Abstract  The antibiotics chloramphenicol (Cm), tetracycline, and erythromycin, which inhibit bacterial protein synthesis and are known to induce the cold shock response, unexpectedly enhance the heterologous expression of P450s and related proteins in Escherichia coli. In contrast, antibiotics that mimic heat shock in E. coli such as puromycin, streptomycin, and kanamycin decrease the expression of the same proteins. A sublethal dose of Cm (1 microgram/ml) effectively enhances the expression of both membrane-bound proteins (microsomal and mitochondrial P450s) and a soluble mitochondrial protein (adrenodoxin) over the range of two- to eightfold. The expression level of N-terminal truncated P450c17 (1600 nmol/liter culture without Cm), for instance, reached 3500 nmol/liter culture by the addition of Cm, approximately 8.4% of the total cellular protein. Cm also enabled expression at useful levels of active P450s previously difficult to express in E. coli. In contrast, the expression of P450scc, a mitochondrial protein, is decreased by Cm but enhanced by ethanol, a powerful elicitor of heat shock response in E. coli. These results show that both the cold shock response induced by some antibiotics and the heat shock response induced by ethanol may lead to enhanced expression of certain heterologous proteins in E. coli. This study also indicates that protein synthesis inhibitors associated with the cold shock response may act as protein synthesis enhancers under certain conditions.

Abstract  An improved method for the enrichment of phenylalanine ammonia lyase (E.C.4.3.1.5-PAL) activity of Rhodotorula yeast is described. Whole cells of various Rhodotorula yeast strains showed low levels of PAL activity on account of membrane permeability barriers for the substrate, L-phenylalanine. Different methods of enriching PAL activity including ultra- sonication, detergent and enzyme solubilization were investigated in this study in an effort to overcome these permeability barriers. Ultra-sonication of the yeast cells was found to be the most effective of the methods and yielded over 10-fold higher enzyme activity as compared to untreated whole cells. Solubilization of the yeast cells involving a synergistic treatment with Triton- X-100 and glucuronidase gave about nine-fold enrichment while cells treated individually with Triton-X-100, glucuronidase and cetyl trimethyl ammonium bromide gave 6.8-, 5.9-and 5.8-fold higher activity, respectively. Homogenization; treatment with chloroform, isopropanol, and toluene; freezing and thawing were ineffective in enhancing the enzyme activity. Of the six Rhodotorula yeast strains employed in this study, Rhodotorula glutinis, NCYC 61 exhibited maximum PAL activity. This simple, rapid and improved method of PAL activity enrichment could serve as a rich enzyme source, especially in the biosynthesis of L-phenylalanine and L-phenylalanine methyl ester.

Abstract  The paper reports oil experimental data oil the extraction of caffeine, coffee oil and chlorogenic acids from green coffee beans using pure supercritical Co-2 and supercritical CO, modified with ethanol (5% w/w) and isopropyl alcohol (5% w/w) at 50 and 60 degrees C and 15.2 24.8 e 35.2 MPa. In this Study extraction kinetics were obtained for all assays i.e. samples were collected at several time intervals for each solvent and mixed solvent. When pure CO, and CO-ethanol mixed solvent were used, all increase in pressure resulted in ail increase in the amount Of Oil extracted. When CO, was modified with isopropyl alcohol, the amount Of coffee oil extracted also increased with pressure. Caffeine extraction initially increased and subsequently decreased with pressure. Chlorogenic acids were only extracted when isopropyl alcohol was used as a cosolvent. An increase in extraction temperature resulted in a decrease of caffeine and oil extraction (retrograde condensation) when only CO, was used as solvent. With the use of co-solvent this retrograde behavior was no longer observed and the increase in temperature resulted in the increase in the extracted amounts of caffeine, coffee oil and chlorogenic acids.

Abstract  Azamacrocycles bearing four arylsulfonyl or arylsulfinyl pendant arms have been synthesised with good yields through nucleophilic addition of 1,4,8,11-tetraazacyclotetradecane (cyclam) to phenylvinylsulfone, phenylvinylsulfoxide or (R)-tolylvinylsulfoxide in isopropanol/ water media. The crystal structure of the tetraethylsulfonylphenyl substituted macrocycle has been determined by X-ray crystallography. Preliminary studies of the coordination properties of these functionalised macrocyles towards Cu(II) and Eu(III) indicate that pendant sulfoxide groups act as oxygen donor coordinating groups. (C) 2007 Elsevier Ltd. All rights reserved.

Abstract  Soluble, truncated mutant and wild-type forms of penicillin-binding protein 5 (sPBP 5) from Escherichia coli were produced in large amounts by placing the dacA gene that encodes PBP 5 under the control of the trp-lac fusion promoter. The 3' end of the dacA gene used in this study contains a stop codon that results in the deletion of 15 amino acids from the carboxyl terminus and the production of a soluble protein. Using oligonucleotide-directed mutagenesis, the role of cysteine 115 in the mechanism of sPBP 5 was investigated. Alkylation of cysteine 115 with sulfhydryl reagents has previously been shown to inhibit severely the D-alanine carboxypeptidase activity of PBP 5. Alkylation also inhibits the hydrolysis of bound penicillin G, with only a slight effect on its binding. Cysteine 115 in sPBP 5 was changed to either a serine (sPBP 5C-S) or an alanine (sPBP 5C-A) residue. The wild-type and mutant sPBPs were purified in milligram amounts from induced cultures by ampicillin affinity chromatography. The mutant PBPs showed only a 2-fold increase in the half-life of the penicilloyl-PBP complex, and had a binding affinity for penicillin G identical to wild-type PBP 5. The Km for the release of D-alanine from the peptide L-Ala-D-gamma-Glu-L-Lys-D-Ala-D-Ala was 5.0, 3.5, and 7.8 mM for PBP 5, PBP 5C-S, and PBP 5C-A, respectively, while the values for Vmax were 2.5, 3.3, and 5.1 mumol/min/mg. From these data it was concluded that the cysteine residue does not directly participate in the enzymatic mechanism.

Abstract  In this research it is demonstrated that, due to the similarities between Entrainer-based Reactive Distillation and azeotropic distillation, the same selection rules can be applied to select a suitable entrainer. From a list of suitable entrainers for the azeotropic distillation of isopropanol and water, cyclohexane and isopropyl acetate are chosen. Residue curve maps, simulations of the distillation section of the column, and simulations of the total Entrainer-based Reactive Distillation concept show that both can be used as an entrainer in Entrainer-based Reactive Distillation. Whether the Entrainer-based Reactive Distillation concept will be feasible, depends strongly on the kinetics of the reaction. (C) 2009 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Abstract  A pressurized liquid extraction (PLE) solid-phase extraction (SPE) gas chromatography-mass spectrometry (GC-MS) method was developed for the determination of two model organophosphorus pesticides (OPPs), i.e. chlorpyrifos (CPF, O,O-diethyl-O-[3,5,6-trichloro-2-pyridyl]-phosphorothioate, CAS no. 2921-88-2) and diazinon (DZN, O,O-diethyl-O-2-isopropyl-4-methyl-6-pyrimidyl thiophosphate, CAS no. 333-41-5) and their two more stable metabolites namely, 3,5,6-trichloro-2-pyridinol (TCP, CAS no. 6515-38-4) and 2-isopropyl-6-methyl-4-pyrimidinol (IMHP, CAS no. 28-14-20-2) in sewage sludge and agricultural soil samples was developed. A PLE procedure was optimized as a less time-consuming and cost-effective extraction approach for sewage sludge/soil samples. Derivatization of metabolites was carried out with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) to form stable derivatives. The method was developed using spiked soil samples at 1mg/kg for the intact insecticides and at 2 mg/kg for their metabolites. Recovery yields for the target pesticides were>92%, meanwhile for the metabolites the recoveries were somewhat low. Limits of quantification (LOQ) were within the range of 4-62 ng/g for the sludge sample and between 3 and 31 ng/g for sludge-fertilized soil samples. The method was applied for the determination of both insecticides in some soils fertilized with sludge from municipal wastewater treatment plants (WWTPs) in the proximity of Barcelona (Catalonia, Spain) and in one representative sludge sample used as fertilizer. Results revealed the presence of CPF in all samples at concentrations within the range of 210.32-38.36 ng/g. Both major metabolites were also found to be present in all samples.

Abstract  The effect of post-induction nutrient feeding strategies on the production of bioadhesive protein using an IPTG inducible expression system in Escherichia coli was investigated. Cells were cultured in an exponential fed-batch mode to the OD600 of ca. 100 (48 gDCW/L) prior to induction. Six different post-induction nutrient feeding strategies (pH-stat, exponential, constant and linear change in feeding rate with three different slopes) were then applied, and bioadhesive protein production was examined. It was found that post-induction cell growth was independent of nutrient feeding rate. However, bioadhesive protein production was significantly affected by post-induction feeding strategies. Linearly changing post-induction feeding rate with a suitable slope allowed production of bioadhesive protein up to 5.3 g/L, which was higher than that obtained by the other post-induction feeding strategies.

Abstract  Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human enteropeptidase (GenBank Accession No. U09860) was synthesized from 26 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. The fusion protein thioredoxin/human enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms. The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor. The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide with kinetic parameters K(m)=0.16 mM and k(cat)=115 s(-1) and small ester Z-Lys-SBzl with K(m)=140 microM, k(cat)=133 s(-1). L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with K(i)(*)=2.3 nM. L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions.

Abstract  PURPOSE: To study the in vivo effects of a thermosensitive retinal adhesive, plasma-polymerized N-isopropyl acrylamide (ppNIPAM), in rabbit eyes.

METHODS: Parylene C(poly(monochloro-p-xylylene)) (20 microm) and poly(dimethyl siloxane) (PDMS) (> or =200 microm) coated with ppNIPAM were used as implant materials. Following pars plana vitrectomy (PPV), ppNIPAM coated parylene (n = 3) and PDMS (n = 3) implants were inserted over the retina in six rabbits. Baseline and follow-up imaging (color fundus photographs, fluorescein angiography, and optic coherence tomography [OCT]) and electroretinogram recordings were performed. Histologic evaluation was performed following enucleation at 6 weeks.

RESULTS: Intraoperative retinal adhesion occurred in all eyes with ppNIPAM coated parylene and PDMS implants. Two eyes developed retinal tears during the implantation procedure and the ppNIPAM coated implants closed the retinal tears successfully preventing retinal detachment. OCT findings confirmed the retinal adhesion in all eyes. Four weeks after implantation one parylene and one PDMS implant detached partly from the retinal surface. Histology showed mild changes at the outer retinal segments. There was no evidence of ocular toxicity and inflammation. None of the eyes that had an implant covering the retinal tear developed a retinal detachment but had some inflammatory changes around the implants.

CONCLUSIONS: ppNIPAM coated implants may provide retinal adhesion in vivo without measurable ocular toxicity in the short term. Covering a retinal tear, the ppNIPAM coated implants may prevent retinal detachment.

Abstract  The uptake of 4-deoxy-4-fluoro-D-glucose (4FG), without subsequent catabolism, by resting cells of Escherichia coli (ATCC 11775) is 0.06 mg/mg dry weight. In frozen-thawed cells of this organism, 4FG is a substrate for the phosphoenolpyruvate phosphotransferase system with a rate of phosphorylation twice that found for the isomeric 3-deoxy-3-fluoro-D-glucose. 4FG is not a carbon source for growth of this organism and it inhibits the extent of growth of cells in the presence of glucose. The inhibition of growth of E. coli K12 on lactose by 4FG is also observed and this is considered to be consistent with the fact that 4FG is an uncompetitive inhibitor of beta-galactosidase (EC 3.2.1.23) activity and that 4FG or 4-deoxy-4-fluoro-D-glucose-6 phosphate repress beta-galactosidase synthesis. These results support the view that catabolite repression may be produced by compounds which are not necessarily metabolised further than hexose-6-phosphates.

Abstract  The protective role of two synthetic organoselenium compounds 1-isopropyl-3-methylbenzimidazole-2-selenone (SeI) and 1, 3-di-p-methoxybenzylpyrimidine-2-selenone (Sell) was examined against the 7,12-dimethylbenz[a]anthracene (DMBA)-induced changes in biochemical parameters in blood of rats. Albino Winstar rats (150-200 g body wt) were treated with single dose of DMBA (50 mg/kg body wt) and organoselenium compounds (25 micromol/kg) for 4 weeks at two days internal. Blood was taken from the anaesthetized rats ventricle from their hearts for biochemical analysis. Administration of DMBA resulted in elevation of urea, uric acid and creatinine levels as well as AST, ALT and LDH activities and decrease in levels of total proteins, albumin and globulin. SeI and SeII caused a significant (p<0.05) decrease in urea, uric acid and creatinine levels and alanine aminotransferase (ALT); aspartate aminotransferase; (AST) and lactate dehydrogenase (LDH) activities and significantly increased the levels of total protein and albumin (p<0.05). These organoselenium compounds are likely to be beneficial in human health.

Abstract  Latanoprost and unoprostone (isopropyl unoprostone) represent the first commercially available prostaglandin analogues to be used for the treatment of glaucoma. Both compounds reduce intraocular pressure by enhancing uveoscleral outflow. Latanoprost, when used once daily in the evening, produces a greater reduction in pressure than timolol. Latanoprost produces mild conjunctival hyperaemia compared with timolol in some patients. Darkening of the irides has been reported, especially in green-brown, yellow-brown and blue/grey-brown irides. Hypertrichosis and hyperpigmentation of the eyelashes have also been demonstrated. Although latanoprost has not been proven to cause uveitis or cystoid macular oedema, case reports of an association exist. Latanoprost does not produce systemic adverse effects nor does it alter routine blood analyses. Unoprostone, when given twice daily, produces less of a reduction in intraocular pressure than timolol or latanoprost. Three times daily use may be required to approach the effectiveness of timolol. Unoprostone may have a similar adverse effect profile to latanoprost, but may to cause more corneal epithelial problems. Unoprostone is also not known to cause systemic adverse effects. Both agents are welcome additions to the treatment of glaucoma. However, additional studies and more experience are needed with each agents.

Abstract  The metabolism and action of chlorpropham (isopropyl N-(3-chlorophenyl)carbamate; CIPC, a post-harvest agent) and its metabolites were studied in freshly isolated rat hepatocytes and isolated rat hepatic mitochondria, respectively. The exposure of hepatocytes to CIPC caused a concentration (0.25-1.0 mM)- and time (0-3h)-dependent cell death accompanied by loss of cellular ATP and adenine nucleotides. CIPC at a weakly toxic level (0.5 mM) was metabolized to isopropyl N-(3-chloro-4-hydroxyphenyl)carbamate (4OH-CIPC) and subsequently to its glucuronide and sulfate conjugates (major metabolites) or alternatively to a minor metabolite 3-chloroaniline (3CA). The addition of SKF-525A (50 microM), an inhibitor of microsomal monooxygenase, enhanced the CIPC (0.5 mM)-induced cytotoxicity accompanied by loss of ATP and 4OH-CIPC and inhibited the decrease in the concentration of the parent compound. CIPC led to a strong decrease in cellular ATP content compared to its metabolites, 4OH-CIPC and 3CA. On the other hand, the exposure of isolated hepatic mitochondria to CIPC reduced State 3 respiration with a FAD-linked substrate (succinate plus rotenone) and/or with a NAD+ -linked substrate (pyruvate plus malate), whereas State 3 respiration with ascorbate plus tetramethyl-p-phenylendiamine (cytochrome oxidase-linked respiration) was not affected markedly by CIPC. Further, the addition of CIPC caused an increase in the rate of State 4 oxygen consumption, indicating an uncoupling effect, and a decrease in the rate of State 3 oxygen consumption in a concentration-dependent manner, respectively. In contrast, the addition of neither 4OH-CIPC nor 3CA markedly affected the rate of states 3 and/or 4 oxygen consumption. These results indicate that CIPC-induced cytotoxicity is mediated by the parent compound rather than by its metabolites such as 4OH-CIPC and 3CA, and that the toxicity is associated with a rapid depletion of ATP via impairment of mitochondrial function related to oxidative phosphorylation.

Abstract  Isopropyl methanesulfonate (IMS), a direct-acting SN1 alkylating agent, induced thymic lymphomas in 29 of 30 female Hsd: (ICR)BR mice by the 139th day following once a week subcutaneous injections at a dose of 10 mumol/mouse. No such neoplasms were observed at the 450th day following 52 once a week subcutaneous injections (20 mumol/mouse) of the borderline SN1/SN2 alkylating agent ethyl methanesulfonate (EMS). Local sarcomas were observed in 2 and pulmonary adenomas in 20 of the 30 EMS-treated mice, 70% of which were alive at the 450th day. Neoplasms were not observed at local or at distant sites other than the thymus gland following treatment with IMS. The results suggest a sensitivity of hemopoietic tissue to the types of DNA lesions induced by IMS.

Abstract  As DNA gyrase is the only enzyme to supercoil DNA actively, we address here the question of whether it does play the expected dominant role in controlling the level of DNA supercoiling and growth rate in Escherichia coli. We modulated the expression of DNA gyrase around its wild-type level, and measured the effect on plasmid supercoiling and growth rate. As both the activity and the transcription rate of DNA gyrase are sensitive to DNA supercoiling we distinguish two types of control (with control defined as the percentage change observed on a 1% modulation of a parameter). The first type of control, here named inherent control, quantifies the effect of a sustained modulation of the transcription rate of gyrase. At its wild-type expression level this inherent control exerted by DNA gyrase on growth rate was very low, i.e. c mu/gyrase = 0.05 - 0.00, as was the inherent control on DNA supercoiling, c aLK/gyrase = 0.2. The second type of control, here named global control, quantifies the effect of a change in gyrase activity whilst allowing the cell to respond by readjusting gyrase transcription. Both types of control are linked via the sensitivity of gyrase transcription to DNA supercoiling, as determined from the inherent control by gyrase of the gyrase promoter activity using a chromosomal gyrB:lacZ fusion. As expected, the latter control was negative, but small, i.e. c gyr promoter/gyrase=-0.3. The global control by gyrase of active linking number was 0.1. These results show that although gyrase is an essential enzyme it does not have a high control, on either growth rate or DNA supercoiling. Homeostatic regulation of physiological DNA structure appears to dominate. At low degrees of DNA supercoiling, the control by DNA gyrase and by the other topoisomerases is much stronger.

Abstract  The kinetics of the reactivation of acetylcholinesterase inhibited by isopropyl methylphosphonofluoridate was studied. The reactivators used include nine bispyridinium monooximes and three bispyridinium dioximes. The dissociation constant (Kd) and the rate constant (k2) of dephosphorylation of the complex formed from the organophosphorus acetylcholinesterase (OP-AChE) and the oxime were measured. The reactivation parameters obtained from the in vitro kinetic studies were used to elucidate the structure-activity relationships. The hydrophobic property of a nonoxime substituent at the 3-position on the pyridinium ring can exert a positive effect on their binding affinity to OP-AChE. However, the rate constants (k2) of the nucleophilic displacement of OP-AChE by oximes depend negatively on these physical and structural factors of the oximes. The correlations of the in vivo antidotal efficacy (ED50) of these bispyridinium oximes have been analyzed with their pharmacological properties, e.g., reactivation potency, antimuscarinic activities, and antinicotinic activities. However, no satisfactory correlations were observed. It may be concluded that the detoxication mechanism of poisoning by isopropyl methylphosphonofluoridate is different from those of pinacolyl methylphosphonofluoridate and paraoxon.

Abstract  In an attempt to examine the ability of benzisothiazole-based drugs to interact with beta-adrenoceptors, a series of 1,2-benzisothiazole derivatives, which were substituted with various propanolamine or oxypropanolamine side chains in the 2 or 3 position, were synthesised and tested. The pharmacological activity of these compounds at the beta-adrenoceptors was examined using isolated rat atria and small intestinal segments, which preferentially express the beta1- and beta3-adrenoceptor-mediated responses, respectively. None of these products showed any beta-adrenoceptor agonistic activity. In contrast, the 2- and 3-substituted isopropyl, tert-butyl, benzyl, and piperonyl derivatives 2a-d and 3a-d elicited surmountable inhibition of the isoprenaline-induced chronotropic effects in the atria, suggesting competitive antagonism at the beta1-recognition site. The pA2 values revealed tert-butyl 3b and the isopropyl substituted piperonyl derivatives 3a to be the most effective. Remarkably, many of the 2-substituted propanolamines were less active than the corresponding 3-substituted oxypropanolamines. With the exception of compound 3b, none of these drugs antagonised the muscle relaxant activity of isoprenaline in the intestine, suggesting no effect on the beta3-adrenoceptors. These results confirm the ability of the benzisothiazole ring to interact with the beta-adrenoceptors, and demonstrate that 2-substitution with propanolamine or 3-substitution with oxypropanolamine groups yields compounds with preferential antagonistic activity at the cardiac beta1-adrenoceptors. The degree of antagonism depends strongly on both the nature of the substituent and its position on the benzisothiazole ring.

Abstract  The removal of four earthy-musty compounds, 2-methylisoborneol (MIB), 2,4,6-trichloroanisole (TCA), 2-isopropyl-3-methoxy-pyrazine (IPMP), and 2-isobutyl-3-methoxy-pyrazine (IBMP), by powdered activated carbon (PAC) were investigated under the simulative condition of Xicun water plant, Guangzhou. The adsorption kinetics of odor compounds by two PACs show that main removal of odor compounds occurs within contact time of 1 h. Compared with the wood-based PAC, the coat-based PAC evidently improves the removal efficiency of poorly adsorbed compounds like MIB. The effects on removal efficiency such as optimum PAC dosage, initial concentration of the organics, chlorine residual, background organics, and changes of water quality were investigated. The percent removal of trace odorants at any given time for a particular carbon dosage is irrelative to the initial concentration of the odor compounds. Adsorptive capacity of PAC for target compounds is reduced by chlorine residual and background organics. Characteristics of raw water have vast influence on the removal of target compounds by PAC.

Abstract  The PCR-amplified beta-subunit of the human chorionic gonadotropin structural gene (betahCG) was cloned under the control of the tac promoter and the heat-labile enterotoxin chain B (LTB) signal sequence (LTBss). BetahCG was successfully produced, processed and exported to the periplasmic space in Escherichia coli. Expression of betahCG was confirmed by immunoblot analysis using an anti-betahCG polyclonal antibody. The processing of the protein was very efficient, as only the processed band could be detected at all time points during the course of induction. Expression was evident soon after the addition of the lactose analogue, IPTG. These results demonstrate that E. coli cells can synthesize, process and export betahCG using the LTBss.

Abstract  This study describes the preparation and characterization of poly(lactic-co-glycolic acid) microspheres containing a novel anticancer agent, taxol (namely, Taxol-PLGA-MS). A solvent evaporation technique was utilized to prepare Taxol-PLGA-MS. The trapping efficiency of taxol in the microspheres was greater than 90% and reproducible. The in vitro release rate of taxol from the microspheres was very low, and less than 15% of the initial amount of taxol was released in three weeks, irrespective of the drug loading level. When a chemical additive, isopropyl myristate (IPM), was introduced at the level of 30% (w/w), the release of taxol increased significantly; approximately 70% of the initial amount of taxol was released at a nearly constant rate for three weeks. Elevation of the loaded IPM level to 50% (w/w) produced a more rapid release of the drug. Scanning electron microscopy showed that Taxol-PLGA-MS were spherical with a smooth surface. More than half (55-65%) of the microspheres had a diameter of 20-45 microns. Incorporation of IPM had no significant influence on the particle size, surface morphology, or degradation behavior of the microspheres. It was strongly suggested that the release of taxol from the microspheres was dominated mainly by the drug diffusion in the matrix. As evaluated from the particle size, drug content, and in vitro release property, IPM-containing Taxol-PLGA-MS may be suitable for chemoembolization therapy of cancer diseases.

Abstract  The structure of a new C15-lactone, (7S)-10-oxo-4xi-methyl-7-isopropyl-5E-undecen-4-olide, isolated from Greek tobacco, has been determined mainly be 1H NMR, 13C NMR and high resolution MS, and its absolute configuration established by degradation to (2S)-5-oxo-2-isopropylhexanal. The carbon skeleton of the new compound indicated that it is derived from a thunbergane precursor and constitutes the first C15 nor-thunberganoid in tobacco.

Abstract  p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.

Abstract  Numerous reports have shown that fine particulates threaten human health. Since their health impact is associated with both mass and number concentrations, it is necessary to evaluate the emission standards for particulate mass accordingly. This study examined the particulate matter characteristics of diesel locomotive engine exhaust at various engine ratings. Diesel engine exhaust was collected via a dilution tunnel and the concentration and size distribution of fine particles were measured by a scanning mobility particle sizer. Exhaust gasses were measured simultaneously by a stack sampler. The maximum carbon monoxide emission was reached at 59% of the maximum rating, after which emissions decreased. The particle count median diameter increased with the engine rating, until a maximum was reached at 40% of the maximum rating. Most exhaust particles were nanoparticles with the nuclei mode range, a particle diameter (D(P))<50 nm. The increase in particles with the accumulation mode range, 50<D(P)<1000 nm, led to a mass concentration increase and number concentration reduction. The count median diameter was within the nuclei mode range at lower engine ratings, and within the accumulation mode range at higher engine ratings. Since diesel engines mainly generate fine particles, exhaust particle mass and size distribution should be considered in emission regulations.