Synthesis of poly-hydroxyalkanoates from activated sludge under various oxidation-reduction potentials

The production of poly-hydroxyalkanoates (PHA) from the activated sludge subjected to conditions with various oxidationreduction potentials (ORPs) was investigated. By controlling the dissolved oxygen concentration in the cultural media, the ORP were kept at preset levels of ?20, ?10, 0, and +10 mV. With glucose as the dedicated carbon source, we have demonstrated a correlating relationship with the ORP’s in the culture media to the PHA accumulation rate, the PHA production-yield, cell growth rate, glucose uptakes and 3-hydroxybutyrate to 3-hydroxyvalerate (HB/HV) mole ratios in the PHA copolymers. The highest PHA production yield of 0.26 g/g with HB/HV mole ratio of 8.03 was achieved at +10 mV ORP. We concluded that oxygen plays an important role in PHA accumulation and HB/HV mole ratio activated sludge-to-copolymer PHA conversion process.     

Vitamin D receptor agonist/histone deacetylase inhibitor molecular hybrids

Incorporation of zinc-binding groups into the side-chain of 1α,25-dihydroxyvitamin D₃ (1,25D) fully bifunctional hybrid molecules which act both as vitamin D receptor agonists and histone deacetylase inhibitors. These bifunctional hybrids display in vitro antiproliferative activity against the AT84 squamous carcinoma cell line while lacking the in vivo hypercalcemic effects of 1,25D.     

Cajanol,a novel anticancer agent from Pigeonpea [Cajanus cajan (L.) Millsp.] roots,induces apoptosis in human breast cancer cells through a ROS-mediated mitochondrial pathway

Cajanol (5-hydroxy-3-(4-hydroxy-2-methoxyphenyl)-7-methoxychroman-4-one) is an isoflavanone from Pigeonpea [Cajanus cajan (L.) Millsp.] roots. As the most effective phytoalexin in pigeonpea, the cytotoxic activity of cajanol towards cancer cells has not been report as yet. In the present study, the anticancer activity of cajanol towards MCF-7 human breast cancer cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of cajanol, cell cycle distribution, DNA fragmentation assay and morphological assessment of nuclear change, ROS generation, mitochondrial membrane potential (ΔΨm) disruption, and expression of caspase-3 and caspase-9, Bax, Bcl-2, PARP and cytochrome c were measured in MCF-7 cells. Cajanol inhibited the growth of MCF-7 cells in a time and dose-dependent manner. The IC₅₀ value was 54.05 μM after 72 h treatment, 58.32 μM after 48 h; and 83.42 μM after 24 h. Cajanol arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of cajanol towards cancer cells in vitro.     

Protective effects of pre-germinated brown rice diet on low levels of Pb-induced learning and memory deficits in developing rat

Lead (Pb) is a known neurotoxicant in humans and experimental animals. Numerous studies have provided evidence that humans, especially young children, and animals chronically intoxicated with low levels of Pb show learning and memory impairments. Unfortunately, Pb-poisoning cases continue to occur in many countries. Because the current treatment options are very limited, there is a need for alternative methods to attenuate Pb toxicity. In this study, the weaning (postnatal day 21, PND21) rats were randomly divided into five groups: the control group (AIN-93G diet, de-ionized water), the lead acetate (PbAC) group (AIN-93G diet, 2 g/L PbAC in de-ionized water), the lead acetate + WR group (white rice diet, 2 g/L PbAC in de-ionized water; PbAC + WR), the lead acetate + BR group (brown rice diet, 2 g/L PbAC in de-ionized water; PbAC + BR) and the lead acetate + PR group (pre-germinated brown rice diet, 2 g/L PbAC in de-ionized water; PbAC + PR). The animals received the different diets until PND60, and then the experiments were terminated. The protective effects of pre-germinated brown rice (PR) on Pb-induced learning and memory impairment in weaning rats were assessed by the Morris water maze and one-trial-learning passive avoidance test. The anti-oxidative effects of feeding a PR diet to Pb-exposed rats were evaluated. The levels of reactive oxygen species (ROS) were determined by flow cytometry. The levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), γ-aminobutyric acid (GABA) and glutamate were determined by HPLC. Our data showed that feeding a PR diet decreased the accumulation of lead and decreased Pb-induced learning and memory deficits in developing rats. The mechanisms might be related to the anti-oxidative effects and large amount of GABA in PR. Our study provides a regimen to reduce Pb-induced toxicity, especially future learning and memory deficits in the developing brain.     

Origins of proton transport behavior from selectivity domain mutations of the aquaporin-1 channel

The permeation free-energy profile and maximum ion conductance of proton transport along the channel of three aquaporin-1 (AQP1) mutants (H180A/R195V, H180A, and R195V) are calculated via molecular dynamics simulations and Poisson-Nernst-Planck theory. The proton dynamics was described by the multistate empirical valence bond (MS-EVB) model. The results reveal three major contributions to the overall free-energy barrier for proton transport in AQP1: 1), the bipolar field, 2), the electrostatic repulsion due to the Arg-195 residue, and 3), the dehydration penalty due to the narrow channel pore. The double mutation (H180A/R195V) drastically drops the overall free-energy barrier by roughly 20 kcal/mol via simultaneously relaxing the direct electrostatic interaction (by R195V) and dehydration effect (by H180A).     

Design, synthesis, and biological activity of novel factor Xa inhibitors: 4-aryloxy substituents of 2,6-diphenoxypyridines

A novel series of triaryloxypyridines have been designed to inhibit factor Xa, a serine protease strategically located in the coagulation cascade. Inhibitor 5e has a K(I) against factor Xa of 0.12nM and is greater than 8000- and 2000-fold selective over two related serine proteases, thrombin and trypsin, respectively. The 4-position of the central pyridine has been identified as a site that tolerates various substitutions without deleterious effects on potency and selectivity. This suggests that the 4-position of the pyridine ring is an ideal site for chemical modifications to identify inhibitors with improved pharmacokinetic characteristics. This investigation has resulted in inhibitor 5d, which has an oral availability of 6% in dogs. The synthesis, in vitro activity, and in vivo profile of this class of inhibitors is outlined.     

The emerging role of Arf/Arl small GTPases in cilia and ciliopathies

Once overlooked as an evolutionary vestige, the primary cilium has recently been the focus of intensive studies. Mounting data show that this organelle is a hub for various signaling pathways during vertebrate embryonic development and pattern formation. However, how cilia form and how cilia execute the sensory function still remain poorly understood. Cilia dysfunction is correlated with a wide spectrum of human diseases, now termed ciliopathies. Various small GTPases, including the members in Arf/Arl, Rab, and Ran subfamilies, have been implicated in cilia formation and/or function. Here we review and discuss the role of one particular group of small GTPase, Arf/Arl, in the context of cilia and ciliopathy.     

Effects of gamma irradiation and repetitive freeze-thaw cycles on the biomechanical properties of human flexor digitorum superficialis tendons

An increasing number of tissue banks have begun to focus on gamma irradiation and freeze-thaw in the reconstruction of anterior cruciate ligaments using allografts. The purpose of this study was to evaluate the biomechanical properties of human tendons after exposure to gamma radiation and repeated freeze-thaw cycles and to compare them with fresh specimens. Forty flexor digitorum superficialis tendons were surgically procured from five fresh cadavers and divided into four groups: fresh tendon, gamma irradiation, freeze-thaw and gamma irradiation+freeze-thaw. The dose of gamma irradiation was 25 kGy. Each freeze-thaw cycle consisted of freezing at -80 °C for 7 day and thawing at 25 °C for 6 h. These tendons underwent 4 freeze-thaw cycles. Biomechanical properties were analyzed during load-to-failure testing. The fresh tendons were found to be significantly different in ultimate load, stiffness and ultimate stress relative to the other three groups. The tendons of the gamma+freeze-thaw group showed a significant decrease in ultimate load, ultimate stress and stiffness compared with the other three groups. Gamma irradiation and repeated freezing-thawing (4 cycles) can change the biomechanical properties. However, no significant difference was found between these two processes on the effect of biomechanical properties. It is recommended that gamma irradiation (25 kGy) and repetitive freeze-thaw cycles (4 cycles) should not be adopted in the processing of the allograft tendons.     

Application of principal component analysis to determine the key structural features contributing to iron superoxide dismutase thermostability

Iron superoxide dismutase (Fe-SOD) is predominantly found in bacteria and mitochondria. The thermal stability of Fe-SOD from different sources can vary dramatically. We have studied the influence of structural parameters on Fe-SOD thermostability by principal component analysis (PCA). The results show that an increased α-helical and turn content, an increased α-helix and loop length, an increase in the number of main-main chains and charged-uncharged hydrogen bonds, a decrease in the 3(10) -helix content, and a decreased β-strand and loop length are all important factors for Fe-SOD thermostability. Interestingly, the use of charged residues to form salt bridges is tendentious in thermophilic Fe-SOD. Negatively charged Arg and positively charged Glu are efficiently used to form salt bridges. The cooperative action of the exposed area, the hydrogen bonds, and the secondary structure plays a crucial role in resisting high temperatures, which demonstrates that the increased stability of thermophilic Fe-SOD is provided by several structural factors acting together.     

Okazaki fragment processing-independent role for human Dna2 enzyme during DNA replication

Dna2 is an essential helicase/nuclease that is postulated to cleave long DNA flaps that escape FEN1 activity during Okazaki fragment (OF) maturation in yeast. We previously demonstrated that the human Dna2 orthologue (hDna2) localizes to the nucleus and contributes to genomic stability. Here we investigated the role hDna2 plays in DNA replication. We show that Dna2 associates with the replisome protein And-1 in a cell cycle-dependent manner. Depletion of hDna2 resulted in S/G(2) phase-specific DNA damage as evidenced by increased γ-H2AX, replication protein A foci, and Chk1 kinase phosphorylation, a readout for activation of the ATR-mediated S phase checkpoint. In addition, we observed reduced origin firing in hDna2-depleted cells consistent with Chk1 activation. We next examined the impact of hDna2 on OF maturation and replication fork progression in human cells. As expected, FEN1 depletion led to a significant reduction in OF maturation. Strikingly, the reduction in OF maturation had no impact on replication fork progression, indicating that fork movement is not tightly coupled to lagging strand maturation. Analysis of hDna2-depleted cells failed to reveal a defect in OF maturation or replication fork progression. Prior work in yeast demonstrated that ectopic expression of FEN1 rescues Dna2 defects. In contrast, we found that FEN1 expression in hDna2-depleted cells failed to rescue genomic instability. These findings suggest that the genomic instability observed in hDna2-depleted cells does not arise from defective OF maturation and that hDna2 plays a role in DNA replication that is distinct from FEN1 and OF maturation.