Metallohelix vectors for efficient gene delivery via cationic DNA nanoparticles

The design of efficient and safe gene delivery vehicles remains a major challenge for the application of gene therapy. Of the many reported gene delivery systems, metal complexes with high affinity for nucleic acids are emerging as an attractive option. We have discovered that certain metallohelices—optically pure, self-assembling triple-stranded arrays of fully encapsulated Fe—act as nonviral DNA delivery vectors capable of mediating efficient gene transfection. They induce formation of globular DNA particles which protect the DNA from degradation by various restriction endonucleases, are of suitable size and electrostatic potential for efficient membrane transport and are successfully processed by cells. The activity is highly structure-dependent—compact and shorter metallohelix enantiomers are far less efficient than less compact and longer enantiomers.     

The Fim and FhaB adhesins play a crucial role in nasal cavity infection and Bordetella pertussis transmission in a novel mouse catarrhal infection model

Pulmonary infections caused by Bordetella pertussis used to be the prime cause of infant mortality in the pre-vaccine era and mouse models of pertussis pneumonia served in characterization of B. pertussis virulence mechanisms. However, the biologically most relevant catarrhal disease stage and B. pertussis transmission has not been adequately reproduced in adult mice due to limited proliferation of the human-adapted pathogen on murine nasopharyngeal mucosa. We used immunodeficient C57BL/6J MyD88 KO mice to achieve B. pertussis proliferation to human-like high counts of 10⁸ viable bacteria per nasal cavity to elicit rhinosinusitis accompanied by robust shedding and transmission of B. pertussis bacteria to adult co-housed MyD88 KO mice. Experiments with a comprehensive set of B. pertussis mutants revealed that pertussis toxin, adenylate cyclase toxin-hemolysin, the T3SS effector BteA/BopC and several other known virulence factors were dispensable for nasal cavity infection and B. pertussis transmission in the immunocompromised MyD88 KO mice. In contrast, mutants lacking the filamentous hemagglutinin (FhaB) or fimbriae (Fim) adhesins infected the nasal cavity poorly, shed at low levels and failed to productively infect co-housed MyD88 KO or C57BL/6J mice. FhaB and fimbriae thus appear to play a critical role in B. pertussis transmission. The here-described novel murine model of B. pertussis-induced nasal catarrh opens the way to genetic dissection of host mechanisms involved in B. pertussis shedding and to validation of key bacterial transmission factors that ought to be targeted by future pertussis vaccines.     

Metabolism of l-threonine and fatty acids and tylosin biosynthesis in Streptomyces fradiae

Abstract In Streptomyces fradiae l -threonine is catabolized by threonine dehydratase or threonine aldolase to 2-ketobutyrate or acetaldehyde and glycine, respectively. Threonine dehydratase synthesis is repressed and its activity is inhibited by NH₄⁺ ions. Threonine aldolase is not repressed by NH₄⁺ ions and its activity is slightly stimulated by these ions. The addition of threonine to the medium increased pronouncedly the fraction of non-branched fatty acids with an even carbon number under conditions when threonine dehydratase was repressed and inhibited. The results indicate that threonine serves as a source of propionyl-CoA and 2-methylbutyryl-CoA and also of acetyl-CoA required for tylosin and fatty acid biosynthesis.     

Manipulation of division symmetry and developmental fate in cultures of potato microspores

The effect of media composition on microspore culture was investigated in one tetraploid and two diploid potatoes. The viability of microspores isolated from 4.5 to 5 mm buds was in the range of 33 to 52%. In media for anther culture, microspores showed no further development and lost viability within 2 days. In M1 medium containing mineral components, sucrose, uridine, cytidine, myo-inositol, glutamine and lactalbumin hydrolysate, 18 to 37% of microspores underwent mitosis within 14 days. Up to 95% of the divisions were symmetric and produced equal nuclei. Some symmetrically divided microspores eventually produced structures with 3 to 10 nuclei. The proportion of the total microspore population producing multinuclear structures reached 9% in diploid clones responsive to anther culture and 1 to 2% in recalcitrant cv. Borka. Symmetric mitoses in M1 medium were induced in the presence of glutamine and lactalbumin hydrolysate. Nucleosides and myo-inositol had no effect on microspore division. In the absence of all organic components except sucrose, most mitoses were asymmetric, formation of multinuclear structures was reduced and most pollen accumulated starch indicative of gametophytic fate. In complete M1 medium, starch accumulation was suppressed. Suppression also occurred in asymmetrically divided microspores, indicating a direct inhibition of pollen development independent of the mode of microspore division. This inhibitory effect of M1 medium might present a stress which triggers the induction of symmetric microspore division and subsequent formation of multinuclear structures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis of hexose-related imidazolidinones: Novel glycation products in the Maillard reaction

Carbohydrate-peptide esters which mimic the reactivity of sugar 6-phosphates in nonenzymatic glycations were used as model compounds for the study of the Maillard reaction in vitro. We found that intramolecular cyclization of the monosaccharide ester in which the sugar moiety (D-glucose or D-galactose) is linked, through the C-6 hydroxy group, to the C-terminal carboxy group of the endogenous opioid pentapeptide leucine-enkephalin, in methanol as the solvent, resulted in the formation of imidazolidinone diastereoisomers having cis or trans relative geometry of the substituents at the imidazolidinone ring moiety. The diastereoisomeric imidazolidinones were separated and each transformed by hydrolysis into the corresponding D-gluco- and D-galacto-related imidazolidinone products of leucine-enkephalin. Along with the previous evidence that, from the same sugar-peptide esters by changing the reaction conditions Amadori rearrangement products could be obtained [Horvat et al. (1998) J Chem Soc Perkin Trans 1:99–13], the presented results point to the possibility that similar carbohydrate-related imidazolidinones may also be generated in the early stage of the Maillard reaction in vivo.     

Neural stem cells transplanted into intact brains as neurospheres form solid grafts composed of neurons, astrocytes and oligodendrocyte precursors

Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential that can give rise to neurons and glia in vivo and in vitro. The aim of this study was to transplant NSCs as whole neurospheres into intact brain and assess the fate and phenotype of their progeny generated in vivo. We isolated NSCs from E14 foetal rat forebrains and cultured them in basic fibroblast and epidermal growth factor-supplemented serum-free medium in the form of neurospheres in vitro. Neurospheres were transplanted into the intact brains of 2 Wistar rats and after a period of 3 weeks, grafted brains were examined immunohistochemically. Neurospheres formed solid grafts that were found in the lateral ventricle and in the velum interpositum under the hippocampus. The majority of cells in the transplanted tissue were identified as beta-III-tubulin(+), NeuN(+), PanNF(+) and synaptophysin(+) neurons and were accumulated throughout the graft centre. GFAP(+) astrocytes were scattered throughout the entire graft and astrocyte processes delimited the outer and perivascular surfaces. A great number of NG2(+) oligodendrocyte precursors was detected. Nestin(+) endothelial cells were found to line capillaries growing in the transplant. These data indicate that nestin(+) NSCs prevailing in neurospheres differentiate following transplantation into nestin(-) neuronal and glial cells which confirms the multipotency of NSCs. Three weeks posttransplantation neuronal and astrocyte cells reached terminal differentiation (formation of synaptic vesicles and superficial and perivascular limiting membranes) while elements of oligodendroglial cell lineage remained immature. Grafting stem cells as non-dissociated neurospheres provide cells with favourable conditions which facilitate cell survival, proliferation and differentiation. However, in the intact brain, grafted neurosphere cells were not found to integrate with the brain parenchyma and formed a compact structure demarcated from its surroundings.     

Macleya cordata and Prunella vulgaris in oral hygiene products - their efficacy in the control of gingivitis

A double-blind, placebo-controlled clinical trial was performed to investigate the effectiveness of a herbal-based dentifrice in the control of gingivitis. Forty volunteers completed the 84-day study. All subjects were balanced for parameters measured - plaque index (PI), community periodontal index of treatment needs (CPITN) and papillary bleeding index (PBI). The dentifrice was effective in reducing symptoms of gingivitis as evaluated by the CPITN and PBI indexes.     

Activation and inhibition of cyclin-dependent kinase-2 by phosphorylation; a molecular dynamics study reveals the functional importance of the glycine-rich loop

Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin-dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160-CDK2/Cyclin A/ATP, inhibited pT14-; pY15-; and pT14,pY15,pT160-CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine-rich loop (G-loop) positioned opposite the activation T-loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G-loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho-group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X-ray data.     

Binding and orientation of conantokins in PL vesicles and aligned PL multilayers

The association of a ligand with its cognate cell surface receptor can be facilitated by interactions between the ligand and the lipid phase of the cell membrane. With respect to the N-methyl-D-aspartate receptor (NMDAR), we have previously established a low affinity, nonreceptor-mediated interaction of the peptidic conantokins with synaptic membranes in conjunction with a high affinity binding to the NMDARs present therein [Klein, R. C., Prorok, M., and Castellino, F. J. (2003) J. Pept. Res. 61, 307-317]. In the current study, several techniques including size-exclusion chromatography, circular dichroism, fluorescence, and NMR spectroscopies were used to investigate the binding, conformation, and orientation of conantokins and their variants to a variety of phospholipid (PL) vesicles and multilayers. We have found that conantokins bind to PLs and that the effectors Ca(2+) and spermine slightly increase this binding ability. The conantokins preserve a high degree of helical conformation when bound to vesicles in the presence of Ca(2+). In the absence of Ca(2+), only conantokin-G (con-G) manifests an increase in conantokin helicity with increasing vesicle concentration. In solution, the conantokins appear to be localized at the headgroup of vesicles and do not insert into the hydrophobic core of the bilayer. On aligned PL films, the helical axis of the conantokins can either reside normal to the membrane surface or partition in a parallel orientation, depending on the nature of the conantokins and the PLs used. These orientation preferences may be conjoined with the biological activities of the conantokins.     

Genomic occurrence of microsatellites containing integral and non-integral repeat numbers

We calculated occurrences of all dinucleotide and trinucleotide microsatellites in the human, mouse, and yeast genomes. The microsatellites were considered separately not only according to the repeated dinucleotide or trinucleotide and the microsatellite length but also according to the starting/terminal nucleotide. The analysis showed that dramatically non-equal amounts occurred in the human genome of microsatellites that differed only by the terminal nucleotides. For example, the 23-mer (TTG)(7)TT occurs 635 times in the human genome whereas (GTT)(7)GT is present only three times in the human genome though the two 23-mers share a 22 nucleotide sequence. The dramatically non-equal occurrences of microsatellites differing only by the terminal nucleotides are observed for most dinucleotide and trinucleotide microsatellites and in all analyzed genomes. We suppose that the strikingly non-equal genomic occurrences of these closely related microsatellites originate from conformational properties of DNA.