Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells.

With our recent success in developing a recombinant human arginase drug against broad-spectrum cancer cell lines, we have explored the potential of a recombinant Bacillus caldovelox arginase mutant (BCA-M) for human cervical cancer treatment. Our studies demonstrated that BCA-M significantly inhibited the growth of human cervical cancer cells in vitro regardless of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) expression. Drug susceptibilities correlate well with the expressions of major urea cycle genes and completeness of L-arginine regeneration pathways. With the expressions of ASS and ASL genes conferring resistance to L-arginine deiminase (ADI) which is undergoing Phase III clinical trial, BCA-M offers the advantage of a broader spectrum of susceptible cancer cells. Mechanistic studies showed that BCA-M inhibited the growth of human cervical cancer cells by inducing apoptosis and cell cycle arrest at S and/or G2/M phases. Our results also displayed that autophagy served as a protective mechanism, while the growth inhibitory effects of BCA-M could be enhanced synergistically by its combination to the autophagy inhibitor, chloroquine (CQ), on human cervical cancer cells.

Mono-PEGylation of a Thermostable Arginine-Depleting Enzyme for the Treatment of Lung Cancer.

L-arginine (L-Arg) depletion induced by randomly PEGylated arginine deiminase (ADI-PEG20) can treat arginosuccinate synthase (ASS)-negative cancers, and ADI-PEG20 is undergoing phase III clinical trials. Unfortunately, ASS-positive cancers are resistant to ADI-PEG20. Moreover, the yield of ADI production is low because of the formation of inclusion bodies. Here, we report a thermostable arginine-depleting enzyme, Bacillus caldovelox arginase mutant (BCA-M: Ser161->Cys161). An abundant amount of BCA-M was easily obtained via high cell-density fermentation and heat treatment purification. Subsequently, we prepared BCA-M-PEG20, by conjugating a single 20 kDa PEG monomer onto the Cys161 residue via thio-chemistry. Unlike ADI-PEG20, BCA-M-PEG20 significantly inhibited ASS-positive lung cancer cell growth. Pharmacodynamic studies showed that a single intraperitoneal injection (i.p). administration of 250 U/mouse of BCA-M-PEG20 induced low L-Arg level over 168 h. The mono-PEGylation of BCA-M prolonged its elimination half-life from 6.4 to 91.4 h (a 14-fold increase). In an A549 lung cancer xenograft model, a weekly administration of 250 U/mouse of BCA-M-PEG20 suppressed tumor growth significantly. We also observed that BCA-M-PEG20 did not cause any significant safety issue in mouse models. Overall, BCA-M-PEG20 showed excellent results in drug production, potency, and stability. Thereby, it has great potential to become a promising candidate for lung cancer therapy.

A bioengineered arginine-depleting enzyme as a long-lasting therapeutic agent against cancer.

L-Arginine (L-Arg) depletion has attracted great attention in cancer therapy. Although two types of arginine-depleting enzymes, arginine deiminase (ADI) and human arginase I, are undergoing clinical trials, random site of PEGylation, low efficacy of heavy metal as co-factor, and immunogenicity limit the performance of these drugs and cause difficulty in a homogeneous production. Here we screened ten catalytic metal ions and have successfully produced a site-specific mono-PEGylated human arginase I mutant by conjugating the Cys45 residue to PEG-maleimide to minimize the decrease in activity and produce a homogeneous product. The catalytic efficiency trend of metal ion-enriched human arginase I mutant (HAI) was Co2+ > Ni2+ ≫ Mn2+. The overall kcat/KM values of Co-HAI and Ni-HAI were higher than Mn-HAI by ~ 8.7- and ~ 5.2-folds, respectively. Moreover, the results of enzyme kinetics and circular dichroism spectrometry demonstrated that the 20 or 40 kDa linear and branched PEG attached on the HAI surface did not affect the enzyme activity and the protein secondary structures. In vitro studies showed that both Co-HAI-PEG20L and Ni-HAI-PEG20L inhibited the growth of eight types of cancer cell lines. The pharmacodynamic study in mice demonstrated that the i.p. administration of Co-HAI-PEG20L at 13 mg/kg and Ni-HAI-PEG20L at 15 mg/kg was able to maintain a L-Arg level below its detection limit for over 120 h after one injection. The body weights of mice could return to normal levels within 5 days after injection, showing that the doses were well-tolerated. Therefore, both the Ni-HAI-PEG20L and Co-HAI-PEG20L are promising candidates for cancer therapy. KEY POINTS: • Mono-PEGylation applied on human arginase I mutant (HAI) successfully. • The catalytic efficiency of Co- and Ni-enriched HAI was higher than the wild type. • At least eight types of cancer cell lines were inhibited by Co- and Ni-HAI-PEG20L. • Co- and Ni-HAI-PEG20L were able to achieve weekly depletion of L-Arg. Graphical abstract.

Ultra-high adsorption of cationic methylene blue on two dimensional titanate nanosheets.

In this work, we examined the performance of 2D titanate nanosheets for dye adsorption. Their adsorption capacity for methylene blue (MB) is up to 3937 mg g-1, which is more than 10 times higher than active carbon and occupies the highest place among all the reports.

An Amyloid-Fibril-Based Colorimetric Nanosensor for Rapid and Sensitive Chromium(VI) Detection.

Appearing with a trace! A new colorimetric nanosensor for CrVI from the amyloid fibrils of hen lysozyme has been developed. This nanosensor, which makes use of hen lysozyme fibrils as CrVI binders and acidified diphenylcarbazide (DPC) as the colorimetric probe, can specifically detect CrVI at the ppb level without sample pretreatment and the use of advanced instruments.

Deprivation of arginine by recombinant human arginase in prostate cancer cells.

BACKGROUND: Recombinant human arginase (rhArg) has been developed for arginine deprivation therapy in cancer, and is currently under clinical investigation. During pre-clinical evaluation, rhArg has exhibited significant anti-proliferative activity in cancer cells deficient in the expression of ornithine carbamoyl transferase (OCT). Interestingly, a variety of cancer cells such as melanoma and prostate cancer deficient in argininosuccinate synthetase (ASS) are sensitive to arginine deprivation by arginine deiminase. In this study, we investigated levels of gene expression of OCT and ASS, and the effects of rhArg in human prostate cancer cells: LNCaP (androgen-dependent), PC-3 and DU-145 (both androgen-independent). RESULTS: Quantitative real-time PCR showed minimal to absent gene expression of OCT, but ample expression of ASS expression in all 3 cell lines. Cell viability assay after 72-h exposure of rhArg showed all 3 lines had half maximal inhibitory concentration less than or equal to 0.02 U/ml. Addition of ornithine to cell culture media failed to rescue these cells from rhArg-mediated cytotoxicity.Decreased phosphorylation of 4E-BP1, a downstream effector of mammalian target of rapamycin (mTOR), was noted in DU-145 and PC-3 after exposure to rhArg. Moreover, there was no significant apoptosis induction after arginine deprivation by rhArg in all 3 prostate cancer cell lines. CONCLUSION: rhArg causes significant cytotoxicity in LNCaP, DU-145 and PC-3 prostate cancer cells which all demonstrate decreased OCT expression. Inhibition of mTOR manifested by hypophosphorylation of 4E-BP1 suggests autophagy is involved as alternative cell death mechanism. rhArg demonstrates a promising novel agent for prostate cancer treatment.

Degradation of chelating agents in aqueous solution using advanced oxidation process (AOP).

This article presents an overview with critical analysis of technical applicability of advanced oxidation process (AOP) in removing chelating agents from aqueous solution. Apart from the effect of metals for chelating agents as a major influencing factor, selected information such as pH, oxidant's dose, concentrations of pollutants and treatment performance is presented. The performance of individual AOP is compared. It is evident from our literature survey that photocatalysis with UV irradiation alone or coupled with TiO(2), ozonation and Fenton's oxidation are frequently applied to mineralize target pollutants. Overall, the selection of the most suitable AOP depends on the characteristics of effluents, technical applicability, discharge standard, regulatory requirements and environmental impacts.

Recombinant human arginase inhibits the in vitro and in vivo proliferation of human melanoma by inducing cell cycle arrest and apoptosis.

Melanoma has been shown to require arginine for growth, thus providing a potential Achilles' heel for therapeutic exploitation. Our investigations show that arginine depletion, using a recombinant form of human arginase I (rhArg), efficiently inhibits the growth of mammalian melanoma cell lines in vitro. These cell lines are consistently deficient in ornithine transcarbamylase (OTC) expression, correlating with their sensitivity to rhArg. Cell cycle distribution of A375 human melanoma cells treated with rhArg showed a remarkable dual-phase cell cycle arrest in S and G2/M phases, in contrast to the G2/M single-phase arrest observed with arginine deiminase (ADI), another arginine-degrading enzyme. rhArg and ADI both induced substantial apoptosis in A375 cells, accompanied by global modulation of cell cycle- and apoptosis-related transcription. Moreover, PEGylated rhArg dramatically inhibited the growth of A375 and B16 melanoma xenografts in vivo. Our results establish for the first time that (PEGylated) rhArg is a promising candidate for effective melanoma treatment, with fewer safety issues than ADI. Insight into the mechanism behind the antiproliferative activity of rhArg could inform us in designing combination therapies for future clinical trials.

Removal of refractory compounds from stabilized landfill leachate using an integrated H2O2 oxidation and granular activated carbon (GAC) adsorption treatment.

This study investigated the treatment performances of H(2)O(2) oxidation alone and its combination with granular activated carbon (GAC) adsorption for raw leachate from the NENT landfill (Hong Kong) with a very low biodegradability ratio (BOD(5)/COD) of 0.08. The COD removal of refractory compounds (as indicated by COD values) by the integrated H(2)O(2) and GAC treatment was evaluated, optimized and compared to that by H(2)O(2) treatment alone with respect to dose, contact time, pH, and biodegradability ratio. At an initial COD concentration of 8000 mg/L and NH(3)-N of 2595 mg/L, the integrated treatment has substantially achieved a higher removal (COD: 82%; NH(3)-N: 59%) than the H(2)O(2) oxidation alone (COD: 33%; NH(3)-N: 4.9%) and GAC adsorption alone (COD: 58%) at optimized experimental conditions (p< or =0.05; t-test). The addition of an Fe(II) dose at 1.8 g/L further improved the removal of refractory compounds by the integrated treatment from 82% to 89%. Although the integrated H(2)O(2) oxidation and GAC adsorption could treat leachate of varying strengths, treated effluents were unable to meet the local COD limit of less than 200 mg/L and the NH(3)-N of lower than 5 mg/L. However, the integrated treatment significantly improved the biodegradability ratio of the treated leachate by 350% from 0.08 to 0.36, enabling the application of subsequent biological treatments for complementing the degradation of target compounds in the leachate prior to their discharge.

Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC).

BACKGROUND: Protein used in medicine, e.g. interferon, are immunogenic and quickly broken down by the body. Pegylation is a recognized way of preserving their integrity and reducing immune reactions, and works well with enzymes used to degrade amino acids, a recent focus of attention in controlling cancer growth. Of the two arginine-degrading enzymes being explored clinically, arginine deiminase is a decidedly foreign mycoplasm-derived enzyme, whereas human arginase 1 is a native liver enzyme. Both have been pegylated, the former with adjuncts of 20 kD, the latter with 5 kD PEG. Pegylation is done by several different methods, not all of which are satisfactory or desirable. METHODS: The preparation of novel polyethylene glycol (PEG) derivatives for modifying proteins is described, but directed specifically at pegylation of recombinant human arginase 1 (rhArg1). rhArg1 expressed in Escherichia coli was purified and coupled in various ways with 5 different PEG molecules to compare their protective properties and the residual enzyme activity, using hepatocellular cell lines both in vitro and in vivo. RESULTS: Methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA 5,000) coupled with very high affinity under mild conditions. The resulting pegylated enzyme (rhArg1-peg5,000 mw) had up to 6 PEG chains of 5K length which not only protected it from degradation and any residual immunogenicity, but most importantly let it retain >90% of its native catalytic activity. It remained efficacious in depleting arginine in rats after a single ip injection of 1,500 U of the conjugate as the native enzyme, plasma arginine falling to >0.05 microM from approximately 170 microM within 20 min and lasting 6 days. The conjugate had almost the same efficacy as unpegylated rhArg1 on 2 cultured human liver cancer (HCC) cell lines. It was considerably more effective than 4 other pegylated conjugates prepared. CONCLUSION: Valuable data on the optimization of the pegylation procedure and choice of ligand that best stabilizes the enzyme arginase 1 are presented, a protocol that should equally fit many other enzymes and proteins. It is a long lasting arginine-depleting enzyme in vivo which will greatly improve its use in anti-cancer therapy.

Pegylated recombinant human arginase (rhArg-peg5,000mw) inhibits the in vitro and in vivo proliferation of human hepatocellular carcinoma through arginine depletion.

Hepatocellular carcinoma (HCC) is believed to be auxotrophic for arginine through the lack of expression of argininosuccinate synthetase (ASS). The successful use of the arginine-depleting enzyme arginine deiminase (ADI) to treat ASS-deficient tumors has opened up new possibilities for effective cancer therapy. Nevertheless, many ASS-positive HCC cell lines are found to be resistant to ADI treatment, although most require arginine for proliferation. Thus far, an arginine-depleting enzyme for killing ASS-positive tumors has not been reported. Here, we provide direct evidence that recombinant human arginase (rhArg) inhibits ASS-positive HCCs. All the five human HCC cell lines we used were sensitive to rhArg but ADI had virtually no effect on these cells. They all expressed ASS, but not ornithine transcarbamylase (OTC), the enzyme that converts ornithine, the product of degradation of arginine with rhArg, to citrulline, which is converted back to arginine via ASS. Transfection of HCC cells with OTC resulted in resistance to rhArg. Thus, OTC expression alone may be sufficient to induce rhArg resistance in ASS-positive HCC cells. This surprising correlation between the lack of OTC expression and sensitivity of ASS-positive HCC cells shows that OTC-deficient HCCs are sensitive to rhArg-mediated arginine depletion. Therefore, pretreatment tumor gene expression profiling of ASS and OTC could aid in predicting tumor response to arginine depletion with arginine-depleting enzymes. We have also shown that the rhArg native enzyme and the pegylated rhArg (rhArg-peg(5,000mw)) gave similar anticancer efficacy in vitro. Furthermore, the growth of the OTC-deficient Hep3B tumor cells (ASS-positive and ADI-resistant) in mice was inhibited by treatment with rhArg-peg(5,000mw), which is active alone and is synergistic in combination with 5-fluorouracil. Thus, our data suggest that rhArg-peg(5,000mw) is a novel agent for effective cancer therapy.

Construction of recombinant Bacillus subtilis for production of polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1 and pBE2C1AB, containing one or two PHA synthse genes, respectively. The two plasmids were inserted into Bacillus subtilis DB104 to generate modified strains, B. subtilis/pBE2C1 and B. subtilis/pBE2C1AB. The two recombinants strains were subjected to fermentation and showed PHA accumulation, the first reported example of mcl-PHA production in B. subtilis. Gas Chromatography analysis identified the compound produced by B. subtilis/pBE2C1 to be a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer whereas that produced by B. subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxydecanoate-co-hydroxydodecanoate (HB-HD-HDD) polymer.

Degradation of recalcitrant compounds from stabilized landfill leachate using a combination of ozone-GAC adsorption treatment.

Laboratory experiments were undertaken to investigate the treatment performances of ozonation alone and/or its combination with granular activated carbon (GAC) adsorption for raw leachate from the NENT landfill (in Hong Kong). To improve its removal of recalcitrant contaminants from the leachate, the surface of GAC was oxidized with ozone prior to treatment. With respect to ozone dose and pH, the removal of COD and/or NH(3)-N from ozonation alone and combined ozone-GAC adsorption were evaluated and compared to those of other physico-chemical treatments in some reported studies. The removal mechanism of recalcitrant compounds by ozone-GAC adsorption treatment was presented. Among the various treatments studied, the combination of ozone-GAC adsorption using ozone-modified GAC had the highest removal for COD (86%) and/or NH(3)-N (92%) compared to ozonation alone (COD: 35%; NH(3)-N: 50%) at the same initial COD and/or NH(3)-N concentrations of 8000 and 2620 mg/L, respectively. Although the integrated treatment was more effective than ozonation alone for treating stabilized leachate, the results suggested that it could not generate treated effluent that complied with the COD limit of lower than 200 mg/L and the NH(3)-N discharge standard of less than 5 mg/L. Therefore, further biological treatments to complement the degradation of the leachate are still required to meet the environmental legislation.

Physico-chemical treatments for removal of recalcitrant contaminants from landfill leachate.

In this paper, the technical applicability and treatment performance of physico-chemical techniques (individual and/or combined) for landfill leachate are reviewed. A particular focus is given to coagulation-flocculation, chemical precipitation, ammonium stripping, membrane filtration and adsorption. The advantages and limitations of various techniques are evaluated. Their operating conditions such as pH, dose required, characteristics of leachate in terms of chemical oxygen demand (COD) and NH3-N concentration and treatment efficiency are compared. It is evident from the survey of 118 papers (1983-2005) that none of the individual physico-chemical techniques is universally applicable or highly effective for the removal of recalcitrant compounds from stabilized leachate. Among the treatments reviewed in this article, adsorption, membrane filtration and chemical precipitation are the most frequently applied and studied worldwide. Both activated carbon adsorption and nanofiltration are effective for over 95% COD removal with COD concentrations ranging from 5690 to 17,000 mg/L. About 98% removal of NH3-N with an initial concentration ranging from 3260 to 5618 mg/L has been achieved using struvite precipitation. A combination of physico-chemical and biological treatments has demonstrated its effectiveness for the treatment of stabilized leachate. Almost complete removal of COD and NH3-N has been accomplished by a combination of reverse osmosis (RO) and an upflow anaerobic sludge blanket (UASB) with an initial COD concentration of 35,000 mg/L and NH3-N concentration of 1600 mg/L and/or RO and activated sludge with an initial COD concentration of 6440 mg/L and NH3-N concentration of 1153 mg/L. It is important to note that the selection of the most suitable treatment method for landfill leachate depends on the characteristics of landfill leachate, technical applicability and constraints, effluent discharge alternatives, cost-effectiveness, regulatory requirements and environmental impact.

Comparisons of low-cost adsorbents for treating wastewaters laden with heavy metals.

In this article, the removal performance and cost-effectiveness of various low-cost adsorbents derived from agricultural waste, industrial by-product or natural material are evaluated and compared to those of activated carbon for the removal of heavy metals (Cd(II), Cr(III), Cr(VI), Cu(II), Ni(II) and Zn(II)) from metals-contaminated wastewater. To highlight their technical applicability, selected information on pH, dose required, initial metal concentration, adsorption capacity and the price of the adsorbents is presented. It is evident from the survey of 102 published studies (1984-2005) that low cost adsorbents derived from agricultural waste have demonstrated outstanding capabilities for the removal of heavy metal (Cr(VI): 170 mg/g of hazelnut shell activated carbon, Ni(II): 158 mg/g of orange peel, Cu(II): 154.9 mg/g of soybean hull treated with NaOH and citric acid, Cd(II): 52.08 mg/g of jackfruit), compared to activated carbon (Cd(II): 146 mg/g, Cr(VI): 145 mg/g, Cr(III): 30 mg/g, Zn(II): 20 mg/g). Therefore, low-cost adsorbents can be viable alternatives to activated carbon for the treatment of metals-contaminated wastewater. It is important to note that the adsorption capacities presented in this paper vary, depending on the characteristics of the individual adsorbent, the extent of surface modification and the initial concentration of the adsorbate. In general, technical applicability and cost-effectiveness are the key factors that play major roles in the selection of the most suitable adsorbent to treat inorganic effluent.

Air-stable Ir-(P-Phos) complex for highly enantioselective hydrogenation of quinolines and their immobilization in poly(ethylene glycol) dimethyl ether (DMPEG).

An air-stable catalyst system Ir-(P-Phos) catalyst was found to be highly effective in the asymmetric hydrogenation of quinoline derivatives. The catalyst immobilized in DMPEG was efficiently recovered and reused eight times, retaining reactivity and enantioselectivity.

Construction of recombinant Escherichia coli strains for production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate).

Plastic wastes constitute a worldwide environmental problem, and the demand for biodegradable plastics has become high. One of the most important characteristics of microbial polyesters is that they are thermoplastic with environmentally degradable properties. In this study, pUC19/PHA was cloned and transformed into three different Escherichia coli strains. Among the three strains that were successfully expressed in the production of polyhydroxyalkanoates (PHA), E. coli HMS174 had the highest yield in the production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-HV]). The cell dry weight and PHA content of recombinant HMS174 reached as high as 10.27 g/L and 43% (w/w), respectively, in fed-batch fermentor culture. The copolymer of PHA, P(HB-HV), was found in the cells, and the biopolymers accumulated were identified and analyzed by gas chromatography, proton nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. We demonstrated clearly that the E. coli host for PHA production has to be carefully selected to obtain a high yield. The results obtained indicated that a superior E. coli with high PHA production can be constructed with a desirable ratio of P(HB-HV), which has potential applications in industry and medicine.

Sphaerotilus natans isolated from activated sludge and its production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Sphaerotilus natans is a sheathed bacterium existing in the activated sludge of wastewater treatment plants. It is one of the filamentous bacteria causing the bulking and foaming difficulties of activated sludge. Isolating the strain and culturing it in an axenic environment could not only provide the metabolic knowledge of the strains that would be useful in the development of wastewater treatment methods, but also could enable us to gain an understanding of the mechanism by which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (poly[3-HB-co-3-HV]) is produced by this strain. This article reports the screening and isolation of the strain from the activated sludge using the Nile blue staining method together with Fourier transform infrared analysis. We investigated the ability of the selected strain to produce poly(3-HB-co-3-HV) copolymer using glucose and peptone, or by adding valeric acid or sodium propionate as precursor. Proper precursor feeding could dramatically enhance its 3HV content in the copolymer P(3HB-co-3HV). By controlling the different feeding times in fed-batch fermentation, different desired copolymers were obtained with 15, 40, and 70% 3HV mole fraction of the copolymer. Polymer properties were analyzed by gas chromatography, differential scanning calorimetry, thermo-gravimetry, and nuclear magnetic resonance analysis.

Synthesis of N-protected peptide alcohols catalyzed by subtilisin or alpha-chymotrypsin in organic solvents.

A series of N-protected peptide alcohols were synthesized using amino alcohols with unprotected hydroxy groups as amino components by the catalysis of subtilisin or alpha-chymotrypsin in organic solvents. N-protected aromatic amino acid esters were more suitable as acyl donors for subtilisin. The influences of different N-protecting groups, organic solvents, and content of water on synthesis of N-protected peptide alcohols were systematically studied.