Carbon Nanotube-Mediated Delivery of PTEN Variants: In Vitro Antitumor Activity in Breast Cancer Cells.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a crucial tumor suppressor protein with frequent mutations and alterations. Although protein therapeutics are already integral to numerous medical fields, their potential remains nascent. This study aimed to investigate the impact of stable, unphosphorylated recombinant human full-length PTEN and its truncated variants, regarding their tumor suppression activity with multiwalled-carbon nanotubes (MW-CNTs) as vehicles for their delivery in breast cancer cells (T-47D, ZR-75-1, and MCF-7). The cloning, overexpression, and purification of PTEN variants were achieved from E. coli, followed by successful binding to CNTs. Cell incubation with protein-functionalized CNTs revealed that the full-length PTEN-CNTs significantly inhibited cancer cell growth and stimulated apoptosis in ZR-75-1 and MCF-7 cells, while truncated PTEN fragments on CNTs had a lesser effect. The N-terminal fragment, despite possessing the active site, did not have the same effect as the full length PTEN, emphasizing the necessity of interaction with the C2 domain in the C-terminal tail. Our findings highlight the efficacy of full-length PTEN in inhibiting cancer growth and inducing apoptosis through the alteration of the expression levels of key apoptotic markers. In addition, the utilization of carbon nanotubes as a potent PTEN protein delivery system provides valuable insights for future applications in in vivo models and clinical studies.

Caecal endometriosis presenting with an acute abdomen in pregnancy.

Endometriosis is defined as the presence of endometrial tissue outside the uterus, which induces a chronic inflammatory response. Its prevalence remains unknown, but it has been estimated to affect up to 10% of women of reproductive age. Although it is a benign oestrogen-dependent gynaecological condition, women may describe painful symptoms such as cyclical pelvic pain, dysmenorrhoea and dyschezia. Intestinal endometriosis may affect the ileum, appendix, sigmoid colon and rectum. It may present with a myriad of symptoms such as abdominal pain, vomiting, diarrhoea, constipation and haematochezia. Caecal endometriosis can present as an acute appendicitis, making the diagnosis challenging to establish in pregnancy. Transmural involvement and acute occlusion are very rare events. The gold standard for diagnosis remains laparoscopy with tissue sampling for histological confirmation. Although endometriosis improves during pregnancy under the effect of progesterone, the ectopic endometrium becomes decidualised with a progressive reduction in size. The authors present the case of a multiparous woman in her mid-30s with acute onset of right-sided abdominal pain at 35 weeks gestation. Physical examination was suggestive of an acute appendicitis and MRI showed an inflamed caecum. She became acutely unwell requiring an emergency caesarean section. A mass in the caecum was observed with impending perforation at the caecal pole. A right hemicolectomy was performed. Histopathological examination confirmed the diagnosis of endometriosis with decidualisation. Although endometriosis improves during pregnancy, this case shows the unexpected complications of the disease and demonstrates the importance of considering endometriosis in the differential diagnosis of an acute abdomen in women of childbearing age to prevent maternal morbidity and fetal loss.

The Synthesis of 1,3,5-triazine Derivatives and JNJ7777120 Analogues with Histamine H4 Receptor Affinity and Their Interaction with PTEN Promoter.

The involvement of histamine and H4 receptor (H4 R) in cancer has been investigated recently using the H4 R agonists and antagonists. The scope of the research project was synthesis and exploration of the consequences of a group of compounds with histamine H4 receptor (H4 R) affinity on the promoter of PTEN gene encoding the antitumor PTEN protein. The series of novel compounds based either on H4 R antagonists JNJ7777120 structure or 1,3,5-triazine scaffold were synthesized, evaluated for histamine H4 R affinity and used in this study. Compounds 5 and 7 belonging to the group of JNJ7777120 analogues showed the highest interaction with the promoter of PTEN gene and weak affinity against H4 R with Ki value >100 µm. These compounds showed no significant effect on neuroblastoma IMR-32 cells viability indicating no correlation between PTEN gene promoter affinity and antitumor activity. Compound 6, another JNJ7777120 analogue, showed the highest effect on IMR-32 viability with calculated IC50 = 23.27 µm. The 1,3,5-triazine derivatives exhibited generally low or medium interaction with PTEN gene promoter. However, the 1,3,5-triazine derivative 11 with the para-bromo substituent showed the highest affinity against H4 R with Ki value of 520 nm and may be considered as a new lead structure.

Tumor suppressor PTEN in breast cancer: heterozygosity, mutations and protein expression.

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is one of the most frequently mutated human tumor suppressor genes, implicated in cell growth and survival and suppressing tumor formation. Loss of PTEN activity, either at the protein or genomic level, has been related to many primary and metastatic malignancies including breast cancer. The present study investigates the heterozygosity, mutation spectrum and protein expression of PTEN in 43 patients with breast cancer or precursor lesions of the breast and 10 healthy individuals. Microsatellite analysis at the PTEN locus using D10S215, D10S541 and D10S579 markers indicated that the observed heterozygosity (Ho) is lower than the expected heterozygosity (Hs) in benign and malignant breast disease. Mutational analysis in exons 1, 5, 7 and 9 of the PTEN gene revealed several mutations, most of which cause truncation of the PTEN protein and consequently loss of activity. Increased circulating levels of PTEN and phosphorylated PTEN protein were also observed by immunostaining in patients with breast cancer and precursor breast lesions. In support, increased PTEN protein expression was detected in corresponding tissue specimens. Our data suggest an association between breast cancer and PTEN mutations, resulting in the production of truncated forms of the corresponding protein, thus indicating that breast carcinogenesis is potentially related to PTEN loss of activity rather than loss of expression. Peripheral blood sampling may provide an advantageous application for the determination of PTEN gene mutations and its protein expression in human cancer.

Mannose binding lectin and ficolin-2 polymorphisms are associated with increased risk for bacterial infections in children with B acute lymphoblastic leukemia.

BACKGROUND: We aimed to investigate whether the presence of mannose binding lectin (MBL2), ficolin 2 (FCN2) polymorphisms or the combined deficiency significantly influence the risk and subsequently the frequency of chemotherapy-induced bacterial infections in children with B acute lymphoblastic leukemia (B-ALL). PROCEDURE: MBL2 polymorphisms for exon 1 and FCN2 polymorphisms for promoter regions -986, -602, -557, -64, -4 and exon 8 regions +6,359, +6,424 were determined in children with B-ALL. FCN2 haplotype was determined by gene sequencing. Number and duration of FN episodes as well as number of bacterial infections were recorded during induction chemotherapy. RESULTS: Forty-four children with B-ALL (median age 4.3 years, 65.9% males) suffered from 142 FN episodes and 92 bacterial infections (40.2% Gram positive and 59.8% Gram negative). MBL2 low-risk genotype was found in 59.1%, medium-risk in 31.8% and high-risk in 9%. FCN2 low-risk haplotypes were detected in 38.2%, medium-risk in 44.1% and high-risk in 17.6%. MBL2 genotype and FCN2 haplotype were not associated with increased frequency of FN episodes. MBL2 medium/high-risk genotype and FCN2 medium/high-risk haplotype were associated with prolonged duration of FN (P = 0.007 and P = 0.001, respectively) and increased number of bacterial infections (P = 0.001 and P = 0.002, respectively). The combined MBL2/FCN2 medium/high-risk genotype was associated with an increased number of bacterial infections (P = 0.001). CONCLUSIONS: MBL2 and FCN2 single or combined deficiencies are associated with increased duration of FN episodes as well as increased number of bacterial infections in children with B-ALL suggesting a prognostic role of these genes.

Regulation of poly-(R)-(3-hydroxybutyrate-co-3-hydroxyvalerate) biosynthesis by the AtoSCDAEB regulon in phaCAB+ Escherichia coli.

AtoSC two-component system (TCS) upregulates the high-molecular weight poly-(R)-3-hydroxybutyrate (PHB) biosynthesis in recombinant phaCAB (+) Escherichia coli strains, with the Cupriavidus necator phaCAB operon. We report here that AtoSC upregulates also the copolymer P(3HB-co-3HV) biosynthesis in phaCAB (+) E. coli. Acetoacetate-induced AtoSC maximized P(3HB-co-3HV) to 1.27 g/l with a 3HV fraction of 25.5 % wt. and biopolymer content of 75 % w/w in a time-dependent process. The atoSC locus deletion in the ∆atoSC strains resulted in 4.5-fold P(3HB-co-3HV) reduction, while the 3HV fraction of the copolymer was restricted to only 6.4 % wt. The ∆atoSC phenotype was restored by extrachromosomal introduction of AtoSC. Deletion of the atoDAEB operon triggered a significant decrease in P(3HB-co-3HV) synthesis and 3HV content in ∆atoDAEB strains. However, the acetoacetate-induced AtoSC in those strains increased P(3HB-co-3HV) to 0.8 g/l with 21 % 3HV, while AtoC or AtoS expression increased P(3HB-co-3HV) synthesis 3.6- or 2.4-fold, respectively, upon acetoacetate. Complementation of the ∆atoDAEB phenotype was achieved by the extrachromosomal introduction of the atoSCDAEB regulon. Individual inhibition of ß-oxidation and mainly fatty acid biosynthesis pathways by acrylic acid or cerulenin, respectively, reduced P(3HB-co-3HV) biosynthesis. Under those conditions, introduction of atoSC or atoSCDAEB regulon was capable of upregulating biopolymer accumulation. Concurrent inhibition of both the fatty acid metabolic pathways eliminated P(3HB-co-3HV) production. P(3HB-co-3HV) upregulation in phaCAB (+) E. coli by AtoSC signaling through atoDAEB operon and its participation in the fatty acids metabolism interplay provide additional perceptions of AtoSC critical involvement in E. coli regulatory processes towards biotechnologically improved polyhydroxyalkanoates biosynthesis.

Calcium channels blockers inhibit the signal transduction through the AtoSC system in Escherichia coli.

Verapamil, diltiazem and nifedipine are Ca(2+)-channel blockers used in cardiovascular diseases. We report here that the Escherichia coli AtoSC signaling is inhibited by those blockers. AtoSC two-component system plays a pivotal role in sophisticated signaling networks in E. coli regulating processes implicated in bacterial homeostasis and pathogenicity. The Ca(2+)-channel blockers abrogated the in vitro full-length AtoS kinase autophosphorylation. However, they demonstrated no effect on the AtoS cytoplasmic form autophosphorylation. AtoC protected AtoS from verapamil or diltiazem but not from nifedipine, when the two constituents formed complex. The blockers did not affect the AtoS≈P to AtoC phosphotransfer. The blockers-mediated AtoSC inhibition was verified in vivo on the atoDAEB expression, which was inhibited only in AtoSC-expressing bacteria upon acetoacetate. The AtoS and AtoC protein or their genes transcription levels were unaffected by the blockers. Blockers demonstrated differential effects in the regulation of both the cytosolic- and most potently the membrane-bound-cPHB. Extracellular Ca(2+) counteracted the verapamil-mediated effect on cPHB only in atoSC(+) cells. Extracellular Ca(2+) reversed the diltiazem-mediated cPHB decreases in cells of both genetic backgrounds, yet a Ca(2+)-concentration dependent reversion was observed only in the AtoSC-regulated cPHB. Nifedipine caused a more pronounced cPHB down-regulation that was not reversed by extracellular Ca(2+). The AtoSC signaling inhibition by Ca(2+)-channel blockers used for human treatment, and their differential effects on cPHB-formed Ca(2+)-channels, signify their implications in bacterial-host interactions through the two-component signaling and could stimulate the design of Ca(2+)-channels blockers derivatives acting as inhibitors of two-component systems.

Molecularly imprinted polymers for histamine recognition in aqueous environment.

Molecularly imprinted polymers (MIP) for histamine using methacrylic acid were developed and recognition mechanisms were thoroughly characterized for the first time in this study. The binding affinity of imprinted polymer with structurally related compounds was studied in organic and aqueous media, at various conditions. In organic media, MIP was found to bind histamine two and six times more than ranitidine and fluoxetine, respectively, whereas higher selectivity was observed in the case of dimentidene or disodium cromoglycate. The specific binding sites of MIP recognized histamine over L-histidine in aqueous conditions, while higher affinity for histamine compared to ranitidine, disodium cromoglycate, putrescine and to a putrescine analogue was observed. A combination of NMR and UV spectroscopy analyses for investigation of imprinting and recognition properties revealed that strong specific interactions between the functional monomer and histamine in the prepolymerization and in the aqueous solutions were probably responsible for histamine recognition. The preparation of histamine MIPs and elucidation of imprinting and recognition mechanism may serve as useful insight for future application of MIPs.

Involvement of the AtoSCDAEB regulon in the high molecular weight poly-(R)-3-hydroxybutyrate biosynthesis in phaCAB(+)Escherichia coli.

AtoSC two-component system plays a pivotal role in many regulatory indispensable Escherichia coli processes. AtoSCDAEB regulon, comprising the AtoSC system and the atoDAEB operon, regulates the short-chain fatty acids catabolism. We report here, that AtoSC up-regulates the high-molecular weight PHB biosynthesis, in recombinant phaCAB(+)E. coli, with the Cupriavidus necator phaCAB operon. PHB accumulation was maximized upon the acetoacetate-mediated induction of AtoSC, under glucose 1% w/v, resulting in a yield of 1.73 g/l with a biopolymer content of 64.5% w/w. The deletion of the atoSC locus, in the ΔatoSC strains, resulted in a 5 fold reduction of PHB accumulation, which was restored by the extrachromosomal introduction of the AtoSC system. The deletion of the atoDAEB operon triggered a significant decrease in PHB synthesis in ΔatoDAEB strains. However, the acetoacetate-induced AtoSC system in those strains increased PHB to 1.55 g/l, while AtoC expression increased PHB to 1.4 g/l upon acetoacetate. The complementation of the ΔatoDAEB phenotype was achieved by the extrachromosomal introduction of the atoSCDAEB regulon. The individual inhibition of ß-oxidation and mainly fatty-acid biosynthesis pathways by acrylic acid or cerulenin respectively, reduced PHB biosynthesis. Under those conditions the introduction of the atoSC locus or the atoSCDAEB regulon was capable to up-regulate the biopolymer accumulation. The concurrent inhibition of both the fatty acids metabolic pathways eliminated PHB production. PHB up-regulation in phaCAB(+)E. coli, by AtoSC signaling through atoDAEB operon and its participation in the fatty acids metabolism interplay, provide additional perceptions of AtoSC critical involvement in E. coli regulatory processes towards the biotechnologically improved polyhydroxyalkanoates biosynthesis.

Flagellin gene (fliC) of Thermus thermophilus HB8: characterization of its product and involvement to flagella assembly and microbial motility.

Thermus thermophilus HB8 flagellin protein (FliC) is encoded by the TTHC004 (fliC) gene, which is located in the pTT8 plasmid of the bacterium. Flagellin monomer and flagella fibres were isolated from a culture of T. thermophilus grown in rich medium, or in mineral salt medium with sodium gluconate as the carbon source. Western blot immunodetection with anti-FliC revealed a stable complex (FliC)(1)(FliS)(2) of flagellin (FliC, 27.7 kDa) with a homodimer of FliS (FliS, 18.2 kDa) that are encoded by TTHC004 and TTHC003 genes, respectively. The complex is dissociable at low pHs and/or by heat treatment. Glycan staining of purified flagella and treatment with N-glycosidase F suggested that flagellin of T. thermophilus is a glycosylated protein. Size exclusion chromatography revealed that flagellar filaments (FliC) have a molecular mass higher than 200 kDa. The formation of flagella is enhanced after prolonged cultivation time where phosphate and other nutrient were depleted, giving in the bacterium considerable swimming motility in low viscosity media.

Involvement of AtoSC two-component system in Escherichia coli flagellar regulon.

The AtoSC two-component system in Escherichia coli is a key regulator of many physiological processes. We report here the contribution of AtoSC in E. coli motility and chemotaxis. AtoSC locus deletion in ΔatoSC cells renders cells not motile or responsive against any chemoattractant or repellent independently of the AtoSC inducer's presence. AtoSC expression through plasmid complemented the ΔatoSC phenotype. Cells expressing either AtoS or AtoC demonstrated analogous motility and chemotactic phenotypes as ΔatoSC cells, independently of AtoSC inducer's presence. Mutations of AtoC phosphate-acceptor sites diminished or abrogated E. coli chemotaxis. trAtoC, the AtoC constitutive active form which lacks its receiver domain, up-regulated E. coli motility. AtoSC enhanced the transcription of the flhDC and fliAZY operons and to a lesser extent of the flgBCDEFGHIJKL operon. The AtoSC-mediated regulation of motility and chemotactic response required also the expression of the CheAY system. The AtoSC inducers enhanced the AtoSC-mediated motility and chemotaxis. Acetoacetate or spermidine further promoted the responses of only AtoSC-expressing cells, while Ca(2+) demonstrated its effects independently of AtoSC. Histamine regulated bacterial chemotaxis only in atoSC (+) cells in a concentration-dependent manner while reversed the AtoSC-mediated effects when added at high concentrations. The trAtoC-controlled motility effects were enhanced by acetoacetate or spermidine, but not by histamine. These data reveal that AtoSC system regulates the motility and chemotaxis of E. coli, participating in the transcriptional induction of the main promoters of the chemotactic regulon and modifying the motility and chemotactic phenotypes in an induction-dependent mechanism.

Histamine in two component system-mediated bacterial signaling.

Histamine is a key mediator governing vital cellular processes in mammals beyond its decisive role in inflammation. Recent evidence implies additional actions in both eukaryotes and prokaryotes. Besides its function in host defense against bacterial infections, histamine elicits largely undefined actions in microorganisms that may contribute to bacteria-host interactions. Bacterial proliferation and adaptation are governed by sophisticated signal transduction networks, including the versatile two-component systems (TCSs) that comprise sensor histidine kinases and response regulators and rely on phosphotransfer mechanisms to exert their modulatory function. The AtoSC TCS regulates fundamental cellular processes such as short-chain fatty acid metabolism, poly-(R)-3-hydroxybutyrate (cPHB) biosynthesis and chemotaxis in Escherichia coli. The implication of exogenous histamine in the AtoSC-mediated cPHB biosynthesis and in E. coli chemotactic behavior is indicative of a putative function of histamine in bacterial physiology. The data raise questions on the significance of histamine actions in bacteria-host symbiosis, dysbiosis and pathogenicity as well as on the possible consequences upon therapeutic administration of histamine receptor-targeting agents and in particular ligands of the recently identified immunomodulatory H4 receptor.

Molecular typing of Brucella melitensis endemic strains and differentiation from the vaccine strain Rev-1.

In the present study forty-four Greek endemic strains of Br. melitensis and three reference strains were genotyped by Multi locus Variable Number Tandem Repeat (ML-VNTR) analysis based on an eight-base pair tandem repeat sequence that was revealed in eight loci of Br. melitensis genome. The forty-four strains were discriminated from the vaccine strain Rev-1 by Restriction Fragment Length Polymorphism (RFLP) and Denaturant Gradient Gel Electrophoresis (DGGE). The ML-VNTR analysis revealed that endemic, reference and vaccine strains are genetically closely related, while most of the loci tested (1, 2, 4, 5 and 7) are highly polymorphic with Hunter-Gaston Genetic Diversity Index (HGDI) values in the range of 0.939 to 0.775. Analysis of ML-VNTRs loci stability through in vitro passages proved that loci 1 and 5 are non stable. Therefore, vaccine strain can be discriminated from endemic strains by allele's clusters of loci 2, 4, 6 and 7. RFLP and DGGE were also employed to analyse omp2 gene and reveled different patterns among Rev-1 and endemic strains. In RFLP, Rev-1 revealed three fragments (282, 238 and 44 bp), while endemic strains two fragments (238 and 44 bp). As for DGGE, the electrophoretic mobility of Rev-1 is different from the endemic strains due to heterologous binding of DNA chains of omp2a and omp2b gene. Overall, our data show clearly that it is feasible to genotype endemic strains of Br. melitensis and differentiate them from vaccine strain Rev-1 with ML-VNTR, RFLP and DGGE techniques. These tools can be used for conventional investigations in brucellosis outbreaks.

Regulation of the Escherichia coli AtoSC two component system by synthetic biologically active 5;7;8-trimethyl-1;4-benzoxazine analogues.

The Escherichia coli AtoSC two component system;upon acetoacetate induction;regulates the expression of the atoDAEB operon;through His→Asp phopshotransfer;thus modulating important cellular processes. In this report the effect of seven 5,7,8-trimethyl-1,4-benzoxazine derivatives on the regulation of the E. coli AtoSC system was studied. The new compounds were tested for their effectiveness on the expression of the atoC and the regulated atoDAEB operon. The non-substituted 5,7,8-trimethyl-1,4-benzoxazine (4a), was the most potent inducer on atoC transcription;resulting in accumulation of AtoC protein. The induction of atoC by 4a was specific;since no effect was observed on the other genes of the system (atoS and atoDAEB). Moreover;compound 4a was shown to significantly up-regulate the in vitro kinase activity of the histidine kinase AtoS without altering the protein levels in the cell. Interestingly;this compound appeared to modulate the acetoacetate-mediated induction of the atoDAEB promoter by the AtoSC system. These data provide the first evidence for a differential modulator role of 5,7,8-trimethyl-1,4-benzoxazine;on the AtoSC two component system mediated signaling.

Escherichia coli genome-wide promoter analysis: identification of additional AtoC binding target elements.

BACKGROUND: Studies on bacterial signal transduction systems have revealed complex networks of functional interactions, where the response regulators play a pivotal role. The AtoSC system of E. coli activates the expression of atoDAEB operon genes, and the subsequent catabolism of short-chain fatty acids, upon acetoacetate induction. Transcriptome and phenotypic analyses suggested that atoSC is also involved in several other cellular activities, although we have recently reported a palindromic repeat within the atoDAEB promoter as the single, cis-regulatory binding site of the AtoC response regulator. In this work, we used a computational approach to explore the presence of yet unidentified AtoC binding sites within other parts of the E. coli genome. RESULTS: Through the implementation of a computational de novo motif detection workflow, a set of candidate motifs was generated, representing putative AtoC binding targets within the E. coli genome. In order to assess the biological relevance of the motifs and to select for experimental validation of those sequences related robustly with distinct cellular functions, we implemented a novel approach that applies Gene Ontology Term Analysis to the motif hits and selected those that were qualified through this procedure. The computational results were validated using Chromatin Immunoprecipitation assays to assess the in vivo binding of AtoC to the predicted sites. This process verified twenty-two additional AtoC binding sites, located not only within intergenic regions, but also within gene-encoding sequences. CONCLUSIONS: This study, by tracing a number of putative AtoC binding sites, has indicated an AtoC-related cross-regulatory function. This highlights the significance of computational genome-wide approaches in elucidating complex patterns of bacterial cell regulation.

Inhibition of the signal transduction through the AtoSC system by histidine kinase inhibitors in Escherichia coli.

AtoSC two-component system participates in many indispensable processes of Escherichia coli. We report here that the AtoSC signal transduction is inhibited by established histidine kinase inhibitors. Closantel, RWJ-49815 and TNP-ATP belonging to different chemical classes of inhibitors, abrogated the in vitro AtoS kinase autophosphorylation. However, when AtoS was embedded in the membrane fractions, higher inhibitor concentrations were required for total inhibition. When AtoS interacted with AtoC forming complex, the intrinsic histidine kinase was protected by the response regulator, requiring increased inhibitors concentrations for partially AtoS autophosphorylation reduction. The inhibitors exerted an additional function on AtoSC, blocking the phosphotransfer from AtoS to AtoC, without however, affecting AtoC~P dephosphorylation. Their in vivo consequences through the AtoSC inhibition were elucidated on atoDAEB operon expression, which was inhibited only in AtoSC-expressing bacteria where AtoSC was induced by acetoacetate or spermidine. The inhibitor effects were extended on the AtoSC regulatory role on cPHB [complexed poly-(R)-3-hydroxybutyrate] biosynthesis. cPHB was decreased upon the blockers only in acetoacetate-induced AtoSC-expressing cells. Increased ATP amounts during bacterial growth reversed the inhibitory TNP-ATP-mediated effect on cPHB. The alteration of pivotal E. coli processes as an outcome of AtoSC inhibition, establish this system as a target of two-component systems inhibitors.

AtoSC two-component system is involved in cPHB biosynthesis through fatty acid metabolism in E. coli.

BACKGROUND: We have shown previously that AtoSC two-component system regulates the biosynthesis of E. coli cPHB [complexed poly-(R)-3-hydroxybutyrate]. METHODS: The AtoSC involvement on fatty acids metabolism, towards cPHB synthesis, was studied using cPHB determination, gene expression, and fatty acid metabolic pathways inhibitors. RESULTS: Deletion of the atoDAEB operon from the E. coli genome resulted in a consistent reduction of cPHB accumulation. When in ΔatoDAEB cells, the atoDAEB operon and the AtoSC system were introduced extrachromosomally, a significant enhancement of cPHB levels was observed. Moreover, the introduction of a plasmid with atoSC genes regulated positively cPHB biosynthesis. A lesser cPHB enhancement was triggered when plasmids carrying either atoS or atoC were introduced. The intracellular distribution of cPHB was regulated by AtoSC or AtoC according to the inducer (acetoacetate or spermidine). Blockage of ß-oxidation by acrylic acid reduced cPHB levels, suggesting the involvement of this pathway in cPHB synthesis; however, the overproduction of AtoSC or its constituents separately resulted in cPHB enhancement. Inhibition of fatty acid biosynthesis by cerulenin resulted to a major cPHB reduction, indicating the contribution of this pathway in cPHB production. Inhibition of both ß-oxidation and fatty acid biosynthesis reduced dramatically cPHB, suggesting the contribution of both pathways in cPHB biosynthesis. CONCLUSIONS: Short fatty acid catabolism (atoDAEB operon) and fatty acids metabolic pathways participate in cPHB synthesis through the involvement of AtoSC system. GENERAL SIGNIFICANCE: The involvement of the AtoSC system in the fatty acids metabolic pathways interplay towards cPHB biosynthesis provides additional perceptions of AtoSC role on E. coli regulatory biochemical processes.

Activation of the AtoSC two-component system in the absence of the AtoC N-terminal receiver domain in E. coli.

The AtoSC two-component system in E. coli consists of the AtoS sensor kinase and the AtoC response regulator. It regulates positively the transcriptional activation of atoDAEB operon, encoding enzymes involved in short-chain fatty acid catabolism upon acetoacetate-mediated induction. AtoSC acting on atoDAEB operon, regulates the biosynthesis and the intracellular distribution of short-chain poly-(R)-3-hydroxybutyrate (cPHB). A phosphorylation-incompetent AtoC form was constructed lacking its N-terminal receiver domain, trAtoC, to study the effects of AtoC domains on cPHB biosynthesis and atoDAEB operon regulation. Both cPHB biosynthesis and atoDAEB gene expression were regulated positively by trAtoC in the absence of any inducer in E. coli of both atoSC (+) and ΔatoSC genotypes. The presence of acetoacetate or spermidine further promoted these trAtoC actions. Competitive regulatory functions between the full length AtoC and trAtoC were observed referring to atoDAEB and cPHB targets as well as growth of trAtoC-overproducing atoSC (+) cells on butyrate as the sole carbon source. trAtoC in contrast to the wild-type AtoC presented different modes of cPHB and atoDAEB regulation in the presence of compounds involved in fatty acid metabolism including CoA-SH, acetyl-CoA, sodium acetate or 3-hydroxybutyryl-CoA. These data provide evidence for a role of the AtoC N-terminal receiver domain in regulating the biological activities of AtoSC as well as additional mechanisms of interactions between the AtoSC constituents including their established inducers or new effectors towards the accomplishment of the AtoSC TCS signal transduction.

Synthesis, Crystal Structures, and DNA Binding Properties of Zinc(II) Complexes with 3-Pyridine Aldoxime.

The employment of 3-pyridine aldoxime, (3-py)CHNOH, in Zn(II) chemistry has afforded two novel compounds: [Zn(acac)(2){(3-py)CHNOH}]·H(2)O (1·H(2)O) [where acac(-) is the pentane-2,4-dionato(-1) ion] and [Zn(2)(O(2)CMe)(4){(3-py)CHNOH}(2)] (2). Complex 1·H(2)O crystallizes in the monoclinic space group P2(1)/n. The Zn(II) ion is five-coordinated, surrounded by four oxygen atoms of two acac(-) moieties and by the pyridyl nitrogen atom of the (3-py)CHNOH ligand. Molecules of 1 interact with the water lattice molecules forming a 2D hydrogen-bonding network. Complex 2 crystallizes in the triclinic P-1 space group and displays a dinuclear paddle-wheel structure. Each Zn(II) exhibits a perfect square pyramidal geometry, with four carboxylate oxygen atoms at the basal plane and the pyridyl nitrogen of one monodentate (3-py)CHNOH ligand at the apex. DNA mobility shift assays were performed for the determination of the in vitro effect of both complexes on the integrity and the electrophoretic mobility of pDNA.

Successful anatomic repair of fetoscopic access sites in the mid-gestational rabbit model using amnion cell engineering.

BACKGROUND/AIM: Our aim was to evaluate the impact of in vitro cultured amnion cells, injected and/or seeded in different scaffolds, on in vivo fetal membrane repair. MATERIALS AND METHODS: Amnion cells, isolated from allogeneic fetal membranes, were cultured on three different scaffolds for 14 to 21 days. In 33 mid-gestational rabbits, fetoscopic access sites were randomly allocated to four closure study groups: conventional collagen plug, as well as collagen plug, collagen foil, and fibrin glue as scaffolds for the cultured amnion cells. All membrane access sites were sealed with fibrin glue, and the myometrium closed with sutures. Fetal survival, amnion membrane integrity, and the presence of amniotic fluid were evaluated one week later. RESULTS: Cultures showed good survival in the collagen scaffolds. The use of collagen plug as a scaffold for the in vitro cultured amnion cells improved the integrity of fetal membranes to 80%, better than that of any other study group. CONCLUSION: Despite the need for additional studies, the present data suggest that amnion cells can be a practical and important source of cells for the engineering of constructs for sealing of the fetal membrane.