Isolation of bacteria able to grow on both polyethylene glycol (PEG) and polypropylene glycol (PPG) and their PEG/PPG dehydrogenases

Two bacterial consortia growing on a random copolymer of ethylene glycol and propylene glycol units were obtained by enrichment cultures from various microbial samples. Six major strains included in both consortia were purified and identified as Sphingomonads, Pseudomonas sp. and Stenotrophomonas maltophilia. Three of them (Sphingobium sp. strain EK-1, Sphingopyxis macrogoltabida strain EY-1, and Pseudomonas sp. strain PE-2) utilized both PEG and polypropylene glycol (PPG) as a sole carbon source. Four PEG-utilizing bacteria had PEG dehydrogenase (PEG-DH) activity, which was induced by PEG. PCR products from DNA of these bacteria generated with primers designed from a PEG-DH gene (AB196775 for S. macrogoltabida strain 103) indicated the presence of a sequence that is the homologous to the PEG-DH gene (99% identity). On the other hand, five PPG-utilizing bacteria had PPG dehydrogenase (PPG-DH) activity, but the activity was constitutive. PCR of a PPG-DH gene was performed using primers designed from a polyvinyl alcohol dehydrogenase (PVA-DH) gene (AB190288 for Sphingomonas sp. strain 113P3) because a PPG-DH gene has not been cloned yet, but both PPG-DH and PVA-DH were active toward PPG and PVA (Mamoto et al. 2006). PCR products of the five strains did not have similarity to each other or to oxidoreductases including PVA-DH. The paper was edited by a native speaker through American Journal Experts (http://www.journalexperts.com).     

Interactions of melatonin and serotonin with lactoperoxidase enzyme

Melatonin is the chief secretory product of the pineal gland and is synthesized enzymatically from serotonin. These indoleamine derivatives play an important role in the prevention of oxidative damage. Lactoperoxidase (LPO; EC 1.11.1.7) was purified from bovine milk with three purification steps: Amberlite CG-50 resin, CM-Sephadex C-50 ion-exchange, and Sephadex G-100 gel filtration chromatography, respectively. LPO was purified with a yield of 21.6%, a specific activity of 34.0 EU/mg protein, and 14.7-fold purification. To determine the enzyme purity, SDS-PAGE was performed and a single band was observed. The R_z (A₄₁₂/A₂₈₀) value for LPO was 0.9. The effect of melatonin and serotonin on lactoperoxidase was determined using ABTS as chromogenic substrate. The half-maximal inhibitory concentration (IC₅₀) values for melatonin and serotonin were found to be 1.46 and 1.29 μM, respectively. Also, the inhibition constants (K_i) for melatonin and serotonin were 0.82?±?0.28 and 0.26?±?0.04 μM, respectively. Both melatonin and serotonin were found to be competitive inhibitors.     

聚乙二醇（PEG）对杜仲胚乳愈伤组织茎芽分化的影响

实验中发现,在培养基中加入适量PEG可以显著提高杜仲胚乳愈伤分化频率。PEG这种促进分化的效果既与PEG的分子量和所用的浓度有关,也与培养基中无机离子的强度和蔗糖浓度有关。效果最佳的培养基配方是：在激素组成为BA（2.0-2.75mg/L）＋NAA0.15mg/L）的基本培养基（MS无机盐＋B5有机物＋3％蔗糖）中,添加浓度为4％－6％PEG 4000或4％－5％PEG6000。在这种培养基上杜仲胚乳愈伤组织的分化频率均超过50％,最高可达70％以上,而在同样的条件下不加PEG时分化频率不到10％。然而,经PEG处理分化出来的胚乳再生植株中,部分苗玻璃化现象严重。     

Effects of microwaves (900 MHz) on peroxidase systems: A comparison between lactoperoxidase and horseradish peroxidase

ABSTRACT

This work shows the effects of exposure to an electromagnetic field at 900?MHz on the catalytic activity of the enzymes lactoperoxidase (LPO) and horseradish peroxidase (HRP). Experimental evidence that irradiation causes conformational changes of the active sites and influences the formation and stability of the intermediate free radicals is documented by measurements of enzyme kinetics, circular dichroism spectroscopy (CD) and cyclic voltammetry.     

Induction of microspore-derived embryos of Brassica napus L. with polyethylene glycol (PEG) as osmoticum in a low sucrose medium

Isolated microspores of Brassica napus were cultured on high concentrations of mannitol or polyethylene glycol (PEG 4000), with only a very limited amount of sucrose (0.08–0.1%) provided as carbohydrate source in the medium. While microspores cultured on high mannitol yielded no embryos and no embryogenic cell divisions were observed, microspores on high PEG developed into embryos within 2 weeks, and the embryo yield appeared comparable to that of the sucrose control. When placed under light, PEG embryos quickly changed color from yellow to dark green, while sucrose embryos first remained yellowish and then slowly changed color to pale green. Three-week-old PEG embryos were strikingly similar to immature zygotic embryos developed in ovulo, dissected at 14–15 days post-anthesis (DPA), while sucrose embryos differed from the latter in the size and shape, color and morphology of their cotyledons. These results demonstrate that in microspore embryogenesis of Brassica napus: (1) the level of metabolizable carbohydrate required for microspore embryo induction and formation appears to be substantially less than commonly used amounts, (2) sucrose as an osmoticum can be replaced with high-molecular-weight PEG. With further improvement the new method described here might be suitable for other Brassica species and would have a great potential application in breeding programs. Received: 29 May 1997 / Revision received: 12 August 1997 / Accepted: 2 September 1997     

Stimulatory effect of polyethylene glycol (PEG) on gene expression in mouse liver following hydrodynamics-based transfection

BACKGROUND: Rapid intravenous injection of a large volume of plasmid DNA (pDNA), i.e. a transfection procedure based on hydrodynamics, is known to be an efficient and liver-specific method of in vivo gene delivery. However, the gene expression is transient. METHODS: We investigated the effect of addition of polyethylene glycol (PEG) to a solution of naked pDNA (luciferase) on the expression of the gene in mouse liver following transfection by the hydrodynamics-based technique. In addition, the mechanism leading to the enhancement of the gene expression was studied. RESULTS: The addition of 1% (w/v) PEG2000 to the pDNA solution enhanced the resulting gene expression in the liver. Increasing the PEG2000 concentration to more than 1 and up to 10% (w/v) rather diminished the gene expression level. By contrast, increasing the molecular weight of PEG to over 2000 up to 10 000 did not affect the level of gene expression. Histopathological and serum-chemistry examinations indicated that hydrostatic or osmotic pressure increased tissue and hepatocellular damage in a PEG-concentration-dependent manner, and resulted in a decrease in gene expression. Quantitative evaluation showed that the enhanced gene expression resulted from stabilization of the pDNA introduced into the hepatocytes and an enhancement of the transport of intact pDNA to the nucleus. CONCLUSIONS: For most gene therapy applications and gene function studies, sustained expression of the introduced gene(s) is necessary. This simple method to achieve enhanced gene expression in liver may have a great potential for a wide variety of laboratory studies in molecular and cellular biology as well as possibly for future clinical applications in humans.     

The chemical modification of papain with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

The reaction of the water-soluble carbodimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), with active papain in the presence of the nucleophile ethyl glycinate results in an irreversible inactivation of the enzyme. This inactivation is accompanied by the derivatization of the catalytically essential thiol group of the enzyme (Cys-25) and by the modification of 6 out of 14 of papain's carboxyl groups and up to 9 out of 19 of the enyzme's tyrosyl residues. No apparent irreversible modification of histidine residues is observed. Mercuripapain is also irreversibly inactivated by EDC/ethyl glycinate, again with the concomitant modification of 6 carboxyl groups, up to 10 tyrosyl residues, and no histidine residues; but in this case there is no thiol derivatization. Treatment of either modified native papain or modified mercuripapain with hydroxylamine results in the complete regeneration of free tyrosyl residues but does not restore any activity. The competitive inhibitor benzamidoacetonitrile substantially protects native papain against inactivation and against the derivatization of the essential thiol group as well as 2 of the 6 otherwise accessible carboxyl groups. The inhibitor has no effect upon tyrosyl modification. These findings are discussed in the context of a possible catalytic role for a carboxyl group in the active site of papain.     

Generation of 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-) mouse line

Human/rodent CYP1A1 and CYP1A2 orthologs are well known to exhibit species-specific differences in substrate preferences and rates of metabolism. This lab previously characterized a BAC-transgenic mouse carrying the human CYP1A1_CYP1A2 locus; in this line, human dioxin-inducible CYP1A1 and basal vs dioxin-inducible CYP1A2 have been shown to be expressed normally (with regard to mRNAs, proteins and three enzyme activities) in every one of nine mouse tissues studied. The mouse Cyp1a1 and Cyp1a2 genes are oriented head-to-head and share a bidirectional promoter region of 13,954 bp. Using Cre recombinase and loxP sites inserted 3' of the stop codons of both genes, we show here a successful interchromosomal excision of 26,173 bp that ablated both genes on the same allele. The Cyp1a1/1a2(-) double-knockout allele was bred with the "humanized" line; the final product is the hCYP1A1_1A2_Cyp1a1/1a2(-/-) line on a theoretically >99.8% C57BL/6J genetic background-having both human genes replacing the mouse orthologs. This line will be valuable for human risk assessment studies involving any environmental toxicant or drug that is a substrate for CYP1A1 or CYP1A2.     

Synthesis and anti-inflammatory activity of 2-(2-aroylaroxy)-4,6-dimethoxy pyrimidines

Abstract-Reaction of 6a–f individually with 2-methylsulfonyl-4,6-dimethoxypyrimidine yielded 7a–f in excellent yield. The newly synthesized heterocycles were characterized by IR, ¹H NMR, and mass spectral data. Compounds 7a–f was screened for their anti-inflammatory activity and were compared with standard drugs. Of the compounds studied, the compound 7e showed more potent activity than the standard drugs at all doses tested.     

(E)-3-(3,4,5-Trimethoxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one,a heterocyclic chalcone is a potent and selective CYP1A1 inhibitor and cancer chemopreventive agent

The overexpression of CYP1 family of enzymes is reported to be associated with development of human carcinomas. It has been well reported that CYP1A1 specific inhibitors prevents carcinogenesis. Herein, thirteen pyridine-4-yl series of chalcones were synthesized and screened for inhibition of CYP1 isoforms 1A1, 1B1 and 1A2 in Sacchrosomes? and live human HEK293 cells. The structure-activity relationship analysis indicated that chalcones bearing tri-alkoxy groups (8a and 8k) on non-heterocyclic ring displayed selective inhibition of CYP1A1 enzyme, with IC₅₀ values of 58 and 65?nM, respectively. The 3,4,5-trimethoxy substituted derivative 8a have shown >10-fold selectivity towards CYP1A1 with respect to other enzymes of the CYP1 sub-family and >100-fold selectivity with respect to CYP2 and CYP3 family of enzymes. The potent and selective CYP1A1 inhibitor 8a displayed antagonism of B[a]P mediated activation of aromatic hydrocarbon receptor (AhR) in yeast cells, and also protected human cells from CYP1A1-mediated B[a]P toxicity in human cells. This potent and selective inhibitor of CYP1A1 enzyme have a potential for development as cancer chemopreventive agent.     

Adsorption of lipoproteins with the aid of carboxymethylchitosan mircrospheres crosslinked with poly(ethylene glycol) bisglycidyl ether

Carboxymethylchitosan microspheres crosslinked with poly(ethylene glycol) bisglycidyl ether were prepared and then tested as an adsorbent for selective removal of low-density lipoprotein (LDL) in human plasma. The microspheres were formed by a method of electrostatic instillation and crosslinked with poly(ethylene glycol) bisglycidyl ether. FTIR spectral analyses and X-ray photoelectron spectroscopy revealed that carboxymethylchitosan was crosslinked through amino groups to poly(ethylene glycol) bisglycidyl ether. The plasma lipoprotein sorption tests showed that the adsorption properties of the crosslinked microspheres for LDL were dependent on the concentrations of carboxymethylchitosan and poly(ethylene glycol) bisglycidyl ether. When the concentrations of carboxymethylchitosan and poly(ethylene glycol) bisglycidyl ether were 3.5% and 6%, respectively, 40% LDL and lower than 10% high density lipoprotein in plasma could be removed and the adsorption could be reach an equilibrium in 30 min.     

Regioselective and stereoselective benzoylation of 2-N-protected 4,6-O-ketal derivatives of D-glucosamines with 1-(benzoyloxy)benzotriazole

A highly regioelective and stereoselective benzoylation of the 2-N-protected 4,6-O-ketal derivatives of D-glucosamines with 1-(benzoyloxy)benzotriazole that affords the corresponding beta anomeric benzoates in excellent yields is described herein.     

Inhibition of tumor growth by poly(ethylene glycol) derivatives of anti-ErbB2 antibodies

Poly(ethylene glycol) (PEG) modification of substances with antitumor activity was shown to enhance penetration into growing solid tumors and extend antitumor effects. Accordingly, PEG was introduced as a modifier to two types of monoclonal antibodies (N12 and L26) specific to the ErbB2 (HER2) oncoprotein. These antibodies suppress the growth of tumors overexpressing ErbB2 (e.g. N87 human tumor) and the effect of PEG on their antitumor activity was evaluated. Methoxy-PEG-maleimide conjugated to sulfhydryl groups at the hinge region of the antibodies impaired their antibody binding to N87 tumor cells and did not enhance the antitumor inhibitory activity in tumor-bearing mice. A branched N-hydroxysuccinimide-activated PEG (PEG2), conjugated through amino groups of the protein, was used for binding to the whole antibody (Ab) or to its monomeric Fab′ fragment. When tested against N87 cells in vitro, the binding activity and antitumor cytotoxic effects of Ab-PEG2 were mostly preserved. PEG2 modification did not seem to alter the tumor-inhibitory activity of the antibodies in vivo and the same pattern of tumor development was observed during the first few weeks following administration. However, the stimulating effects of PEG were observed at later stages of tumor growth since tumor development was either slowed down or completely arrested. Furthermore, a second tumor implanted into the same mice during this later stage was significantly or completely inhibited, as compared to results in mice injected with the unmodified antibody. The Fab′-PEG2 monomeric derivative was also shown to be effective in inhibiting the growth of a second tumor. The extended and prolonged enhancing effect of PEG on the antitumor activity of antibodies or Fab′ fragments directed against ErbB2 may be of importance in the treatment of ErbB2-overexpressing neoplasms. Received: 2 September 1999 / Accepted: 19 February 2000     

2-(Cyclohexylamino)-1-(4-cyclopentylpiperazin-1-yl)-2-methylpropan-1-one, a novel compound with neuroprotective and neurotrophic effects in vitro

Focusing on development of novel drug candidates for the treatment of neurodegenerative diseases, we developed and synthesized a new compound, 2-(cyclohexylamino)-1-(4-cyclopentylpiperazin-1-yl)-2-methylpropan-1-one (amido-piperizine 1). The compound demonstrated robust neuroprotective properties after both glutamate excitotoxicity and peroxide induced oxidative stress in primary cortical cultures. Furthermore, amido-piperizine 1 was found to significantly induce neurite outgrowth in vitro which could suggest central reparative and regenerative potential of the compound.With these potential beneficial effects in CNS, the ability of the amido-piperizine 1 to penetrate the blood-brain barrier was tested using MDR1-MDCK cells. Amido-piperizine 1 was found not to be a P-gp substrate and to have a high blood-brain barrier penetration potential, indicating excellent availability to the CNS. Moreover, amido-piperizine 1 had a fast metabolic clearance rate in vitro, suggesting that parenteral in vivo administration seems preferable.As an attempt to elucidate a possible mechanism of action, we found that amido-piperizine 1 bound in nano-molar range to the sigma-1 receptor, which could explain the observed neuroprotective and neurotrophic properties, and with a 100-fold lower affinity to the sigma-2 receptor. These results propose that amido-piperizine 1 may hold promise as a drug candidate for the treatment of stroke/traumatic brain injury or other neurodegenerative diseases.     

Effects of protease activated receptor (PAR)2 blocking peptide on endothelin-1 levels in kidney tissues in endotoxemic rat mode

Aims

Septic shock, the severe form of sepsis, is associated with development of progressive damage in multiple organs. Kidney can be injured and its functions altered by activation of coagulation, vasoactive-peptide and inflammatory processes in sepsis. Endothelin (ET)-1, a potent vasoconstrictor, is implicated in the pathogenesis of sepsis and its complications. Protease-activated receptors (PARs) are shown to play an important role in the interplay between inflammation and coagulation. We examined the time-dependent alterations of ET-1 and inflammatory cytokine, such as tumor necrosis factor (TNF)-α in kidney tissue in lipopolysaccharide (LPS)-induced septic rat model and the effects of PAR2 blocking peptide on the LPS-induced elevations of renal ET-1 and TNF-α levels.

Main methods

Male Wistar rats at 8 weeks of age were administered with either saline solution or LPS at different time points (1, 3, 6 and 10 h). Additionally, we treated LPS-administered rats with PAR2 blocking peptide for 3 h to assess whether blockade of PAR2 has a regulatory role on the ET-1 level in septic kidney.

Key findings

An increase in ET-1 peptide level was observed in kidney tissue after LPS administration time-dependently. Levels of renal TNF-α peaked (around 12-fold) at 1 h of sepsis. Interestingly, PAR2 blocking peptide normalized the LPS-induced elevations of renal ET-1 and TNF-α levels.

Significance

The present study reveals a distinct chronological expression of ET-1 and TNF-α in LPS-administered renal tissues and that blockade of PAR2 may play a crucial role in treating renal injury, via normalization of inflammation, coagulation and vaso-active peptide.     

A dinuclear europium(III) complex with thenoyltrifluoroacetonato and 1-(2-pyridylzao)-2-naphtholato ligands and its optical properties

Dinuclear lanthanide complexes of the general for Ln₂(TTA)₄(PAN)₂ (Ln = Eu, Gd, Tb, Yb; TTA and monodeprotonated thenoyltrifluoroacetone and PAN 1-(2-pyridylazo)-2-naphthol, respectively) were prepared and structurally characterized. These novel complexes, representing the first examples of crystallographically characterized lanthanide-PAN complexes, each feature a dinuclear core with the metal atoms bridged by the phenolato O atoms of the chelating-bridging PAN ligands. Electronic spectroscopic and photoluminescence studies were carried out for the Eu(III) complex, and the results are consistent with ligand-mediated energy transfer and ligand-sensitized luminescence characteristic of Eu(III). The Eu(III) complex doped into a polymeric film was shown to effectively limit a nanosecond 523-nm laser pulse, and the limiting effect is rationalized in terms of reverse saturable absorption due to the strong absorption of the metal’s excited triplet states that are populated by intersystem crossing.     

Association of hydrophobically-modified poly(ethylene glycol) with fusogenic liposomes

We present results on using cooperative interactions to shield liposomes by incorporating multiple hydrophobic anchoring sites on polyethylene glycol (PEG) polymers. The hydrophobically-modified PEGs (HMPEGs) are comb-graft polymers with strictly alternating monodisperse PEG blocks (M_w=6, 12, or 35 kDa) bonded to C18 stearylamide hydrophobes. Cooperativity is varied by changing the degree of oligomerization at a constant ratio of PEG to stearylamide. Fusogenic liposomes prepared from N-C12-DOPE:DOPC 7:3 (mol:mol) were equilibrated with HMPEGs. Affinity for polymer association to liposomes increases with the degree of oligomerization; equilibrium constants (given as surface coverage per equilibrium concentration of free polymer) for 6 kDa PEG increased from 6.1±0.8 (mg/m²)/(mg/ml) for 2.5 loops to 78.1±12.2 (mg/m²)/(mg/ml) for 13 loops. In contrast, the equilibrium constant for distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG5k) was 0.4±0.1 (mg/m²)/(mg/ml).The multi-loop HMPEGs demonstrate higher levels of protection from complement binding than DSPE-PEG5k. Greater protection does not correlate with binding strength alone. The best shielding was by HMPEG6k-DP3 (with three 6 kDa PEG loops), suggesting that PEG chains with adequate surface mobility provide optimal protection from complement opsonization. Complement binding at 30 min and 12 h demonstrates that protection by multi-looped PEGs is constant whereas DSPE-PEG5k initially protects but presumably partitions off of the surface at longer times.     

PIAS1 enhances SUMO-1 modification and the transactivation activity of the major immediate-early IE2 protein of human cytomegalovirus

The protein inhibitor of activated STAT1 (PIAS1), known to be a small ubiquitin-like modifier (SUMO) E3 ligase, was found to interact with the human cytomegalovirus IE2 protein. We found that the sumoylation of IE2 was markedly enhanced by wild-type PIAS1 but not by a mutant containing a Cys to Ser substitution at position 351 (C351S) within the RING finger-like domain. In target reporter gene assays, wild-type PIAS1, but not the C351S mutant, enhanced the IE2-mediated transactivations of viral polymerase promoter and cellular cyclin E promoter and this augmentation required the intact sumoylation sites of IE2. Our results suggest that PIAS1 acts as a SUMO E3 ligase toward IE2 and that it may regulate the transactivation function of IE2. To our knowledge, IE2 is the first viral target found to be regulated by a SUMO E3 ligase.