Presence Of Sodium Butyrate Research Articles

Chemically Defined Organoid Culture System for Cholangiocyte Differentiation.

Cholangiocyte organoids provide a powerful platform for applications ranging from in vitro modeling to tissue engineering for regenerative medicine. However, their expansion and differentiation are typically conducted in animal-derived hydrogels, which impede the full maturation of organoids into functional cholangiocytes. In addition, these hydrogels are poorly defined and complex, limiting the clinical applicability of organoids. In this study, a novel medium composition combined with synthetic polyisocyanopeptide (PIC) hydrogels to enhance the maturation of intrahepatic cholangiocyte organoids (ICOs) into functional cholangiocytes is utilized. ICOs cultured in the presence of sodium butyrate and valproic acid, a histone deacetylase inhibitor, and a Notch signaling activator, respectively, in PIC hydrogel exhibit a more mature phenotype, as evidenced by increased expression of key cholangiocyte markers, crucial for biliary function. Notably, mature cholangiocyte organoids in PIC hydrogel display apical-out polarity, in contrast to the traditional basal-out polarization of ICOs cultured in Matrigel. Moreover, these mature cholangiocyte organoids effectively model the biliary pro-fibrotic response induced by transforming growth factor beta. Taken together, an animal-free, chemically defined culture system that promotes the ICOs into mature cholangiocytes with apical-out polarity, facilitating regenerative medicine applications and in vitro studies that require access to the apical membrane, is developed.

Multi-omics Data Reveal the Effect of Sodium Butyrate on Gene Expression and Protein Modification in Streptomyces

Streptomycetes possess numerous gene clusters and the potential to produce a large amount of natural products. Histone deacetylase (HDAC) inhibitors play an important role in the regulation of histone modifications in fungi, but their roles in prokaryotes remain poorly understood. Here, we investigated the global effects of the HDAC inhibitor, sodium butyrate (SB), on marine-derived Streptomycesolivaceus FXJ 8.021, particularly focusing on the activation of secondary metabolite biosynthesis. The antiSMASH analysis revealed 33 secondary metabolite biosynthetic gene clusters (BGCs) in strain FXJ 8.021, among which the silent lobophorin BGC was activated by SB. Transcriptomic data showed that the expression of genes involved in lobophorin biosynthesis (ge00097–ge00139) and CoA-ester formation (e.g., ge02824), as well as the glycolysis/gluconeogenesis pathway (e.g., ge01661), was significantly up-regulated in the presence of SB. Intracellular CoA-ester analysis confirmed that SB triggered the biosynthesis of CoA-ester, thereby increasing the precursor supply for lobophorin biosynthesis. Further acetylomic analysis revealed that the acetylation levels on 218 sites of 190 proteins were up-regulated and those on 411 sites of 310 proteins were down-regulated. These acetylated proteins were particularly enriched in transcriptional and translational machinery components (e.g., elongation factor GE04399), and their correlations with the proteins involved in lobophorin biosynthesis were established by protein–protein interaction network analysis, suggesting that SB might function via a complex hierarchical regulation to activate the expression of lobophorin BGC. These findings provide solid evidence that acetylated proteins triggered by SB could affect the expression of genes involved in the biosynthesis of primary and secondary metabolites in prokaryotes.

Immuno-regulatory and anti-inflammatory actions of phycocyanin on Caco-2/U937 cells co-culture as a model of the intestinal barrier

Background: Phycocyanin, a photosynthetic pigment protein of cyanobacteria Spirulina platensis, has been used as a health or functional food because of its health promoting and medicinal effects such as its antioxidant, anti-inflammatory, immunomodulatory, and anticancer effects.Methods: Under the co-culture system using a human colorectal cancer cell line, Caco-2 cells, and differentiated U937 macrophages in the presence of sodium butyrate (SB) and/or lipopolysaccharide (LPS), immune-regulatory and anti-inflammatory activities of phycocyanin were examined.Results: Phycocyanin attenuated a certain damage in the tight junction of co-cultured Caco-2 cells induced by LPS in the presence of SB, and suppressed IL-6 and IL-8 and conversely enhanced TGF-β1 productions. Phagocytic and bactericidal abilities of the differentiated U937 cells were also regulated under the co-cultivation.Conclusions: Phycocyanin contributes to protecting against inflammation and to regulating macrophages in the mucosal immune responses partly through release of cytokines in the presence of butyrate.Keywords: Spirulina, phycocyanin, Caco-2 cells, U937 cells, IL-6, IL-8, TGF-β1

Synthesis, Crystal Structure, and Magnetic Properties of a Family of Undecanuclear [Cu II 9 Ln III 2 ] Nanoclusters

The syntheses, crystal structures, and magnetic properties of a novel family of heterometallic aggregates [CuII9LnIII2(μ3-OH)8(mea)4(C3H7COO)4(NO3)2Cl6(MeOH)4(H2O)2]·3MeOH·2H2O [Ln = Eu (1), Gd (2), Tb (3), Dy (4)] are reported. The compounds were obtained by the direct reaction of monoethanolamine (Hmea) with CuCl2·2H2O and Ln(NO3)3·6H2O in the presence of sodium butyrate (C3H7COONa) in MeOH/MeCN solution. Compounds 1–4 are isomorphous and have an intriguing heterometallic undecanuclear [Cu9Ln2] core that contains a [3 × 3] [Cu9] grid and two [LnCu4] tetragonal pyramids that share a common [Cu4] vertex. Compounds 1–4 are the first high-nuclearity 3d-4f aggregates bearing a monoethanolamine (Hmea) ligand to be reported. The magnetic properties of 1–4 have been investigated by dc and ac susceptibility measurements. A detailed magnetic analysis revealed that antiferromagnetic Cu···Cu interactions and ferromagnetic Cu···Gd couplings exist in [Cu9Gd2] (2), which exhibits a significant magnetocaloric effect. Furthermore, [Cu9Tb2] (3) and [Cu9Dy2] (4) display slow relaxation of the magnetization.

Studying the enucleation process, DNA breakdown and telomerase activity of the K562 cell lines during erythroid differentiation in vitro

During erythropoiesis, some organelles such as mitochondria and nucleus are lost by autophagy and enucleation processes in the presence of macrophages in vivo. In vitro production of erythrocytes has raised many questions about the mechanism of enucleation. The aim of this work was to study the DNA breakdown, enucleation, hemoglobin synthesis and telomerase activity of K562 cells during erythroid differentiation. For these purposes, K562 cells were induced to differentiate by erythropoietin + rhGM-CSF, DMSO, and sodium butyrate separately up to 14 d. In different time intervals, hemoglobin synthesis was evaluated by benzidine staining and RT-PCR for γ-globin gene expression. DNA breakdown was analyzed by 4',6-diamidino-2-phenylindole (DAPI) staining, DNA ladder electrophoresis and comet assay. The telomerase activity was evaluated by TRAP assay. Our result indicated that, sodium butyrate and DMSO inhibited K562 cell growth about 50-60% in comparison to untreated control cells. The percentage of benzidine-positive cells was about 45% in the presence of sodium butyrate after 10 d. Densitometric analysis of RT-PCR and calculated data indicated a 1.5-fold increase in relative γ-globin gene expression at 96 h, in the presence of 1 mM sodium butyrate in comparison with untreated cells. DAPI staining did not reveal any evidence of internal lysis of the nucleus during erythroid differentiation at first wk, but this was obvious in the second wk. DNA laddering pattern was not observed in differentiated cells during 14 d. In comet assay, the percentage of DNA in tail, tail length, and tail moment were significantly different between untreated and treated cells (p < 0.05). Telomerase activity was inhibited up to 90.3% during erythroid differentiation of these cells.

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate (1-3). We used this method to reprogram late passage (>p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged (4,5). These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals( 6), human newborn fibroblasts, as well as human keratinocytes.

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (>p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

Sodium butyrate inhibits pathogenic yeast growth and enhances the functions of macrophages

Butyrate is a short-chain fatty acid that is produced by several human commensal bacteria, such as Clostridium and Lactobacillus species. Butyrate is also known to inhibit histone deacetylase. In this study we assessed the antifungal activity of sodium butyrate (SB) against the human pathogenic yeasts Candida albicans, Candida parapsilosis and Cryptococcus neoformans. The growth and virulence traits of the yeasts were assayed in vitro and during interaction with macrophages in the presence of SB. SB strongly inhibited yeast growth in a concentration-dependent manner, inhibited virulence traits such as filamentation in C. albicans and melanization and capsule formation in C. neoformans and, importantly, significantly decreased yeast biofilm formation. SB also enhanced the antifungal activity of azole drugs. Notably, SB augmented the antifungal activity of macrophages by enhancing the production of reactive oxygen species. The phagocytic rate and killing activity of macrophages significantly increased in the presence of SB, which coincided with an increase in nitric oxide production. These results demonstrate that SB exerts significant antifungal activity on pathogenic yeasts and enhances the antimicrobial actions of macrophages in response to these microbes.

Sodium butyrate enhances the expression of baculovirus-mediated sodium/iodide symporter gene in A549 lung adenocarcinoma cells

Increased expression of sodium/iodide symporter (NIS) is required for reporter gene imaging and effective radioiodine treatment of tumor. We investigated whether increased accumulation of iodine can be induced by sodium butyrate through a newly developed baculoviral transfer of the human NIS (hNIS) gene in A549 human lung adenocarcinoma. A recombinant baculovirus [Bac-cytomegalovirus (CMV)-hNIS] encoding hNIS gene under the control of the CMV promoter was constructed. After A549 cells were transfected with Bac-CMV-hNIS in the presence of sodium butyrate, the expression of hNIS protein was detected by immunofluorescence and western blot analysis. The uptake and efflux of iodine were determined after the incubation of the transfected cells with I-iodide in the presence or absence of sodium butyrate. Immunocytochemical staining and western blot analysis showed increased hNIS protein expression in A549 cells transfected with Bac-CMV-hNIS after sodium butyrate treatment. Bac-CMV-hNIS transfected A549 cells accumulated up to about nine times more I than nontransfected cells; the amount of I uptake increased in a sodium butyrate in dose-dependent manner (P<0.001). However, rapid efflux of radioactivity was observed, with 50% lost during the first 2 min after I-containing medium had been replaced by a nonradioactive medium. Our results indicated that an improved efficiency of baculovirus-mediated hNIS reporter gene imaging in lung adenocarcinoma is possible with treatment with sodium butyrate. However, additional conditions need to be defined to reduce the rapid efflux of radioiodine for the purpose of radionuclide therapy.

Aqueous foam stabilized by plate-like particles in the presence of sodium butyrate

The addition of salt promotes the adsorption of layered double hydroxide (LDH) particles onto the air–water interface, but stable foams cannot be prepared from LDH dispersions at all the concentration of NaCl or sodium acetate. We generated stable foams using positively charged plate-like LDH particles in the presence of sodium butyrate. The effects of adding sodium butyrate to LDH on the particle zeta potential, adsorption behavior and the adsorption of modified particles at the air–water interface were studied. At a fixed LDH particle concentration, adding of a trace amount of sodium butyrate maximizes flocculation of the aqueous particle dispersion. Foams prepared under this condition of particle dispersion are most stable to coalescence and halt completely disproportionation. Also, the size of the bubbles is the smallest. The bubbles are stable when drying at 80 °C with little change in size. Laser-induced fluorescent confocal micrographs and scanning electron microscopy observations clearly confirm the adsorption of LDH particles on the foam surfaces, and the bubbles are armored by an interfacial particle multilayer.

Development of a Hybrid Baculoviral Vector for Sustained Transgene Expression

Baculovirus is a promising gene delivery vector but its widespread application is impeded as it only mediates transient transgene expression in mammalian cells. To prolong the expression, we developed a dual baculovirus system whereby one baculovirus expressed FLP recombinase while the other harbored an Frt-flanking cassette encompassing the transgene and oriP/EBNA1 derived from Epstein-Barr virus. After cotransduction of cells, the expressed FLP cleaved the Frt-flanking cassette off the baculovirus genome and catalyzed circular episome formation, then oriP/EBNA1 within the cassette enabled the self-replication of episomes. The excision/recombination efficiency was remarkably enhanced by sodium butyrate, reaching 75% in human embryonic kidney-293 (HEK293) cells, 85% in baby-hamster kidney (BHK) cells, 77% in primary chondrocytes, and 48% in mesenchymal stem cells (MSCs). The hybrid baculovirus substantially prolonged the transgene expression to approximately 48 days without selection and >63 days with selection, thanks to the maintenance of replicons and transgene transcription. In contrast to the replicating episomes, the baculovirus genome was rapidly degraded. Furthermore, an osteoinductive growth factor gene was efficiently delivered into MSCs using this system, which not only prolonged the growth factor expression but also potentiated the osteogenesis of MSCs. These data collectively implicate the potential of this hybrid baculovirus system in gene therapy applications necessitating sustained transgene expression.

Selective enrichment of hepatocytes from mouse embryonic stem cells with a culture system containing cholestatic serum

There is increasing evidence indicating that embryonic stem (ES) cells are capable of differentiating into hepatocyte-like cells in vitro. However, it is necessary to improve the differentiation efficiency so as to promote the clinical application. Here, we report an efficient culture system to support hepatocyte differentiation from ES cells by utilizing cholestatic serum. One week after the induction of E14 mouse ES cells into hepatocytes with sodium butyrate, cholestatic serum was added into the culture system at various concentrations and hepatocyte-like cells were induced to proliferate. The morphological and phenotypic markers of hepatocytes were characterized using light microscopy, immunocytochemistry, and RT-PCR, respectively. The function of glycogen storage of the differentiated cells was detected by Periodic acid-Schiff (PAS) reaction, and the ratio of hepatic differentiation was determined by counting the albumin and PAS-positive cells. In the presence of conditional selective medium containing cholestatic serum, numerous epithelial cells resembling hepatocytes were observed. The RT-PCR analysis showed that undifferentiated ES cells did not express any hepatic-specific markers; however, in the presence of sodium butyrate and conditional selective medium containing cholestatic serum, hepatic differentiation markers were detected. Immunofluorescence staining showed that those ES-derived hepatocytes were alpha-fetoprotein, albumin, and cytokeratin 18 positive, with the ability of storing glycogen. Further determination of the hepatic differentiation ratio showed that the application of cholestatic serum efficiently enriched ES-derived hepatocyte-like cells by inducing lineage differentiation and enhancing lineage proliferation. The conditional selective medium containing cholestatic serum is optimal to selectively enrich hepatocyte-like cells from mixed differentiated ES cells, which may provide a novel method to improve the hepatic differentiation ratio of ES cells.

Directed Differentiation of Mouse Embryonic Stem Cells into Pancreatic-Like or Neuronal- and Glial-Like Phenotypes

The derivation of definitive endoderm and in particular endocrine cell types from undifferentiated embryonic stem (ES) cells remains difficult to achieve. In this study, we investigated the potential to regulate the differentiation of ES cells into endodermal derivatives using extracellular factors previously associated with various aspects of pancreatic development. Feeder-free-cultured mouse ESD3 cells were manipulated to form embryoid bodies (EBs) in the presence of retinoic acid (RA). RA-treated EBs were subsequently exposed to sodium butyrate (SB), betacellulin (BTC) or activin A (AA). A comparative analysis was performed on these models of directed differentiation in parallel with a model of spontaneous differentiation. Lineage differentiation was determined by profiling multilineage marker transcript expression (neuronal, myogenic, exocrine and endocrine pancreas, extraembryonic and apoptotic) and subsequent protein expression within ES-derived cultures. Using a two-stage differentiation protocol developed during this study, we successfully demonstrated the derivation of an intermediate multipotential population (RA_EBs) from undifferentiated ES cells that preferentially gives rise to pancreatic endocrine insulin-expressing cell types in the presence of SB, and neuronal- and glial-like cell types in the presence of AA or BTC.

Protective effect of sodium butyrate on the cell culture model of Huntington disease

Abstract This study aimed to develop a cell culture model of Huntington disease and observe the effect of sodium butyrate on this cell culture model. Exon 1 of both a wild type and a mutant IT15 gene from the genomic DNA of a healthy adult and a patient with Huntington disease was amplified and cloned into the eukaryotic expression vector pEGFP-C1. Human neuroblastoma SH-SY5Y cells were transiently transfected with these recombinant plasmids in the absence and presence of sodium butyrate (0.1,0.2,0.5,1.0 mmol L). The MTT assay was used to measure cell viability. The results indicated that the N-terminal fragment of mutant huntingtin formed perinuclear and intranuclear aggregates and caused a decrease SH-SY5Y cell viability. Sodium butyrate inhibited the decrease of SH-SY5Y cell viability caused by the N-terminal fragment of mutant huntingtin. This suggests that sodium butyrate has a protective effect on this cell culture model of Huntington disease.

Targeted Molecular Therapy for Inherited Glycosylphosphatidylinositol Deficiency.

Inherited glycosylphosphatidylinositol (GPI) deficiency (IGD) is an autosomal recessive disease characterized by splanchnic vein thrombosis and epilepsy. The partial GPI deficiency in the affected children is due to disrupted addition of the first mannose residue onto the GPI intermediate PI-GlcN, a step catalyzed by the α1>4 mannosyltranferase PIGM. IGD is caused by a −270C>G substitution in the core promoter of PIGM which disrupts binding of the transcription factor (TF) Sp1 to its cognate motif resulting in markedly reduced transcription. Sp1 regulates transcription through interactions an with the basal transcriptional complex or with other TF. In some genes, Sp1 is also required for locus histone acetylation. We investigated whether histone acetylation of the PIGM locus was Sp1-dependent and whether histone deacetylase (HDAC) inhibitors such as sodium butyrate (SB) could be used to enhance PIGM transcription even in the presence of the mutated Sp1 binding motif associated with IGD. Using ChIP assays we first showed that histone 4 (H4) at the PIGM locus is constitutively acetylated in GPI+ EBV B cell lines (LBCL), consistent with the housekeeping function of PIGM. By contrast, the same H4 in GPI- LBCL from individuals with IGD was hypoacetylated indicating that efficient PIGM promoter acetylation requires an intact Sp1 core promoter motif. Upon exposure to SB, acetylation was completely restored in the affected cell lines. Consistent with this, in luciferase reporter assays performed in the presence of mithramycin, an agent that specifically inhibits Sp binding, the enhancing effect of SB on transcription driven by both wild type and mutant (−270C>G) promoter constructs was largely (but not exclusively) Sp1-dependent. The increased transcriptional activity of the mutated PIGM promoter in the presence of SB was accompanied by a more than 100-fold increase in the PIGM mRNA and almost complete restoration of surface GPI expression in the affected LBCL.Over a period of 10 years, one of the children with IGD developed progressive, intractable and incapacitating seizures, despite a multitude of different anti-epileptic treatments including sodium valproate. At the age of 14, she was wheelchair-bound with global hypotonia, extreme drowsiness, poorly responsive, unable to feed herself and almost permanently in minor status with approximately 5 tonic-clonic seizures/day. Extensive CNS imaging showed no structural abnormalities or evidence of thrombosis. An EEG was consistent with minor status epilepticus. Guided by the effectiveness of SB in restoring PIGM transcription and GPI expression in vitro, the patient was commenced on sodium phenylbutyrate (SPB) at an oral dose of 20mg/kg tds. SPB treatment induced a progressive increase in the proportions of GPI+ granulocytes and an increase in PIGM mRNA levels in primary mononuclear blood cells from a pre-SPB level of 1.65% to 26%. A marked clinical improvement was noted after only 2 weeks of treatment, in that the patient could walk, interact with others and feed herself and became entirely seizure-free; she remains so 6 months later. No side effects were noted. These data suggest that SPB may be an effective therapeutic option for diseases caused by Sp1-dependent histone hypoacetylation and also for diseases caused by non-inactivating mutations in the coding region of genes where transcription is regulated by Sp1-dependent histone acetylation.

Identification of Determinants of Ligand Binding Affinity and Selectivity in the Prostaglandin D2 Receptor CRTH2

The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D(2) (PGD(2)) generated in allergic inflammation. The CRTH2 receptor shares greatest sequence similarity with chemoattractant receptors compared with prostanoid receptors. To investigate the structural determinants of CRTH2 ligand binding, we performed site-directed mutagenesis of putative mCRTH2 ligand-binding residues, and we evaluated mutant receptor ligand binding and functional properties. Substitution of alanine at each of three residues in the transmembrane (TM) helical domains (His-106, TM III; Lys-209, TM V; and Glu-268, TM VI) and one in extracellular loop II (Arg-178) decreased PGD(2) binding affinity, suggesting that these residues play a role in binding PGD(2). In contrast, the H106A and E268A mutants bound indomethacin, a nonsteroidal anti-inflammatory drug, with an affinity similar to the wild-type receptor. HEK293 cells expressing the H106A, K209A, and E268A mutants displayed reduced inhibition of intracellular cAMP and chemotaxis in response to PGD(2), whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE(2) by the E268A mutant was enhanced compared with the wild-type receptor, suggesting that Glu-268 plays a role in determining prostanoid ligand selectivity. Replacement of Tyr-261 with phenylalanine did not affect PGD(2) binding but decreased the binding affinity for indomethacin. These results provided the first details of the ligand binding pocket of an eicosanoid-binding chemoattractant receptor.

The effect of sodium butyrate in combination with ATRA on the proliferation/differentiation of SKM-1.

To explore the molecular mechanisms of sodium butyrate working on SKM-1 cell proliferation/differentiation and to study its synergistic effect with all-trans retinoic acid (ATRA), SKM-1 cells were grown in the absence or presence of sodium butyrate and/or ATRA. The percentage of viable cells was determined by trypan blue exclusion. Differentiation was determined by nitroblue tetrazolium (NBT) reduction and cell surface adhesion molecules was analyzed by FACS. Cell cycle distribution was examined after DNA staining by propidium iodide. D-type cyclins, cdks and P21 mRNA were studied by reverse transcription-polymerase chain reaction. Our results showed that sodiun butyrate and/or ATRA blocked cells mainly in the G0/G1 phase of the cell cycle. ATRA inhibited the mRNA expression of CDK6, CDK4, cyclinD3 and cyclinD1. Sodium butyrate inhibited the mRNA expression of CDK2, cyclinD2 and cyclinD1. ATRA and sodium butyrate inhibited the mRNA expression of CDK6, CDK4, CDK2, cyclinD1, cyclinD2 and cyclinD3. Both ATRA and/or sodium butyrate stimulated p21 expression at the mRNA levels. Our results suggest that the effect of sodium butyrate on cell proliferation/differentiation might be linked to its ability to induce expression of p21 mRNA and inhibit the cyclin-cdk complexes. Our observations support the notion that the sodium butyrate works synergistically with ATRA.

Ultrasound imaging of apoptosis in vivo: effects of subcellular nuclear structure and cell membrane morphology

Objective: We have discovered that high-frequency ultrasound imaging may be used to detect apoptosis. Apoptotic cells have striking morphological characteristics including an altered nuclear chromatin structure and an altered cell membrane topology. In this study, we investigated the effects of chromatin structure and cell membrane morphology on ultrasound backscatter signal intensity and spectra. Methods: To investigate the importance of subcellular nuclear structure and chromatin structure on ultrasound backscatter signal formation, we used acute myeloid leukemia cells and their nuclei prepared in over 10 different ionic environments. Ionic environments ranged from 4 mM to over 2000 mM NaCl and were recognized to cause specific configurational changes in chromatin structure. Cells were also grown in the presence of sodium butyrate, which is a histone-deacetylase inhibitor that promotes the accumulation of chromatin in a unraveled structural state consistent with gene expression. Other disruptions of chromatin structure were carried out by enzymatic digestions of DNA. To study the effects of cell membrane morphology changes, red blood cells were used as this provided a cell system devoid of a nucleus. Erythrocytes were placed into different ionic and pH environments causing them to adopt different sizes and shapes ranging from echinocytic to stomatocytic. Light and electron microscopy were used to assess cell and chromatin morphology. Results: Results indicated an inverse correlation between nuclear size and ultrasound backscatter intensity. Results were also dependent on chromatin configuration. Red blood cell experiments indicated that the absence of a cell nucleus membrane morphology can also have an important influence on ultrasound backscatter signals. Conclusions: Results indicated an inverse correlation between nuclear size and ultrasound backscatter intensity. Backscatter signals were also dependent on chromatin configuration as assessed from ionic strength, butyrate, and DNA digestion experiments. Red blood cell experiments indicated cell nucleus membrane morphology and can also have an important influence on ultrasound backscatter signals.

Calmodulin-Like Skin Protein: A New Marker of Keratinocyte Differentiation

The expression of the calmodulin-like skin protein, a recently discovered new skin-specific calcium binding protein, was studied in cultured keratinocytes, reconstructed human epidermis, and normal human skin. Using a calmodulin-like skin protein specific polyclonal antibody and Western blot analysis we could show that in cultured keratinocytes calmodulin-like skin protein expression is strongly induced after stimulating cell differentiation by increasing the medium calcium concentration. Known modulators of epidermal differentiation such as sodium butyrate and the synthetic retinoid CD 367 strongly affected calmodulin-like skin protein expression. A more than 10-fold increase was observed in the presence of sodium butyrate, whereas CD 367 abolished almost completely calmodulin-like skin protein expression already at nanomolar concentrations. Calmodulin, another calcium binding protein that is expressed throughout the living layers of the epidermis, is not affected by these modulators. In normal human skin, calmodulin-like skin protein expression is restricted to the stratum granulosum and the lower layers of the stratum corneum. From these results we conclude that calmodulin-like skin protein is a new marker of late keratinocyte differentiation with a role distinct from calmodulin.

Butyrate metabolism upstream and downstream acetyl-CoA synthesis and growth control of human colon carcinoma cells.

Butyrate is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibers in the colon lumen which severely affects the proliferation of colon cancer cells in in vitro experiments. Although butyrate is able to interfere with numerous cellular targets including cell cycle regulator expression, little is known about butyrate metabolism and its possible involvement in its effect upon colon carcinoma cell growth. In this study, we found that HT-29 Glc-/+ cells strongly accumulated and oxidized sodium butyrate without producing ketone bodies, nor modifying oxygen consumption nor mitochondrial ATP synthesis. HT-29 cells accumulated and oxidized sodium acetate at a higher level than butyrate. However, sodium butyrate, but not sodium acetate, reduced cell growth and increased the expression of the cell cycle effector cyclin D3 and the inhibitor of the G1/S cdk-cyclin complexes p21/WAF1/Cip1, demonstrating that butyrate metabolism downstream of acetyl-CoA synthesis is not required for the growth-restraining effect of this SCFA. Furthermore, HT-29 cells modestly incorporated the 14C-labelled carbon from sodium butyrate into cellular triacylglycerols and phospholipids. This incorporation was greatly increased when D-glucose was present in the incubation medium, corresponding to the capacity of hexose to circulate in the pentose phosphate pathway allowing NADPH synthesis required for lipogenesis. Interestingly, when HT-29 cells were cultured in the presence of sodium butyrate, their capacity to incorporate 14C-labelled sodium butyrate into triacylglycerols and phospholipids was increased more than twofold. In such experimental conditions, HT-29 cells when observed under an electronic microscope, were found to be characterized by an accumulation of lipid droplets in the cytosol. Our data strongly suggest that butyrate acts upon colon carcinoma cells upstream of acetyl-CoA synthesis. In contrast, the metabolism downstream of acetyl-CoA [i.e. oxidation in the tricarboxylic acid (TCA) cycle and lipid synthesis] likely acts as a regulator of butyrate intracellular concentration.