Development and in vitro evaluation of thiolated chitosan--Poly(methacrylic acid) nanoparticles as a local mucoadhesive delivery system

The main objective of this study was to develop a local, oral mucoadhesive metronidazole benzoate (MET) delivery system that can be applied and removed by the patient for the treatment of periodontal diseases. The results of present study revealed that the retention time of MET at its absorption site could be increased by formulating it into nanoparticles using thiolated chitosan (TCS)-poly(methacrylic acid) (PMAA). The nanoparticles of MET prepared from TCS-PMAA may represent a useful approach for targeting its release at its site of absorption, sustaining its release and improving its oral availability.     

Stepwise adaptations to low temperature as revealed by multiple mutants of psychrophilic α-amylase from Antarctic Bacterium

The mutants Mut5 and Mut5CC from a psychrophilic α-amylase bear representative stabilizing interactions found in the heat-stable porcine pancreatic α-amylase but lacking in the cold-active enzyme from an Antarctic bacterium. From an evolutionary perspective, these mutants can be regarded as structural intermediates between the psychrophilic and the mesophilic enzymes. We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: compactness; kinetically driven stability; thermodynamic stability; resistance toward chemical denaturation, and the kinetics of unfolding/refolding. Concomitantly to this improved stability, both mutants have lost the kinetic optimization to low temperature activity displayed by the parent psychrophilic enzyme. These results provide strong experimental support to the hypothesis assuming that the disappearance of stabilizing interactions in psychrophilic enzymes increases the amplitude of concerted motions required by catalysis and the dynamics of active site residues at low temperature, leading to a higher activity.     

Methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase as a target for bis-indole alkaloids with antibacterial activities

Novel classes of antimicrobials are needed to address the emergence of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). We have recently identified pyruvate kinase (PK) as a potential novel drug target based upon it being an essential hub in the MRSA interactome (Cherkasov, A., Hsing, M., Zoraghi, R., Foster, L. J., See, R. H., Stoynov, N., Jiang, J., Kaur, S., Lian, T., Jackson, L., Gong, H., Swayze, R., Amandoron, E., Hormozdiari, F., Dao, P., Sahinalp, C., Santos-Filho, O., Axerio-Cilies, P., Byler, K., McMaster, W. R., Brunham, R. C., Finlay, B. B., and Reiner, N. E. (2011) J. Proteome Res. 10, 1139-1150; Zoraghi, R., See, R. H., Axerio-Cilies, P., Kumar, N. S., Gong, H., Moreau, A., Hsing, M., Kaur, S., Swayze, R. D., Worrall, L., Amandoron, E., Lian, T., Jackson, L., Jiang, J., Thorson, L., Labriere, C., Foster, L., Brunham, R. C., McMaster, W. R., Finlay, B. B., Strynadka, N. C., Cherkasov, A., Young, R. N., and Reiner, N. E. (2011) Antimicrob. Agents Chemother. 55, 2042-2053). Screening of an extract library of marine invertebrates against MRSA PK resulted in the identification of bis-indole alkaloids of the spongotine (A), topsentin (B, D), and hamacanthin (C) classes isolated from the Topsentia pachastrelloides as novel bacterial PK inhibitors. These compounds potently and selectively inhibited both MRSA PK enzymatic activity and S. aureus growth in vitro. The most active compounds, cis-3,4-dihyrohyrohamacanthin B (C) and bromodeoxytopsentin (D), were identified as highly potent MRSA PK inhibitors (IC(50) values of 16-60 nM) with at least 166-fold selectivity over human PK isoforms. These novel anti-PK natural compounds exhibited significant antibacterial activities against S. aureus, including MRSA (minimal inhibitory concentrations (MIC) of 12.5 and 6.25 μg/ml, respectively) with selectivity indices (CC(50)/MIC) >4. We also report the discrete structural features of the MRSA PK tetramer as determined by x-ray crystallography, which is suitable for selective targeting of the bacterial enzyme. The co-crystal structure of compound C with MRSA PK confirms that the latter is a target for bis-indole alkaloids. It elucidates the essential structural requirements for PK inhibitors in "small" interfaces that provide for tetramer rigidity and efficient catalytic activity. Our results identified a series of natural products as novel MRSA PK inhibitors, providing the basis for further development of potential novel antimicrobials.     

CDIP, a novel pro-apoptotic gene, regulates TNFalpha-mediated apoptosis in a p53-dependent manner

We have identified a novel pro-apoptotic p53 target gene named CDIP (Cell Death Involved p53-target). Inhibition of CDIP abrogates p53-mediated apoptotic responses, demonstrating that CDIP is an important p53 apoptotic effector. CDIP itself potently induces apoptosis that is associated with caspase-8 cleavage, implicating the extrinsic cell death pathway in apoptosis mediated by CDIP. siRNA-directed knockdown of caspase-8 results in a severe impairment of CDIP-dependent cell death. In investigating the potential involvement of extrinsic cell death pathway in CDIP-mediated apoptosis, we found that TNF-alpha expression tightly correlates with CDIP expression, and that inhibition of TNF-alpha signaling attenuates CDIP-dependent apoptosis. We also demonstrate that TNF-alpha is upregulated in response to p53 and p53 inducing genotoxic stress, in a CDIP-dependent manner. Consistently, knockdown of TNF-alpha impairs p53-mediated stress-induced apoptosis. Together, these findings support a novel p53 --> CDIP --> TNF-alpha apoptotic pathway that directs apoptosis after exposure of cells to genotoxic stress. Thus, CDIP provides a new link between p53-mediated intrinsic and death receptor-mediated extrinsic apoptotic signaling, providing a novel target for cancer therapeutics aimed at maximizing the p53 apoptotic response of cancer cells to drug therapy.     

N-Terminal domain of phosphodiesterase-11A4 (PDE11A4) decreases affinity of the catalytic site for substrates and tadalafil, and is involved in oligomerization

The phosphodiesterase-11A (PDE11) family consists of four splice variants (PDE11A1-PDE11A4) that contain a conserved carboxyl-terminal (C-terminal) catalytic domain that hydrolyzes cAMP and cGMP; the amino-termini (N-termini) vary in length and amino acid sequence. PDE11A2, PDE11A3, and PDE11A4 contain one or more GAF (cGMP-binding phosphodiesterase, Anabaena adenylyl cyclase, and Escherichia coli FhlA) subdomains. In the present study, PDE11A1 and PDE11A2 demonstrated higher affinity for cAMP and cGMP when directly compared to that of the longest isoform, PDE11A4. Moreover, PDE11A3, PDE11A2, and PDE11A1, which contain progressively shorter N-termini, were more sensitive than PDE11A4 to inhibition by two structurally unrelated inhibitors, tadalafil (Cialis) and vardenafil (Levitra). The substrate and inhibitor affinity differences among the PDE11 isozymes could not be ascribed to differences in their quaternary structure since PDE11A4, PDE11A3, and PDE11A2 were determined to be dimers, and PDE11A1 was a tetramer. These data also demonstrate that PDE11 isozymes containing at least 123 C-terminal amino acids of the GAF-B domain are stable oligomers and that GAF-A is not required for oligomerization. The isolated PDE11 catalytic domain (Met-563-Asn-934) displayed both monomeric and dimeric forms, and upon dilution, this domain was primarily monomeric, indicating that the main oligomerization contacts are within the N-termini of PDE isozymes. This report is the first to describe an inhibitory effect of the N-terminal region of PDE11A4 on the affinity of the catalytic domain for both substrates and inhibitors and the first to define the quaternary structure and the regions that contribute to this structure within the human PDE11A family.     

Towards optimization of odonto/osteogenic bioengineering: in vitro comparison of simvastatin,sodium fluoride,melanocyte-stimulating hormone

Tissue engineering has emerged as a potential therapeutic option for dental problems in recent years. One of the policies in tissue engineering is to use both scaffolds and additive factors for enhancing cell responses. This study aims to evaluate and compare the effect of three types of biofactors on poly-caprolactone-poly-ethylene glycol-poly caprolactone (PCL-PEG-PCL) nanofibrous scaffold on human dental pulp stem cell (hDPSCs) engineering. The PCL-PEG-PCL copolymer was synthesized with ring opening polymerization method, and its nanofiber scaffold was prepared by electrospinning method. Nanofibrous scaffold-seeded hDPSCs were treated with sodium fluoride (NaF), melanocyte-stimulating hormone (MSH), or simvastatin (SIM). Non-treated nanofiber seeded cells were utilized as control. The viability, biocompatibility, adhesion, proliferation rate, morphology, osteo/odontogenic potential, and the expression of tissue-specific genes were studied. The results showed that significant higher results demonstrated significant higher adhesive behavior, viability, alizarin red activity, and dentin specific gene expression in MSH- and SIM-treated cells (p < 0.05). This study is unique; in that, it compares the effects of different treatments for optimization of dental tissue engineering.     

Structural and functional features in human PDE5A1 regulatory domain that provide for allosteric cGMP binding, dimerization, and regulation

The cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains a catalytic domain that hydrolyzes cGMP and a regulatory (R) domain that contains two GAFs (a and b; GAF is derived from the proteins mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and Escherichia coli (FhlA)). The R domain binds cGMP allosterically, provides for dimerization, and is phosphorylated at a site regulated by allosteric cGMP binding. Quaternary structures and cGMP-binding properties of 10 human PDE5A1 constructs containing one or both GAFs were characterized. Results reveal that: 1) high affinity homo-dimerization occurs between GAF a modules (K(D) < 30 nM) and between GAF b modules (K(D) = 1-20 pM), and the sequence between the GAFs (Thr322-Asp403) contributes to dimer stability; 2) 176 amino acids (Val156-Gln331) in GAF a are adequate for cGMP binding; 3) GAF a has higher affinity for cGMP (K(D) < 40 nM) than does the isolated R domain (K(D) = 110 nM) or holoenzyme (K(D) = 200 nM), suggesting that the sequence containing GAF b and its flanking amino acids autoinhibits GAF a cGMP-binding affinity in intact R domain; 4) a mutant (Met1-Glu321) containing only GAF a has high affinity, biphasic cGMP-binding kinetics consistent with structural heterogeneity of GAF a, suggesting that the presence of GAF b is not required for biphasic cGMP-dissociation kinetics observed in holoenzyme or isolated R domain; 5) significant cGMP binding by GAF b was not detected; and 6) the sequence containing GAF b and its flanking amino acids is critical for cGMP stimulation of Ser102 phosphorylation by cyclic nucleotide-dependent protein kinases. Results yield new insights into PDE5 functions, further define boundaries that provide for allosteric cGMP binding, and identify regions that contribute to dimerization.     

Total branched-chain amino acids requirement in patients with maple syrup urine disease by use of indicator amino acid oxidation with L-[1-13C]phenylalanine

Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by defects in the mitochondrial multienzyme complex branched-chain alpha-keto acid dehydrogenase (BCKD; EC 1.2.4.4), responsible for the oxidative decarboxylation of the branched-chain ketoacids (BCKA) derived from the branched-chain amino acids (BCAA) leucine, valine, and isoleucine. Deficiency of the enzyme results in increased concentrations of the BCAA and BCKA in body cells and fluids. The treatment of the disease is aimed at keeping the concentration of BCAA below the toxic concentrations, primarily by dietary restriction of BCAA intake. The objective of this study was to determine the total BCAA requirements of patients with classical MSUD caused by marked deficiency of BCKD by use of the indicator amino acid oxidation (IAAO) technique. Five MSUD patients from the MSUD clinic of The Hospital for Sick Children participated in the study. Each was randomly assigned to different intakes of BCAA mixture (0, 20, 30, 50, 60, 70, 90, 110, and 130 mg.kg(-1).day(-1)), in which the relative proportion of BCAA was the same as that in egg protein. Total BCAA requirement was determined by measuring the oxidation of l-[1-(13)C]phenylalanine to (13)CO(2). The mean total BCAA requirement was estimated using a two-phase linear regression crossover analysis, which showed that the mean total BCAA requirement was 45 mg.kg(-1).day(-1), with the safe level of intake (upper 95% confidence interval) at 62 mg.kg(-1).day(-1). This is the first time BCAA requirements in patients with MSUD have been determined directly.