regenerated shoots of G. cruciata and G. purpurea gave rise to plants, that displayed the typical phenotypes of A. rhizogenes-transformed plants: short internodes and rolled leaves. In the roots of G. acaulis and G. cruciata, transformed with A. rhizogenes A4M70GUS, a positive reaction with X-gluc indicated the activity of β-glucuronidase. The DNA extracted from hairy roots and from the roots of transgenic plants hybridized with the appropriate genomic probes in Southern blotting. This is taken as evidence of the stable genetic transformation in the 4 Gentiana species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Protein Kinase B Localization and Activation Differentially Affect S6 Kinase 1 Activity and Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 Phosphorylation

Recent studies indicate that phosphatidylinositide-3OH kinase (PI3K)-induced S6 kinase (S6K1) activation is mediated by protein kinase B (PKB). Support for this hypothesis has largely relied on results obtained with highly active, constitutively membrane-localized alleles of wild-type PKB, whose activity is independent of PI3K. Here we set out to examine the importance of PKB signaling in S6K1 activation. In parallel, glycogen synthase kinase 3beta (GSK-3beta) inactivation and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation were monitored as markers of the rapamycin-insensitive and -sensitive branches of the PI3K signaling pathway, respectively. The results demonstrate that two activated PKBalpha mutants, whose basal activity is equivalent to that of insulin-induced wild-type PKB, inhibit GSK-3beta to the same extent as a highly active, constitutively membrane-targeted wild-type PKB allele. However, of these two mutants, only the constitutively membrane-targeted allele of PKB induces S6K1 activation. Furthermore, an interfering mutant of PKB, which blocks insulin-induced PKB activation and GSK-3beta inactivation, has no effect on S6K1 activation. Surprisingly, all the activated PKB mutants, regardless of constitutive membrane localization, induce 4E-BP1 phosphorylation and the interfering PKB mutant blocks insulin-induced 4E-BP1 phosphorylation. The results demonstrate that PKB mediates S6K1 activation only as a function of constitutive membrane localization, whereas the activation of PKB appears both necessary and sufficient to induce 4E-BP1 phosphorylation independently of its intracellular location.     

The First Homozygote Mutation c.499G>T (Asp167Tyr) in the RPE65 Gene Encoding Retinoid Isomerohydrolase Causing Retinal Dystrophy

RPE65, an abundant membrane-associated protein present in the retinal pigment epithelium (RPE), is a vital retinoid isomerase necessary for regenerating 11-cis-retinaldehyde from all-trans retinol in the visual cycle. In patients with inherited retinal dystrophy (IRD), precise genetic diagnosis is an indispensable approach as it is required to establish eligibility for the genetic treatment of RPE65-associated IRDs. This case report aims to report the specific phenotype–genotype correlation of the first patient with a homozygous missense variant RPE65 c.499G>T, p. (Asp167Tyr). We report a case of a 66-year-old male who demonstrated a unique phenotype manifesting less severe functional vision deterioration in childhood and adolescence, and extensive nummular pigment clusters. The underlying causes of the differences in the typical bone spicule and atypical nummular pigment clumping are unknown, but suggest that the variant itself influenced the rate of photoreceptor death. Functional studies are needed to define whether the substitution of aspartate impairs the folding of the tertiary RPE65 structure only and does not lead to the complete abolishment of chromophore production, thus explaining the less severe phenotype in adolescence.     

Neuroprotective Potential and Chemical Profile of Alternatively Cultivated Ganoderma lucidum Basidiocarps

Various neurodegenerative diseases are the main challenges to the modern medicine and there is a great need for novel, natural, neuroprotective agents. Ganoderma lucidum is a well‐known medicinal mushroom, which health benefits have been confirmed by numerous studies. As demand for its basidiocarps is increased and traditional cultivation on hardwoods is not environmentally friendly and economically justified, finding of alternative substrates is necessary. The aim of the study was to assess the effect of alternative cultivation substrates on the chemical profile of G. lucidum basidiocarps and their capacity to inhibit acetylcholinesterase and tyrosinase, which higher activity is directly associated with neurodegenerative processes. Extracts of basidiocarps cultivated on alternative substrates, especially on clear wheat straw, showed significantly higher inhibition capacities than extracts of commercially‐grown ones. These extracts were considerably different chemically from commercial basidiocarps extracts and even nine new compounds were isolated from them. Our results suggest that cultivation substrate greatly affect the chemical profile and neuroprotective capacity of obtained basidiocarps and wheat straw is a promising cultivation substrate.     

Proteolipid/DM-20 proteins bearing the paralytic tremor mutation in peripheral nerves and transfected Cos-7 cells

Paralytic tremor (Plp-pt) is a missense mutation of the myelin proteolipid gene (Plp) in rabbits. The myelin yield in the Plp-pt brain is reduced and the protein and lipid composition of central nervous system (CNS) myelin is abnormal. We studied the intracellular transport of the normal and Plp-pt mutant PLP and DM-20 in transiently transfected Cos-7 cells. While the mutant PLP accumulates in the rough endoplasmic reticulum and does not reach the plasma membrane, the spliced isoform of PLP, mutant DM-20, is normally transported to the cell surface and integrated into the membrane. Analysis of rabbit sciatic nerves revealed that concentration of peripheral nervous system (PNS) myelin proteins is normal in Plp-pt myelin. In the PNS like in the CNS, the level of Plp gene products is subnormal. But this does not affect myelination, in the PNS where PLP, present in low concentration, is not a structural component of compact myelin. The normal level of Plp gene expression in Schwann cells is low and these results suggest that, in the Plp-pt PNS, Schwann cell function is not affected by the deficiency in PLP and/or the impairment of intracellular PLP transport. Special issue dedicated to Dr Marion E. Smith.     

New gold carbene complexes as candidate anticancer agents

Three structurally related gold(I) carbene complexes with bulky hydrophobic ligands i.e. 1–3 were investigated in solution for further consideration as candidate anticancer agents. Cytotoxic assays were subsequently conducted on bone marrow-derived preosteoclast cell line of human origin (FLG 29.1) and human colon cancer cells (HCT-116). A far greater cytotoxic activity was measured for compound 1 against HCT-116 cells compared to 2 and 3; conversely, all compounds were highly and similarly active against FLG 29.1 cells. Results obtained for the reaction of complexes 1 and 2 with RNase A documented the occurrence of a weak interaction with this model protein and the formation of a tiny amount of the corresponding adduct. Moreover, a certain reactivity of the complex 2 was also detected toward GSH. The general implications of the obtained results are discussed.     

Profile and multidrug resistance determinants of Chryseobacterium indologenes from seawater and marine fauna

The aim of this study was to investigate the prevalence and genetic basis of multidrug resistance in Chryseobacterium indologenes from seawater and marine invertebrates used for human consumption, in Ka?tela Bay, Adriatic Sea, Croatia. Out of 16 samples of seawater, Mediterranean mussel (Mytilus galloprovincialis Lam.), Rayed Mediterranean limpets (Patella caerulea L.) and Purple sea urchins (Paracentrotus lividus Lam.) collected, 15 were positive for C. indologenes. In total, 41 isolates were randomly selected and tested for antibiotic susceptibility by disc-diffusion and broth microdilution methods. PCR was used to detect alleles encoding extended-spectrum (ESBLs) and metallo-β-lactamases (MBLs). The clonality of β-lactamase-producing strains was evaluated by random amplified polymorphic DNA (RAPD) analysis. All C. indologenes isolates showed multiple resistance to at least 9 out of 16 antibiotics tested. Lowest resistance rates were found for piperacillin (9.7 %) and ciprofloxacin (24.4 %), whereas only piperacillin/tazobactam and trimethoprim/sulfamethoxazole showed 100 % activity. More than half of isolates carried bla _IND-type gene, including 2 isolates carrying bla _IND-2 and 21 carrying bla _IND-7, that was identified as a major MBL genotype in isolates from Adriatic Sea. RAPD typing of IND-producing isolates revealed 6 major groups with no predominant clone in population. The presence of multidrug resistant and IND-producing C. indologenes in marine environment, including marine fauna, pose a risk for transmitting this opportunistic pathogen to humans through recreation or consummation of seafood. In addition, the antibiotic susceptibility test results have practical relevance for empirical treatment of C. indologenes infections.