Hanna Kozłowska

Neurogenic potential of human umbilical cord blood: Neural-like stem cells depend on previous long-term culture conditions

In vitro studies conducted by our research group documented that neural progenitor cells can be selected from human umbilical cord blood (HUCB‐NPs). Due to further expansion of these cells we have established the first human umbilical cord blood‐derived neural‐like stem cell line (HUCB‐NSC) growing in serum‐free (SF) or low‐serum (LS) medium for over 3 years. The purpose of the study was to evaluate the neurogenic potential of HUCB‐NSCs cultured in SF and LS condition in different in vitro settings before transplantation. We have shown that the number of cells attaining neuronal features was significantly higher for cultures expanded in LS than in SF condition. Moreover, the presence of neuromorphogens, cultured rat astrocytes or hippocampal slices promoted further differentiation of HUCB‐NSCs into neural lineage much more effectively when the cells had derived from LS cultures. The highest response was observed in the case of co‐cultures with rat primary astrocytes as well as hippocampal organotypic slices. However, the LS cells co‐cultured with hippocampal slices expressed exclusively a set of early and late neuronal markers whereas no detection of cells with glial‐specific markers was possible. In conclusion, certain level of stem/progenitor cell commitment is important for optimal response of HUCB‐NSC on the neurogenic signals provided by surrounding environment in vitro.

Affinity of S100A1 protein for calcium increases dramatically upon glutathionylation

S100A1 is a typical representative of a group of EF‐hand calcium‐binding proteins known as the S100 family. The protein is composed of two α subunits, each containing two calcium‐binding loops (N and C). At physiological pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca2+‐titration curves of Trp90 fluorescence for both the native protein and its Glu32 → Gln mutant with an inactive N‐loop. Using a chelator method, we also determined the calcium‐binding constant for the S100A1 Glu73 → Gln mutant with an inactive C‐loop. The protein binds four calcium ions in a noncooperative way with binding constants of K1 =4 ± 2 × 103 m−1 (C‐loops) and K2≈ 102 m−1 (N‐loops). Only when both loops are saturated with calcium does the protein change its global conformation, exposing to the solvent hydrophobic patches, which can be detected by 2‐p‐toluidinylnaphthalene‐6‐sulfonic acid – a fluorescent probe of protein‐surface hydrophobicity. S‐Glutathionylation of the single cysteine residue (85) of the α subunits leads to a 10‐fold increase in the affinity of the protein C‐loops for calcium and an enormous – four orders of magnitude – increase in the calcium‐binding constants of its N‐loops, owing to a cooperativity effect corresponding to ΔΔG = −6 ± 1 kcal·mol−1. A similar effect is observed upon formation of the mixed disulfide with cysteine and 2‐mercaptoethanol. The glutathionylated protein binds TRTK‐12 peptide in a calcium‐dependent manner. S100A1 protein can act, therefore, as a linker between the calcium and redox signalling pathways.

Excitotoxic neuronal injury in acute homocysteine neurotoxicity: Role of calcium and mitochondrial alterations

In this study we tested if calcium imbalance and mitochondrial dysfunction, which have been implicated in the conventional mechanisms of excitotoxicity induced by glutamate (Glu), are also involved in homocysteine (Hcy) neurotoxicity. Primary cultures of rat cerebellar granule cells were incubated for 30 min in the presence of 25 mM D,L-Hcy or 1mM Glu. At these concentrations both amino acids induced comparable neurodegeneration and chromatin condensation, evaluated after 24 h using the propidium iodide and Hoechst 33258 staining. These effects were partially prevented by cyclosporin A (CsA), but not FK506. Hcy-induced release of [(3)H]inositol phosphates and increase in intracellular calcium level (evaluated with fluo-3 fluorescent probe) were weakly expressed. Hcy- and Glu-induced mitochondrial swelling was visualized under electron microscope, and the release of Cytochrome c was evaluated using immunocytochemical method and confocal microscopy. Comparing to Glu, the effects of Hcy were slightly less expressed and less sensitive to CsA, while FK506 did not modify mitochondrial alterations. These data indicate that mitochondrial alterations play a similar role in acute Hcy and Glu neurotoxicity, although the mechanisms triggering Glu- and Hcy-evoked mitochondrial dysfunction seem to differ, Hcy toxicity being less dependent on calcium.

Structural and functional characteristic of a model for deep-seated lacunar infarct in rats

Deep-brain lacunar infarct represents a significant clinical problem as it produces severe symptoms highly resistant to rehabilitation. The limited area of necrosis may facilitate neurorepair via the action of various novel neuroprotective strategies including cell-based therapies. The lesion was induced by stereotactic injection of ouabain into adult rat brains. Subsequent behavioral testing involved beam walking task, rotarod, visual discrimination task and apomorphine rotation. For morphological and topographical analysis brain slices were stained with H-E and evaluated under light microscopy. Lesion size was measured in absolute terms and in relation to the whole brain volume. Immunohistochemical analysis for the co-localization of BrdU with specific cell-type markers (PSA-NCAM, NG2, beta-tubulin III, GFAP, ED1) have has been performed, to determine the fate of newly generated cells with emphasis on evidence of neurogenesis. The lesion involved the basal ganglia, basal forebrain nuclei, internal capsule and striatum (just 1-2% of total brain volume). Significant and relatively stable behavioral deficits were observed up to 30 days. Furthermore, large numbers of cells are seen to be newly generated in response to injury with a significant proportion of these being present on account of neurogenesis.

Developmental stage dependent neural stem cells sensitivity to methylmercury chloride on different biofunctional surfaces.

Sensitivity of neural stem cells viability, proliferation and differentiation upon exposure to methylmercury chloride (MeHgCl) was investigated on different types of biofunctional surfaces. Patterns of biodomains created by microprinting/microspotting of poly-l-lysine or extracellular matrix proteins (fibronectin and vitronectin) allowed for non-specific electrostatic or specific, receptor mediated interactions, respectively, between stem cells and the surface. The neural stem cell line HUCB-NSC has been previously shown to be susceptible to MeHgCl in developmentally dependent manner. Here we demonstrated that developmental sensitivity of HUCB-NSC to MeHgCl depends upon the type of adhesive biomolecules and the geometry of biodomains. Proliferation of HUCB-NSC was diminished in time and MeHgCl concentration dependent manner. In addition, the response to MeHgCl was found to be cell-type dependent. Undifferentiated cells were the most sensitive independently of the type of bioactive domain. Significant decrease of GFAP+ cells was detected among cells growing on poly-l-lysine, while on fibronectin and vitronectin, this effect was observed only in the highest (1μM) concentration of MeHgCl. β-Tubulin III expressing cells were most sensitive on fibronectin domains. In addition, limited bioactive domains to μm in size, as compared to non-patterned larger area of the same adhesive substrate, exerted protective role. Thus, the surface area and type of cell/biofunctional surface interaction exerted significant influence on developmental stage and cell-type specific response of HUCB-NSC to MeHgCl.

Neonatal desensitization does not universally prevent xenograft rejection

1. Gray, V.E., Kukurba, K.R. & Kumar, S. Bioinformatics (2012). 2. González-Perez, A. & Lopez-Bigas, N. Am. J. Hum. Genet. 88, 440–449 (2011). 3. Adzhubei, I.A. et al. Nat. Methods 7, 248–249 (2010). 4. Tennessen, J.A. et al. Science 337, 64–69 (2012). 5. Ng, S.B. et al. Nature 461, 272–276 (2009). 6. Matthews, B.W. Biochim. Biophys. Acta 405, 442–451 (1975). efficient predictive statistical model (Supplementary Methods and Supplementary Figs. 2–5). A web server for evaluating novel variants with EvoD is available at http://barn.asu.edu/EvoD/. At ultraconserved sites, EvoD led to large reductions in the FPR: 55% for Condel and 41% for PolyPhen-2. We retrieved the population allele frequency of the neutral HumVar nSNVs at ultraconserved sites from a 5,400-exome data set4 and found that EvoD improved diagnoses across the spectrum of rare (<0.1%) to common (>5%) alleles (Fig. 1a). The balanced accuracy of EvoD was also significantly higher than that of Condel and PolyPhen-2 at ultraconserved sites (Table 1; P < 10-10). Furthermore, EvoD’s performance was consistent across ultra-, welland less-conserved sites, whereas Condel and PolyPhen-2 showed uneven performance across these classes (Table 1). For each nSNV, the EvoD statistical model also produced an impact score that reflected the degree of neutrality: most neutral = 0 and most non-neutral = 100. In an analysis of 244,272 nSNVs from the 1000 Genomes Project, we found that the population frequency of nSNVs decayed with increasing impact score (Fig. 1b). We therefore used the empirical distribution of EvoD scores to determine the statistical significance of a neutral or non-neutral diagnosis (P value) adaptively for variants at ultra-, welland less-conserved sites (Supplementary Fig. 2). Using EvoD, we analyzed nSNVs in an example set of eight personal HapMap exomes5 containing a total of 13,372 nSNVs at ultraconserved sites. Of these, 4% were predicted to be non-neutral (P < 0.05). An overwhelming majority (94%) of these non-neutral nSNVs were found in heterozygous genotypes that would neutralize the negative effects of recessive alleles. Similar results were observed for 35,367 nSNVs at welland less-conserved sites, which were also reflected in the neutrality heat maps showing the EvoD impact scores of nSNVs in heterozygous genotypes (Fig. 1c). In contrast, the fraction of homozygous nSNVs with high EvoD impact scores was much smaller at ultraconserved sites (Fig. 1c). EvoD predicted no more than one homozygous nSNV per exome to be non-neutral (P < 0.05) in ultraconserved sites, which is consistent with the fact that individuals contributing to HapMap sequencing do not suffer from any known Mendelian disease. Our results show that an evolution-aware approach to training and testing computational tools leads to better functional predictions for nSNVs, particularly at the most functionally important positions.

Interactive 3D stereoscopic digital‐image analysis of the basilar artery bifurcation

The goal of this study was to analyze morphometrical variations of the basilar artery bifurcation (BAB), so that physicians could map out a patients anatomical structure prior to delicate neurosurgical procedures. The CT‐angio files of 98 patients ranging from 12 to 78 years of age were retrieved. These files were evaluated using Gradual Angiographic Image Data Analyzer (GAIDA) software, where a new interactive three‐dimensional (3D) stereoscopic visualization method was used to reconstruct computer images of the BAB complex. Subsequently the measurements of the BAB angles and BAB distances in relation to the dorsum sellae (DS), posterior clinoid processes (PCPs), and posterior biclinoid plane (PBP) were carried out. The average BAB angle was determined to be 117.7° (30.93°–172.2°). The three types of BAB were classified as type T with a BAB angle greater than 145° (mean 154.4°), type Y for an angle being equal or less than 145° but greater than 100° (mean 121.5°) and type V for angles less than 100° (mean 83.28°). The mean distances between BAB and DS (9.55 mm), BAB and left PCP (12.97 mm), and BAB and right PCP (13.01 mm), BAB and PBP (2.2 mm) were evaluated. Furthermore, the BAB is of great importance when examining basilar artery aneurysm development, particularly at the point of greatest hemodynamic stress, as well as the BAB distances in relation to the bony landmarks used for different approach methods in neurosurgical procedures. Clin. Anat. 21:127–137, 2008.

Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method. II. Heteroassociation between 2-aminopurine and thymidine.

Heteroassociation between A and B compounds in liquid solution was considered. Provided that concentration of A molecules is low, a general equation describing fluorescence quantum yield and lifetime of compound A as a function of B molecules concentration was derived. The heteroassociation between 2-aminopurine and thymidine in aqueous solutions was examined within the range of temperatures 0 to 90 degrees C. The equilibrium constants of the first step of association, namely heterodimer formation, were determined and its thermodynamic parameters (deltaH equals - 2.76 kcal/mol, deltaS equals - 5.9 e.u.) were calculated. The observed changes of the stacking rate constants with temperature confirm the two-step mechanism of the reaction. The activation energy (approximately 10.7 angstroms) are only slightly larger than in the case of 2-aminopurine autoassociation, most probably because of a stronger solvation of thymidine molecules.

Synthetic Bastadins Modify the Activity of Ryanodine Receptors in Cultured Cerebellar Granule Cells

Although the interactions of several natural bastadins with the RyR1 isoform of the ryanodine receptor in sarcoplasmic reticulum has been described, their structure-dependent interference with the RyR2 isoform, mainly expressed in cardiac muscle and brain neurons, has not been studied. In this work, we examined calcium transients induced by natural bastadin 10 and several synthetic bastadins in cultured cerebellar granule cells known to contain RyR2. The fluorescent calcium indicator fluo-3 and confocal microscopy were used to evaluate changes in the intracellular Ca2+ concentration (Cai), and the involvement of ryanodine receptors was assessed using pharmacological tools. Our results demonstrate that apart from the inactive BAST218F6 (a bisdebromo analogue of bastadin 10), synthetic bastadin 5, and synthetic analogues BAST217B, BAST240 and BAST268 (at concentrations >20 µM) increased Cai in a concentration-dependent, ryanodine- and FK-506-sensitive way, with a potency significantly exceeding that of 20 mM caffeine. Moreover, the same active bastadins at a concentration of 5 µM in the presence of ryanodine prevented a thapsigargin-induced increase in Cai. These results indicate that bastadins, acting in a structure-dependent manner, modify the activity of RyR2 in primary neuronal culture and provide new information about structure-related pharmacological properties of bastadins.

Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method: I. Autoassociation of 2-aminopurine

A general equation was derived, describing fluorescence quantum yield and lifetime of an autoassociating compound in liquid solutions. The autoassociation of 2-aminopurine in aqueous solution was examined within the range from 0 to 90 degrees C. The compound seemed to associate cooperatively. The thermodynamic parameters of polymerization change with temperature, so that its free enthalypy deltaG = 0.0797 T2 + 45.4 T - 7893. The dimerization enthalpy and entropy are approximately temperature-independent (deltaH2 = -4.17 kcal/mol deltas2 = -10.9 e.u.), although the function: delta g2 = -0.0308T2 +30.3T - 7213 fits experimental points better. The observed dependences can be explained by the increasing role of the hydrophobic effect with temperature and size of the aggregates. The association rate constants were determined, and a two-step reaction mechanism was demonstrated. The first step is diffusion-controlled. The second is characterized by an activation energy of approximately 2 kcal/mol and an encounter distance of approximately 8.3 angstroms.