Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells

In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized P-mercaptoethanol, to the permeabil ization buffer (6 M urea + 3 mM ethylenediaminetetra-acetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. in the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. (C) 1999 John Wiley & Sons, Inc.

Chemical treatment of Escherichia coli .1. Extraction of intracellular protein from uninduced cells

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetetraacetate (EDTA) (greater than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic-phase and stationary-phase E. coli cells at levels equivalent to mechanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kinetics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. (C) 1997 John Wiley & Sons, Inc.

Development of disabled, replication-defective gene transfer vectors from the Jembrana disease virus, a new infectious agent of cattle

Jembrana disease virus (JDV) is a newly isolated and characterised bovine lentivirus. It causes an acute disease in Ball cattle (Bos javanicus). which can be readily transmitted to susceptible cattle with 17% mortality. There is as yet no treatment or preventive vaccine. We have developed a gene transfer vector system based on JDV that has three components. The first of the components is a bicistronic transfer vector plasmid that was constructed to contain cis-sequences from the JDV genome, including 5 '- and 3 ' -long terminal repeats (LTRs), 0.4 kb of truncated gag and 1.1 kb of 3 ' -env, a multiple cloning site to accommodate the gene(s) of interest for transfer, and an internal ribosome entry site plus the neomycin phosphotransferase (Neo) gene cassette for antibiotic selection. The second element is a packaging plasmid that contains trans-sequences. including gag, pol. vif, tar and rev: but without the env and packaging signals. The third is a plasmid encoding the G glycoprotein of vesicular stomatitis virus (VSV-G) to supply the vector an envelope for pseudotyping. Cotransfection of 293T cells with these three plasmid components produced VSV-G pseudotyped. disabled, replication defective, bicistronic JDV vectors encoding the green fluorescent protein (EGFP) and the Neo resistance selection maker simultaneously with a titre range of (0.4-1.2) x 10(6) CFU/ml. Transduction of several replicating primary and transformed cells from cattle, primate and human sources and importantly growth-arrested cells with the JDV vectors showed high efficiency of EGFP gene transfer at 35-75%, which was stable and the expression of EGFP was long term. Furthermore, these JDV vectors were designed to suit the inclusion and expression of genes corresponding to JDV specific proteins, such as gag or env, for the development of vaccines for Jembrana disease. This strategy should also be applicable to other bovine diseases as wall. The design and construction of the JDV vector system should facilitate the study of the lentivirology and pathogenesis of the diseases associated with JDV or other bovine virus infections. To our knowledge, this is the first such vector system developed from a cattle virus. (C) 2001 Elsevier Science B.V. All rights reserved.

Binder treatment and lubricant system for aluminium P/M

Organic binders are used in premixes for powder metallurgy applications to prevent dusting and segregation. This is a particular problem for aluminium powder metallurgy because the dust is a potential safety hazard. The binder must also burn out completely at low temperatures in an inert environment and not react with the metal powders. It is demonstrated that cellulose acetate, polyvinyl acetate and polyvinyl alcohol are effective dedusting agents but they react with the metal powders during sintering and decrease the sintered density. Paraffin wan is an effect dedusting agent that provided die wall lubricity, does not interfere with sintering and increases tensile strength and ductility.

NPS-2143 - Treatment of osteoporosis - Calcilytic

Efforts to discover new treatments for osteoporosis led to the identification of the potent and selective, small-molecule calcium receptor antagonist NPS-2143. NPS-2143 is the prototype calcilytic drug, designed to act on calcium receptors on the surface of parathyroid glands, stimulating the release of the body's own stores of native parathyroid hormone (PTH). In osteopenic ovariectomized rats, daily oral administration of NPS-2143 resulted in moderate but sustained increases in plasma PTH levels and marked increases in bone formation and resorption, with no net bone gain or loss. The combination of NPS-2143 and estrogen increases bone formation and density to a greater extent than either agent alone. These results suggest that NPS-2143 may be useful in the treatment of established osteoporosis.

Plasma concentrations of tafenoquine, a new long-acting antimalarial agent, in Thai soldiers receiving monthly prophylaxis

We measured plasma tafenoquine concentrations in Thai soldiers given a monthly regimen of tafenoquine to determine whether these concentrations adequately suppressed malarial infections on the Thai- Cambodian border. After receiving a treatment course of artesunate and doxycycline, 104 male soldiers were administered a loading dose of tafenoquine ( 400 mg daily for 3 days), followed by tafenoquine monthly ( 400 mg every 4 weeks) for 5 months. Consecutive monthly mean ( +/- standard deviation) trough plasma tafenoquine concentrations were 223 +/- 41, 127 +/- 29, 157 +/- 51. 120 +/- 24, and 88 +/- ng/ mL. Only 1 soldier developed malaria during the study. At the time of malaria diagnosis, his plasma tafenoquine concentration was 40 ng/ mL, which was similar to 3- fold lower than the trough concentrations of the other soldiers. Although low tafenoquine concentrations appear to be uncommon, additional investigations are needed to determine the relationship between plasma tafenoquine concentrations and suppression of malaria.

Farnesoid X receptor agonism - a new approach to the treatment of cholesterol gallstone disease

Gallstone disease is very common among native Americans and Hispanics, and similar to 20 million patients are treated for this disease annually in the US. The nuclear farnesoid X receptor (FXR) is the receptor for bile acids, and GW4064 is a synthetic agonist at the FXR. FXR-/- mice fed a lithogenic diet (high fat, cholesterol and cholic acid) are more susceptible to gallstone disease than wild-type mice with the same mixed background, thus establishing that the ablation of FXR is associated with this disease. The C57L mouse is susceptible to gallstone formation. When C57L mice are fed a lithogenic diet for a week, the bile contains large aggregates of cholesterol precipitates, and two of five C57L mice had macroscopic cholesterol crystals. in contrast, when C57L mice were fed the lithogenic diet and administered GW4064 100 mg/kg/day by oral gavage, there was no precipitation of cholesterol. Treatment with this agent also increased bile salt and phospholipid concentration, and prevented gallbladder epithelium damage. As FXR agonism with GW4064 has been shown to be useful in a mouse model of cholesterol gallstone disease, it should undergo further development for the treatment of this condition.

Depolymerisation and biodegradation of a synthetic tanning agent by activated sludges, the bacteria Arthrobacter globiformis and Comamonas testosteroni, and the fungus Cunninghamella polymorpha

Degradation of a synthetic tanning agent CNSF (a condensation product of 2-naphthatenesulfonic acid (2-NSA) and formaldehyde) by four activated sludges, two previously characterised bacterial strains, Arthrobacter sp. 2AC and Comamonas sp. 4BC, and the fungus Cunninghamella polymorpha, was studied in batch culture at 25 degrees C by determining the changes in the concentrations of CNSF and its component monomers and oligomers (n2-n11). The loss of individual oligomers was correlated with the length of the NSA-CH2 chain. Approximately 25% of the total CNSF was degraded (i.e. mineralised) by the microbes contained in the four activated sludges and by the two bacterial isolates but with different lag phases and at different overall rates. The decline in CNSF concentration was due almost entirely to the biodegradation of the monomers (34.3% of CNSF) and, in particular, 2-NSA (27% of CNSF). There was no change in the n2-n 11 components. The growth of C. polymorpha, on the other hand, arose from extracellular depolymerisation of CNSF oligomers and the biodegradation of the lower molecular mass products. Between 38% and 42% of total CNSF was degraded by C. polymorpha at 25 degrees C. The order of oligomer degradation was inversely related to degree of polymerisation. Eighty percent and 90% of the n4 and n5 and 100% oligomers n6-n11 were degraded after 120 h. At a higher temperature (37 degrees C) oligomers n4-n11 were degraded completely after 120 h. A combination of biodegradation (75%) and sorption to fungal biomass (25%) accounted for the measured loss of all oligomers from the solution phase. The CNSF degradation rates and the volume of fungal biomass produced (and therefore the extent of biosorption) were dependent on the presence of a second carbon source (both optimum at glucose 5 g/l). This is the first report that identifies and distinguishes between depolymerisation, sorption and biodegradation processes in the removal of CNSF and its component oligomers. The use of combinations of the depolymerising fungus C. polymorpha, and the monomer-degrading bacteria, Arthrobacter sp. 2AC and Comamonas sp. 4BC, have potential for wastewater treatment.

Cost-effectiveness analysis of a kidney and cardiovascular disease treatment program in an Australian aboriginal population

The objective of the study was to assess, from a health service perspective, whether a systematic program to modify kidney and cardiovascular disease reduced the costs of treating end-stage kidney failure. The participants in the study were 1,800 aboriginal adults with hypertension, diabetes with microalbuminuria or overt albuminuria, and overt albuminuria, living on two islands in the Northern Territory of Australia during 1995 to 2000. Perindopril was the primary treatment agent, and other medications were also used to control blood pressure. Control of glucose and lipid levels were attempted, and health education was offered. Evaluation of program resource use and costs for follow-up periods was done at 3 and 4.7 years. On an intention-to-treat basis, the number of dialysis starts and dialysis-years avoided were estimated by comparing the fate of the treatment group with that of historical control subjects, matched for disease severity, who were followed in the before the treatment program began. For the first three years, an estimated 11.6 person-years of dialysis were avoided, and over 4.7 years, 27.7 person-years of dialysis were avoided. The net cost of the program was $1,210 more per person per year than status quo care, and dialyses avoided gave net savings of $1.0 million at 3 years and $3.4 million at 4.6 years. The treatment program provided significant health benefit and impressive cost savings in dialysis avoided. (C) 2005 by the National Kidney Foundation, Inc.

Exercise augments the effects of cognitive-behavioural therapy in the treatment of binge eating

To evaluate the effects of adding exercise and maintenance to cognitive-behavior therapy (CBT) for binge eating disorder (BED) in obese women. One hundred fourteen obese female binge eaters were randomized into four groups: CBT with exercise and maintenance, CBT with exercise, CBT with maintenance, and CBT only. Eighty-four women completed the 16-month study. Subjects who received CBT with exercise experienced significant reductions in binge eating frequency compared with subjects who received CBT only. The CBT with exercise and maintenance group had a 58% abstinence rate at the end of the study period and an average reduction of 2.2 body mass index (BMI) units (approximately 14 lb). BMI was significantly reduced in the subjects in both the exercise and maintenance conditions. The results suggest that adding exercise to CBT, and extending the duration of treatment, enhances outcome and contributes to reductions in binge eating and BMI.

Negative stress and the outcome of treatment for binge eating

This study investigated the ability of negatively versus positively perceived stress to predict outcome of treatment for binge eating disorder (BED). Participants were 62 obese women satisfying the DSMIV research criteria for BED. Stress was measured using an instrument based on the Recent Life Change Questionnaire (RLCQ). Participants experiencing high negative stress during the study period reported a binge eating frequency three times greater than that reported by subjects experiencing low negative stress (2.14 vs. 0.65 binge-days/week). Negative stress predicted how fast an individual would reduce binge eating and demonstrated more predictive power than positive stress.

DR-NEGOTIATE - A system for automated agent negotiation with defeasible logic-based strategies

This paper reports on a system for automated agent negotiation, based on a formal and executable approach to capture the behavior of parties involved in a negotiation. It uses the JADE agent framework, and its major distinctive feature is the use of declarative negotiation strategies. The negotiation strategies are expressed in a declarative rules language, defeasible logic, and are applied using the implemented system DR-DEVICE. The key ideas and the overall system architecture are described, and a particular negotiation case is presented in detail.

The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics

Bacteria have been implicated in the pathogenesis and progression of pulp and periapical diseases. The primary aim of endodontic treatment is to remove as many bacteria as possible from the root canal system and then to create an environment in which any remaining organisms cannot survive. This can only be achieved through the use of a combination of aseptic treatment techniques, chemomechanical preparation of the root canal, antimicrobial irrigating solutions and intracanal medicaments. The choice of which intracanal medicament to use is dependent on having an accurate diagnosis of the condition being treated, as well as a thorough knowledge of the type of organisms likely to be involved and. their mechanisms of growth and survival. Since the disease is likely to have been caused by the presence of bacteria within the root canal, the use of an antimicrobial agent is essential. Many medicaments have been used in an attempt to achieve the above aims, but no single preparation has been found to be completely predictable or effective. Commonly used medicaments include calcium hydroxide, antibiotics; non-phenolic biocides, phenolic biocides and iodine compounds. Each has advantages and disadvantages, and further research is required to determine which is best suited for root canal infections.

Engineered ecosystem for sustainable on-site wastewater treatment

A Pilot-Scale Engineered Ecosystem (PSEE) operated for over two years in sub-tropical conditions, produced an effluent with COD (median 38 mg/L) and TSS (median 3 mg/L) levels comparable to that required by the AS/NZS 1547:2000 Onsite Domestic Wastewater Management standard. Only partial nitrification was achieved as dissimilatory nitrate reduction to ammonia occurred; however the level of NH4-N was reduced by 75% and total inorganic nitrogen by 53%. Phosphorus was not removed by the system due to the lack of regular sludge removal. Mass balances around the system showed that bacteria removed 36% of the influent nitrogen and 76% of the influent COD. Algae and plants were shown to remove 5% of the influent nitrogen, and 6% of the influent phosphorus. Challenges in developing a sustainable on-site wastewater treatment system were largely met by minimising chemical, energy and labour inputs, eliminating the need for frequent sludge handling, and creating an effluent quality suitable for re-use in non-potable applications. However, the sludge removal from the system needs to be adequately managed to avoid excessive accumulation as this can cause a range of negative impacts.