Polyelectrolyte membrane scaffold sustains growth of neuronal cells.

Cell immobilization within nano-thin polymeric shells can provide an optimal concentration of biological material in a defined space and facilitate its directional growth. Herein, polyelectrolyte membrane scaffolds were constructed using a layer-by-layer approach to determine the possibility of promoting improved growth of rat cortical neuronal cells. Membrane presence was confirmed by Fourier transform infrared spectroscopy, Zeta potential, and atomic force and scanning electron microscopy. Scaffold performance toward neuronal cell growth was assessed in vitro during a 14-day culture. Cell conditions were analyzed immunocytochemically. Furthermore, western blot and real-time PCR analyses were used to validate the presence of neuronal and glial cells on the scaffolds. We observed that alginate/chitosan, alginate/polylysine, and polyethyleneimine/chitosan scaffolds promote neuronal growth similarly to the control, poly-d-lysine/laminin. We conclude that membranes maintaining cell viability, integrity and immobilization in systems supporting neuronal regeneration can be applied in neurological disease or wound healing treatment. © 2018 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 839-850, 2019.

SIRT1, miR-132 and miR-212 link human longevity to Alzheimer's Disease.

Alzheimer's Disease (AD) is the most common cause of dementia in the elderly. Centenarians - reaching the age of >100 years while maintaining good cognitive skills - seemingly have unique biological features allowing healthy aging and protection from dementia. Here, we studied the expression of SIRT1 along with miR-132 and miR-212, two microRNAs known to regulate SIRT1, in lymphoblastoid cell lines (LCLs) from 45 healthy donors aged 21 to 105 years and 24 AD patients, and in postmortem olfactory bulb and hippocampus tissues from 14 AD patients and 20 age-matched non-demented individuals. We observed 4.0-fold (P = 0.001) lower expression of SIRT1, and correspondingly higher expression of miR-132 (1.7-fold; P = 0.014) and miR-212 (2.1-fold; P = 0.036), in LCLs from AD patients compared with age-matched healthy controls. Additionally, SIRT1 expression was 2.2-fold (P = 0.001) higher in centenarian LCLs compared with LCLs from individuals aged 56-82 years; while centenarian LCLs miR-132 and miR-212 indicated 7.6-fold and 4.1-fold lower expression, respectively. Correlations of SIRT1, miR-132 and miR-212 expression with cognitive scores were observed for AD patient-derived LCLs and postmortem AD olfactory bulb and hippocampus tissues, suggesting that higher SIRT1 expression, possibly mediated by lower miR-132 and miR-212, may protect aged individuals from dementia and is reflected in their peripheral tissues.

Identification of tetrahydrocarbazoles as novel multifactorial drug candidates for treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder and the most frequent cause of dementia. To date, there are only a few approved drugs for AD, which show little or no effect on disease progression. Impaired intracellular calcium homeostasis is believed to occur early in the cascade of events leading to AD. Here, we examined the possibility of normalizing the disrupted calcium homeostasis in the endoplasmic reticulum (ER) store as an innovative approach for AD drug discovery. High-throughput screening of a small-molecule compound library led to the identification of tetrahydrocarbazoles, a novel multifactorial class of compounds that can normalize the impaired ER calcium homeostasis. We found that the tetrahydrocarbazole lead structure, first, dampens the enhanced calcium release from ER in HEK293 cells expressing familial Alzheimer's disease (FAD)-linked presenilin 1 mutations. Second, the lead structure also improves mitochondrial function, measured by increased mitochondrial membrane potential. Third, the same lead structure also attenuates the production of amyloid-beta (Aß) peptides by decreasing the cleavage of amyloid precursor protein (APP) by ß-secretase, without notably affecting α- and γ-secretase cleavage activities. Considering the beneficial effects of tetrahydrocarbazoles addressing three key pathological aspects of AD, these compounds hold promise for the development of potentially effective AD drug candidates.

Postnatal isoform switch and protein localization of LEF1 and TCF7L2 transcription factors in cortical, thalamic, and mesencephalic regions of the adult mouse brain.

ß-Catenin signaling, leading to the activation of lymphoid enhancer-binding factor 1/T cell factor (LEF1/TCF) transcription factors, plays a well-established role in transcription regulation during development and tissue homeostasis. In the adult organism, the activity of this pathway has been found in stem cell niches and postmitotic thalamic neurons. Recently, studies show that mutations in components of ß-catenin signaling networks have been associated with several psychiatric disorders, indicating the involvement of ß-catenin and LEF1/TCF proteins in the proper functioning of the brain. Here, we report a comprehensive analysis of LEF1/TCF protein localization and the expression profile of their isoforms in cortical, thalamic, and midbrain regions in mice. We detected LEF1 and TCF7L2 proteins in neurons of the thalamus and dorsal midbrain, i.e., subcortical regions specialized in the integration of diverse sources of sensory information. These neurons also exhibited nuclear localization of ß-catenin, suggesting the involvement of ß-catenin/LEF1/TCF7L2 in the regulation of gene expression in these regions. Analysis of alternative splicing and promoter usage identified brain-specific TCF7L2 isoforms and revealed a developmentally coordinated transition in the composition of LEF1 and TCF7L2 isoforms. In the case of TCF7L2, the typical brain isoforms lack the so-called C clamp; in addition, the dominant-negative isoforms are predominant in the embryonic thalamus but disappear postnatally. The present study provides a necessary framework to understand the role of LEF1/TCF factors in thalamic and midbrain development until adulthood and predicts that the regulatory role of these proteins in the adult brain is significantly different from their role in the embryonic brain or other non-neural tissues.

Conformationally altered p53: a novel Alzheimer's disease marker?

The identification of biological markers of Alzheimer's disease (AD) can be extremely useful to improve diagnostic accuracy and/or to monitor the efficacy of putative therapies. In this regard, peripheral cells may be of great importance, because of their easy accessibility. After subjects were grouped according to diagnosis, the expression of conformationally mutant p53 in blood cells was compared by immunoprecipitation or by a cytofluorimetric assay. In total, 104 patients with AD, 92 age-matched controls, 15 patients with Parkinson's disease and 9 with other types of dementia were analyzed. Two independent methods to evaluate the differential expression of a conformational mutant p53 were developed. Mononuclear cells were analyzed by immunoprecipitation or by flow-cytometric analysis, following incubation with a conformation-specific p53 antibody, which discriminates unfolded p53 tertiary structure. Mononuclear cells from AD patients express a higher amount of mutant-like p53 compared to non-AD subjects, thus supporting the study of conformational mutant p53 as a new putative marker to discriminate AD from non-AD patients. We also observed a strong positive correlation between the expression of p53 and the age of patients. The expression of p53 was independent from the length of illness and from the Mini Mental State Examination value.

The Alzheimer disease-related calcium-binding protein Calmyrin is present in human forebrain with an altered distribution in Alzheimer's as compared to normal ageing brains.

The EF-hand calcium binding protein Calmyrin (also called CIB-1) was shown to interact with presenilin-2 (PS-2), suggesting that this interaction might play a role in the pathogenesis of Alzheimer's disease (AD). Here we have investigated the distribution of Calmyrin in normal human and AD brain. In normal brain Calmyrin immunoreactivity was unevenly distributed with immunostaining in pyramidal neurones and interneurones of the palaeo-cortex and neocortex, cerebellar granule cells and hypothalamic neurones of the paraventricular, ventromedial and arcuate nuclei. Moderate immunoreactivity was present in hippocampal pyramidal cells and stronger in dentate gyrus neurones. Thalamic and septal neurones were devoid of immunoreactivity. No apparent differences were visible between stainings of brain sections from younger and older nondemented patients. In AD brain a substantial loss of Calmyrin-immunopositive neurones was observed in all regions, especially in cortical areas. Still immunoreactive neurones, however, displayed stronger staining that was especially concentrated in perinuclear regions. Calmyrin immunosignals were in part associated with diffuse and senile plaques. Thus, although protein levels of Calmyrin are low in human forebrain, its cellular localization as well as its altered distribution in AD brain suggest that it may be involved in the pathogenesis of AD.

The -22c/t polymorphism in presenilin 1 gene is not connected with late-onset and early-onset familial Alzheimer's disease in Poland.

The -22c/t polymorphism in the promoter of the presenilin 1 gene is associated with increased risk for Alzheimer's disease (AD) in some populations. It was shown that -22c allele is connected with two-fold decrease in promoter activity. We studied the impact of the polymorphism in groups of Polish late-onset and early-onset AD patients. Our results suggest that -22c/t polymorphism is not connected with AD in Polish population. The -22t allele showed a high degree of linkage disequilibrium with -2797 insertion of 13 bp. An additional -2923g/t polymorphism is also not connected with -22c/t and is not a risk factor for AD.

Structural and biochemical characterization of neuronal calretinin domain I-II (residues 1-100). Comparison to homologous calbindin D28k domain I-II (residues 1-93).

This study characterizes the calcium-bound CR I-II domain (residues 1-100) of rat calretinin (CR). CR, with six EF-hand motifs, is believed to function as a neuronal intracellular calcium-buffer and/or calcium-sensor. The secondary structure of CR I-II, defined by standard NMR methods on 13C,15N-labeled protein, contains four helices and two short interacting segments of extended structure between the calcium-binding loops. The linker between the two helix-loop-helix, EF-hand motifs is 12 residues long. Limited trypsinolysis at K60 (there are 10 other K/R residues in CR I-II) confirms that the linker of CR I-II is solvent-exposed and that other potential sites are protected by regular secondary structure. 45Ca-overlay of glutathione S-transferase (GST)-CR(1-60) and GST-CR(61-100) fusion proteins confirm that both EF-hands of CR I-II have intrinsic calcium-binding properties. The primary sequence and NMR chemical shifts, including calcium-sensitive glycine residues, also suggest that both EF-hand loops of CR I-II bind calcium. NMR relaxation, analytical ultracentrifugation, chemical cross-linking and NMR translation diffusion measurements indicate that CR I-II exists as a monomer. Calb I-II (the homologous domain of calbindin D28k) has the same EF-hand secondary structures as CR I-II, except that helix B is three residues longer and the linker has only four residues [Klaus, W., Grzesiek, S., Labhardt, A. M., Buckwald, P., Hunziker, W., Gross, M. D. & Kallick, D. A. (1999) Eur. J. Biochem. 262, 933-938]. In contrast, Calb I-II binds one calcium cation per monomeric unit and exists as a dimer. Despite close homology and similar secondary structures, CR I-II and Calb I-II probably have distinct tertiary structure features that suggest different cellular functions for the full-length proteins.

Upstream stimulatory factor is involved in the regulation of the human calcyclin (S100A6) gene.

Calcyclin (S100A6) is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity. The calcyclin gene promoter fragment -361/-167 activates transcription several fold when compared to the basal -167/+134 promoter fragment indicating the presence of enhancer element within -361/-167 bp region. By means of the electrophoretic mobility shift assay (EMSA) we found that this region contains a protein-binding site and mapped it to an E-box sequence at position -283/-278. Using antibodies against USF1 we identified the upstream stimulatory factor as the transcription factor bound to the E-box sequence in EMSA. This factor was also enriched in protein fractions obtained from Ehrlich ascites tumor cells nuclear extract by affinity chromatography using the E-box sequence as a ligand. Cotransfection of the USF1 expression vector with a plasmid carrying the luciferase gene under control of the -361/+134 calcyclin gene promoter fragment resulted in several fold activation of luciferase activity. On the other hand, mutations within the E-box led to a marked decrease in the efficiency of calcyclin gene promoter fragment. The results indicate that USF1 binds to an E-box sequence of the calcyclin gene promoter and enhances its transcription activity. This mechanism might be responsible for the upregulation of calcyclin gene expression in response to various stimuli and in tumors.

Calcyclin (S100A6) binding protein (CacyBP) is highly expressed in brain neurons.

The expression of a novel calcyclin (S100A6) binding protein (CacyBP) in different rat tissues was determined by Western and Northern blotting. Polyclonal antibodies against recombinant CacyBP purified from E. coli exhibited the highest reaction in the brain and weaker reaction in liver, spleen, and stomach. CacyBP immunoreactivity was also detected in lung and kidney. Densitometric analysis showed that the concentration of CacyBP in the soluble fractions of total brain and cerebellum is approximately 0.17 and 0. 34 ng/microg protein, respectively. Northern blotting with a specific cDNA probe confirmed the high level of CacyBP expression in the rat brain and lower levels in other tissues examined. Immunohistochemistry and in situ hybridization of rat brain sections revealed strong expression of CacyBP in neurons of the cerebellum, hippocampus, and cortex. The in situ hybridization detected CacyBP in hippocampus as early as P7 (postnatal day 7) and a peak of expression at P21, and the expression signal was preserved until adulthood. In the entorhinal cortex, the peak of expression was observed at P7, whereas in the cerebellum it was seen at P21. The results presented here show that CacyBP is predominantly a neuronal protein. (J Histochem Cytochem 48:1195-1202)

Characterization of the interaction of calcyclin (S100A6) and calcyclin-binding protein.

Calcyclin (S100A6) is an S100 calcium-binding protein whose expression is up-regulated in proliferating and differentiating cells. A novel 30-kDa protein exhibiting calcium-dependent calcyclin-binding (calcyclin-binding protein, CacyBP) had been identified, purified, and cloned previously (Filipek, A., and Kuznicki, J. (1998) J. Neurochem. 70, 1793-1798). Here, we have defined the calcyclin binding region using limited proteolysis and a set of deletion mutants of CacyBP. A fragment encompassing residues 178-229 (CacyBP-(178-229)) was capable of full binding to calcyclin. CacyBP-(178-229) was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified. The protein fragment cleaved from the glutathione S-transferase fusion protein was shown by CD to contain 5% alpha-helix, 15% beta -sheet, and 81% random coil. Fluorescence spectroscopy was used to determine calcyclin dissociation constants of 0.96 and 1.2 microm for intact CacyBP and CacyBP-(178-229), respectively, indicating that the fragment can be used for characterization of calcyclin-CacyBP interactions. NMR analysis of CacyBP-(178-229) binding-induced changes in the chemical shifts of (15)N-enriched calcyclin revealed that CacyBP binding occurs at a discrete site on calcyclin with micromolar affinity.

Application and maintenance habits do make a difference in adhesion of Alora transdermal systems.

OBJECTIVES: To explore and evaluate Alora placebo patch application and maintenance habits of women in order to identify the factors that influence adhesion success. METHODS: This single-center, open-label, placebo, randomized, multiple-application, parallel-group study involved 99 healthy naïve users of transdermal patches. Participants applied and wore an Alora placebo patch for ten consecutive applications of approximately 3.5 days each and evaluated adhesion of the patches twice-daily. Three subgroups comprising participants achieving low, moderate or high adhesion success took part in focus groups to discuss their wear habits, practices and attitudes regarding transdermal patches. RESULTS: There was a significant behavioral component involved in patch adhesion. The habits, practices and attitudes of high achievers were clearly different from the other two subgroups. The three most important issues identified to improve adhesion were: mastering the removal of the patch liner, identifying the best site of application, and developing and implementing techniques to maintain patch adhesion. The Alora placebo patch was well tolerated throughout the study. CONCLUSION: The data showed that there is a learning curve involved in achieving maximal adhesion with a transdermal patch. During the study, a novel patch application method ('press, fold and slide') was demonstrated for the participants. This method was very well received by all participants and was more easily executed than the previous method. An adaptation of this method was incorporated into the Alora patient information leaflet, together with several other changes to help improve adhesion success.

Regulation of cell specific expression of calcyclin (S100A6) in nerve cells and other tissues.

Many of the small, acidic, calcium binding S100 proteins present in the brain specifically map different anatomical regions and cell types and their overexpression is implicated in pathological changes. Similarly to other members of the S100 protein family, calcyclin (S100A6) is expressed in a cell specific manner and is found in subpopulations of neurons and astrocytes in the brain and in epithelial cells and fibroblasts. In this article we review data concerning the cell specific expression of S100 protein genes and present experimental results on the regulation of the calcyclin gene. We have performed promoter deletion studies to locate regions within the calcyclin gene promoter responsible for transcriptional regulation. The results demonstrate that the 3 kb long calcyclin gene promoter lacks a cell specific cis-acting element and drives the expression of the reporter gene also in cells that do not express endogenous calcyclin. The expression is modulated by positive and negative elements acting uniformly in the four different cell lines studied. The first intron of the calcyclin gene was found to have an inhibitory influence on expression regardless of cell type. It was also shown that calcyclin expression can be induced in calcyclin-negative cells by treatment with 5-azacytidine suggesting the involvement of gene methylation in its cell specific expression. The results are discussed in light of the data available on the regulation of other S100 genes.

Use of Pichia pastoris for the expression, purification, and characterization of rat calretinin "EF-hand" domains.

Calretinin (CR) is a calcium-binding, neuronal protein of undefined function. Related proteins either buffer intracellular calcium concentrations or are involved in calcium-signaling pathways. We transformed three CR gene fragment sequences, corresponding to its three complementary domains (I-II, III-IV, and V-VI), into Pichia pastoris. High yields of extracellular expression, of more than 200 mg/liter, were achieved. Simple purification protocols provide high yields of homogenous proteins: dialysis and DEAE-cellulose chromatography for domains I-II and III-IV or ammonium sulfate precipitation and octyl-Sepharose chromatography for domain V-VI. To our knowledge, this is the first report of the expression of an EF-hand protein using P. pastoris. Direct comparison of the purified yields of domain I-II indicates a approximately 20-fold improvement over Escherichia coli. N-terminal amino acid sequencing confirmed our gene products and two anti-calretinin antibodies recognized the appropriate domains. All three CR domains bind (45)Ca and the domain containing EF-hands V and VI seems to have a lower calcium capacity than the other domains. Circular dichroism indicates a high helix content for each of the domains. Calcium-induced structural changes in the first two domains, followed by tryptophan fluorescence, correspond with previous studies, while tyrosine emission fluorescence indicates calcium-induced structural changes also occur in domain V-VI. The methods and expression levels achieved are suitable for future NMR labeling of the proteins, with (15)N and (13)C, and structure-function studies that will help to further understand CR function.

Calretinin--sensor or buffer--function still unclear.

Calretinin (CR) is a 31.5 kDa EF-hand calcium binding protein. CR has a general neuronal localization and is a predominantly cytosolic protein. The biochemical properties of this protein are well characterized but its function is still unclear. It is often postulated that the presence of CR correlates with an increased survival ability of cells under pathological conditions connected with increased intracellular calcium levels. However, not all studies confirm such a relationship and they indicate that the presence of CR does not protect cells against calcium overload and that CR does not act as an intracellular calcium buffer. Recent results concerning CR function are discussed and the conclusion is proposed that CR acts as a calcium modulator rather than a calcium buffer.

[Regulation of protein S100 expression during transcription]. / Regulacja ekspresji bialek S100 na poziomie transkrypcji.

The S100 protein family is a group of homologous, small, calcium-binding proteins expressed in a cell specific manner. In man, the genes coding the majority of these proteins are localized in a cluster on chromosome 1. The cell specific expression of these proteins is mainly attained at the transcriptional level. It appears that the promoters of some S100 genes (rat S100A4, mouse and human S100B and rat CALB3) contain inhibitory sequences which reduce transcription level. Their effect may be counteracted by an interaction with cell specific transcription factor(s), which allows transcription in certain cell types. In other cases (human S100A2, mouse S100A4 and human S100A6), the main mechanism controlling cell specific expression seems to be methylation of the promoter or intronic sequences leading to gene silencing in some cells. The level of protein expression might be further modulated by different regulatory sequences that respond, via interactions with specific transcription factors, to various extracellular stimuli.

Measurements of [Ca2+] using fura-2 in glioma C6 cells expressing calretinin with GFP as a marker of transfection: no Ca2+-buffering provided by calretinin.

Glioma C6 cells were transfected with a plasmid containing the calretinin (CR) and green fluorescent protein (GFP) coding regions to analyze the effect of CR's presence on [Ca2+]i. Positive transfectants were identified by the detection of GFP and [Ca2+]i was measured using fura-2 as a probe. We found that neither the basic [Ca2+]i nor activated [Ca2+]i achieved by exposure to ionomycin, ADP or thapsigargin were affected by CR's presence in transfected cells, despite the ability of CR to bind Ca2+ as part of fusion protein. The level of expressed CR was estimated as at least 1 microM. The presented results suggest that CR's function is unlikely to be an intracellular Ca2+-buffer and support the hypothesis that CR might be involved in a specific Ca2+-dependent process. The results of this work also show that the S65T mutant of GFP is compatible with fura-2 measurements of intracellular [Ca2+]. We have demonstrated that the presence of GFP, as a transfection marker of glioma C6 cells, does not disturb fura-2 fluorescence, the basal or activated [Ca2+]i in these cells.

Molecular cloning and expression of a mouse brain cDNA encoding a novel protein target of calcyclin.

A protein target of mouse calcyclin, p30, which we call calcyclin-binding protein (CacyBP), was identified in mouse brain and Ehrlich ascites tumor (EAT) cells. The amino acid sequence of the CacyBP chymotryptic peptide was used to prepare synthetic oligonucleotides that served as a probe to screen the mouse brain cDNA library. A 1.4-kb positive clone was detected, isolated, and sequenced. The analyzed clone contains an open reading frame encoding a protein of a molecular mass of approximately 26 kDa. The nucleotide and predicted amino acid sequences indicate that CacyBP is a novel protein. The results obtained from northern blots show that the CacyBP gene is expressed predominantly in mouse brain and EAT cells. Using a pGEX vector the recombinant CacyBP was expressed in Escherichia coli, and its properties were analyzed. The recombinant protein interacts with calcyclin at a physiologically relevant range of Ca2+ in solution during affinity chromatography and on blots. Because CacyBP, like calcyclin, is present in the brain, the interaction of these two proteins might be involved in calcium signaling pathways in neuronal tissue.

A model for target protein binding to calcium-activated S100 dimers.

S100 proteins are a family of dimeric calcium-binding proteins implicated in several cancers and neurological diseases. Calbindin D9k is an unusual monomeric member of the S100 family. A calbindin D9k mutant containing a novel calcium-induced helix is characterized. Based on sequence comparison, this helix could be a component of other S100 proteins and a factor in target protein binding. The origin of structural differences between three reported apo S100 dimer structures is verified. We conclude that the differences are a result of modeling rather than a function of different target binding properties. A mechanism for target protein binding is suggested.

The mouse calretinin gene promoter region: structural and functional components.

The 5' flanking region of the mouse calretinin gene was cloned and a 1.8 kbp region adjacent to exon 1 was sequenced. Putative upstream promoter and enhancer elements were identified, including appropriately positioned TATA and CAAT boxes (positions -50 and -68, respectively). There was considerable sequence and structural homology between mouse and human upstream elements. Neuron-restrictive activity was demonstrated via transfection of calretinin promoter-reporter constructs into primary embryonic mouse brain cultures expressing calretinin. In promoterless reporter constructs, the proximal upstream 1.5 kbp of the mouse calretinin gene boosted luciferase activity (up to 100-fold) exclusively in the neuronal population. Deletion analysis revealed the minimal promoter to be within the 95-bp proximal to the transcription start site. Transfections with SV40 promoter constructs in these cultures resulted in reporter gene expression predominantly in non-neuronal cells. Inserting the proximal 1.5 kbp of mouse calretinin upstream in SV40 promoter-reporter constructs reduced luciferase activity. Thus, calretinin upstream sequences increased reporter expression in cultured neurons and decreased expression from the SV40 promoter in non-neuronal cultured brain cells. The calretinin promoter contained relevant regulatory element consensus motifs and demonstrated in vitro neuron-restrictive bioactivity.