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.     

Maintenance of the inner cell mass in human blastocysts from fragmented embryos

The degree of fragmentation during early cleavage is universally used as an indicator of embryo quality during human in vitro fertilization treatment. Extensive fragmentation has been associated with reduced blastocyst formation and implantation. We examined the relationship between early fragmentation and subsequent allocation of cells to the trophectoderm and inner cell mass in the human blastocyst. We retrospectively analyzed data from 363 monospermic human embryos that exhibited varying degrees of fragmentation on Day 2. Embryos were cultured from Day 2 to Day 6 in Earle balanced salt solution with 1 mM glucose and human serum albumin. Rates of development and blastocyst formation were measured. The number of cells in the trophectoderm and inner cell mass and the incidence of apoptosis were assessed following differential labeling with polynucleotide-specific fluorochromes. Increasing fragmentation resulted in reduced blastocyst formation and lower blastocyst cell numbers. For minimal and moderate levels of fragmentation, the reduction in cell numbers was confined largely to the trophectoderm and a steady number of inner cell mass cells was maintained. However, with extensive fragmentation of more than 25%, cell numbers in both lineages were reduced in the few embryos that formed blastocysts. Apoptotic nuclei were present in both the trophectoderm and inner cell mass, with the lowest incidence in blastocysts that had developed from embryos with minor (5-10%) fragmentation. Paradoxically, higher levels of apoptosis were seen in embryos of excellent morphology, suggesting a possible role in regulation of cell number.     

New cationic lipids form channel-like pores in phospholipid bilayers

Two representatives of a new class of cationic lipids were found to have high pore-forming activity in planar bilayer membranes. These molecules, called BHHD-TADC and BHTD-TADC, have qualitatively similar effects on phospholipid membranes. Addition of 2.5-5 micro M of either of them to the membrane bathing solutions resulted in formation of long-lived anion-selective pores with conductance in the range 0.1-2 nS in 0.1 M KCl. Pore formation was found to be dependent on the potential applied to the membrane. When negative potential was applied to membrane at the side of addition, the rate of pore formation was much lower compared to when the positive potential was applied. Dependence of pore formation on compound concentration was highly nonlinear, indicating that this process requires assembly of molecules in the membrane. Addition of any of these compounds on both sides of the membrane increased the efficiency of pore formation by one to two orders of magnitude. Pore formation was strongly pH dependent. Although pores were formed with high efficiency at pH 6.5, only occasional fluctuations of membrane conductance were observed at pH 7.5. Possible mechanisms of new compounds biological activity are discussed.     

Separation of magnetic affinity biopolymer adsorbents in a Davis tube magnetic separator

A Davis tube (a matrix-free, flow-through magnetic separator used mainly in mineral processing) has been tested for separation of magnetic affinity biopolymer adsorbents from larger volumes of suspensions. Both magnetic chitosan and magnetic cross-linked erythrocytes could be efficiently separated from litre volumes of suspensions. Up to 90% adsorbent recovery was achieved under optimised separation conditions.     

Dynamics and binding modes of free cdk2 and its two complexes with inhibitors studied by computer simulations

This article presents a molecular dynamics (MD) study of the cdk2 enzyme and its two complexes with the inhibitors isopentenyladenine and roscovitine using the Cornell et al. force field from the AMBER software package. The results show that inserting an inhibitor into the enzyme active site does not considerably change enzyme structure but it seemingly changes the distribution of internal motions. The inhibitor causes differences in the domain motions in free cdk2 and in its complexes. It was found out that repulsion of roscovitine N9 substituent causes conformational change on Lys 33 side chain. Isopentenyladenine forms with Lys 33 side chain terminal amino group a hydrogen bond. It implies that the cavity, where N9 substituent of roscovitine is buried, can adopt larger substituent due to Lys 33 side chain flexibility. The composition of electrostatic and van der Waals interactions between the inhibitor and the enzyme were also calculated along both cdk2/inhibitor MD trajectories together with MM-PB/GBSA analysis. These results show that isopentenyladenine-like inhibitors could be more effective after modifications leading to an increase in their van der Waals contact with the enzyme. We suggest that a way leading to better inhibitors occupying isopentenyladenine binding mode could be: to keep N9 and N7 purine positions free, to keep 3,3-dimethylallylamino group at C6 position, and to add, e.g., benzylamino group at C2 position. The results support the idea that the isopentenyladenine binding mode can be used for cdk2 inhibitors design and that all possibilities to improve this binding mode were not uncovered yet.     

A fucosylated chondroitin sulfate from echinoderm modulates in vitro fibroblast growth factor 2-dependent angiogenesis

Fucosylated chondroitin sulfate (FucCS), a glycosaminoglycan obtained from sea cucumber, has the same structure as mammalian chondroitin sulfate, but some of the glucuronic acid residues display sulfated fucose branches. This new polysaccharide has a more favorable effect than heparin on vascular cell growth. It inhibits smooth muscle cell proliferation as heparin, and it has a potent enhancing effect on endothelial cell proliferation and migration in the presence of heparin-binding growth factors. We now extend our studies to the effect of this glycosaminoglycan on endothelial cells to an in vitro angiogenesis model on Matrigel. FucCS, in the presence of fibroblast growth factor-2 (FGF-2), strongly increases the capacity of endothelial cells to form vascular tubes on Matrigel with a well-organized capillary-like network and typical closed structures. Comparison between the activity of native and chemically modified chondroitin sulfate from sea cucumber reveals that the sulfated fucose branches are the structural motif for the proangiogenic activity. Heparin does not induce angiogenesis in this experimental model. We also have evidence for the proposition that endothelial cell proliferation is not the sole event involved in the in vitro FGF-2-induced angiogenesis. It implies a variety of other modifications of the endothelial cells and of their interaction with the extracellular matrix, such as integrin expression and actin cytoskeleton reorganization. Finally, the proangiogenic effect of FucCS, concomitant with its capacity to prevent venous and arterial thrombosis, in animal models makes this new glycosaminoglycan a promising molecule with possible beneficial effects in pathological conditions affecting blood vessels such as the neovascularization of ischemic areas.