Simplifying Hansen Solubility Parameters for Complex Edible Fats and Oils

Hansen solubility parameters (HSPs), often used to predict the miscibility between two compounds, are an alternative tool in evaluating the ability of the solvent to interact via dispersion, dipole-dipole, and hydrogen bonding interactions. The aim of this paper is to find a simple way to calculate HSPs for complex mixtures of triglycerides (TAGs). HSPs were calculated using two approaches: the first assumes that the contributions to the dispersion, dipole-dipole, and hydrogen bonding interactions may be subdivided into larger functional moieties (i.e., fatty acids and fatty acid methyl esters) that are additive, while the second approach assumes that vegetable oils are comprised of mixtures of simple TAGs in the same mass fractions as the fatty acids. The HSPs obtained using the two approaches are compared to reference values determined using the “Hansen Solubility Parameters in Practice” software (HSPiP) considering the complex TAG profile for each vegetable oil.HSPs for vegetable oils, obtained with the HSPiP software, did not correspond well to the HSPs obtained from the group contribution approach, when using fatty acids, fatty acids + glycerol or fatty acid methyl esters. In contrast, the HSPs calculated for vegetable oils, assuming that all TAGs are simple and in the same mass fractions as the fatty acids, provide similar values to the HSPs obtained from the HSPiP software. Therefore, it is possible to calculate the HSPs for complex oils by simply knowing the fatty acid composition. Knowledge of HSPs may be used to rationalize the ability of certain low molecular weight molecules to develop organogels in vegetable oils as well as the crystallization of triglycerides.     

The metabolism of abscisic acid in flacca,a wilty mutant of tomato

The wilty tomato mutant flacca and the normal variety Rheinlands Ruhm were used in this research. The mutant phenotype was explained mainly by hormonal changes. One of these, the decrease in abscisic acid level, was suggested as the hormonal change closest to the mutated gene. The cause of the lower abscisic acid level in the mutant, which may be enhanced breakdown or inactivation, or inhibited biosynthesis, was investigated. The first possibility was studied by comparing mutant and normal plants treated with t-abscisic acid-2-C¹⁴ for (1) rate of production of labeled methanol-extractable metabolites and (2) radioactivity remaining in the methanol-unextractable fraction. The level of trans, trans-abscisic acid relative to that of cis,trans-abscisic acid was studied in untreated plants. Only two radioactive regions containing metabolites of abscisic acid were detected from either of the plant types, and their rates of production relative to total radioactivity was equal. The radioactivity in the methanolunextractable fraction and the level of trans,trans-abscisic acid were very low in both mutant and normal plants. The second possibility was studied partly by comparing the levels of various xanthophylls in mutant and normal plants and their effect after illumination on cress seed germination. Xanthophylls of both plant types were identical in their absorption spectra, but their levels were higher in the mutant. Of these xanthophylls, illuminated neoxanthin inhibited seed germination in both plant types, but more effectively in the mutant. The most probable explanation for the low level of cis,trans-abscisic acid in flacca is that the conversion of farnesyl PiP to abscisic acid is inhibited in this plant.     

Second International Conference on Accelerating Biopharmaceutical Development: March 9–12, 2009, Coronado,CA USA

The Second International Conference on Accelerating Biopharmaceutical Development was held in Coronado, California. The meeting was organized by the Society for Biological Engineering (SBE) and the American Institute of Chemical Engineers (AIChE); SBE is a technological community of the AIChE. Bob Adamson (Wyeth) and Chuck Goochee (Centocor) were co-chairs of the event, which had the theme “Delivering cost-effective, robust processes and methods quickly and efficiently.” The first day focused on emerging disruptive technologies and cutting-edge analytical techniques. Day two featured presentations on accelerated cell culture process development, critical quality attributes, specifications and comparability, and high throughput protein formulation development. The final day was dedicated to discussion of technology options and new analysis methods provided by emerging disruptive technologies; functional interaction, integration and synergy in platform development; and rapid and economic purification process development.

• MAbs. 2009 May-Jun; 1(3): 190–209.
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• March 10, 2009 Day 1, Emerging Disruptive Technologies and Cutting-Edge Analytical Techniques

MAbs. 2009 May-Jun; 1(3): 190–209.

March 10, 2009 Day 1, Emerging Disruptive Technologies and Cutting-Edge Analytical Techniques

Janice M ReichertAuthor information Article notes Copyright and License information DisclaimerTufts Center for the Study of Drug Development; Boston, MA USACorresponding author.Correspondence to: Janice M. Reichert; Tufts Center for the Study of Drug Development; 75 Kneeland Street; Suite 1100; Boston, MA 02111 USA; Email: ude.stfut@trehcier.ecinajReceived 2009 Mar 20; Accepted 2009 Mar 20.Copyright © 2009 Landes BioscienceThe meeting was opened by Bob Adamson (Wyeth) who remarked that it is the responsibility of biological engineers to develop technologies that will produce drug products rapidly and cost effectively. On average, protein therapeutics cost more than small molecule drugs. However, technological advances can help to drive down the cost of these products. For example, penicillin was scarce in the 1930s and 1940s because of production issues, but this drug is easily and cheaply obtained today.Recent developments in biosimilars, a potentially disrutive group of products, were discussed by Rob Garnick (Lone Mountain Biotechnology). He first noted the various names by which biosimilars are known. While the term biosimilars is favored in Europe, the US Food and Drug Administration (FDA) uses the term ‘follow-on biologics,’ and Health Canada prefers the phrase ‘subsequent entry biologics.’ The term biogenerics is not usually used because this word implies that the products are identical to approved innovator biologics. The European Medicines Agency and Health Canada have issued regulatory guidances for approval of biosimilars, and some of these products have been approved in Europe.¹ However, the process has gotten stalled in the US for various reasons, including questions surrounding the reliability of sourcing, magnitude of price reduction, need for clinical trials done against a national comparator and comparability issues. On the other hand, the global economic problems have focused political attention on healthcare reform and unsustainable increases in the cost of healthcare.Dr. Garnick noted that in the US the general consensus is that the Drug Price Competition and Patent Term Restoration Act of 1984, also known as the Hatch-Waxman Act, has been successful in promoting generics while still providing financial incentives for research and development by innovators. In addition, Congress believes that scientific issues surrounding biosimilars are addressable and so a regulatory pathway can be established for approval of biosimilars. Due to the progress in defining a regulatory pathway, some major pharmaceutical firms, including Pfizer, AstraZeneca, Novartis and Merck, have recently indicated that they will develop these products. Biosimilars development is attractive because the success rates should be 100% if the products are developed correctly, the manufacturing processes are well-understood and can be out-sourced, and the markets are potentially large. With a global market over US $5 billion, rituximab will certainly be targeted as a biosimilar. As with the Hatch-Waxman Act, the key to success of any US biosimilars legislation will be the maintenance of incentives to innovate.There are still numerous scientific and legal problems to address,² including the exact nature of legislation, patent issues, design of clinical trials, substitutability, interchangeability, safety and post-approval surveillance. The challenge lies in the details, e.g., establishing product specifications and test methods, and defining comparability. Dr. Garnick noted that cautionary tales on comparability come from the experience of a number of innovator companies. For example, efalizumab produced by XOMA was found to have differences when compared to efalizumab produced by Genentech. The differences, which included minor changes in acidic forms, galactosylation, charge heterogeneity and an increase in C-terminal processing, were expected to be inconsequential, but translated into different clinical study results. This experience suggests that a combination of written procedures, training, analytical testing and regulatory agency inspections are needed to control the production of biological products. Quality control release tests need to be supported by rigorous product and process characterization and process control.Outside the US, the reality is that biosimilars are being marketed in Europe, India and China as well as other countries. Marketed biosimilars span a broad range of complexities and include monoclonal antibodies. Reditux, a rituximab biosimilar, was approved in India in April 2007 for non-Hodgkin lymphoma and rheumatoid arthritis. However, the clinical trials included relatively few patients and limited analytical data has been made available. In conclusion, Dr. Garnick remarked that the world is gaining experience with biosimilars, and the products will likely become a reality in the US by 2009. FDA will need input to develop effective guidance documents for assessment of biologics. Immunogenicity will be a key concern of regulators. Comparability studies will be required, product differences will need to be investigated and appropriate clinical studies must be done, but biosimilars will come to market.Global trends in antibody development by innovator companies were presented by Janice Reichert (Tufts University). Clinical development of protein therapeutics is on the rise worldwide.^3–5 Approximately 120 recombinant proteins and 240 monoclonal antibodies (mAbs) are currently in clinical studies. While recombinant proteins have historically entered the clinic at a rate of fewer than 20 candidates per year, mAbs are now approaching the 40 candidate per year mark. Recombinant proteins have somewhat higher success rates on average (approximately 30% vs. 20% for mAbs) and have been studied in a wider array of therapeutic categories compared to mAbs, but, because of their versatility as therapeutics, mAbs are clearly the focus of the biopharmaceutical industry''s attention. A total of 22 mAbs are approved in the US, and eight of these products have global markets over US$1 billion. Six additional candidates are currently undergoing regulatory review.Dr. Reichert noted that the ascendancy of mAbs is due to technologies that addressed immunogenicity, affinity, specificity, stability and production challenges. While murine versions dominated in the 1980s, the less immunogenic humanized versions comprised 45% of the total mAbs in clinical study in the 1990s. In the 2000s, the human versions have comprised the largest contingent. Historically, mAbs have not been discontinued while in regulatory review. Assuming the six mAbs in review are approved, then cumulative FDA approval success rates for humanized and human mAbs will be nearly identical (19 and 18%, respectively). mAbs are commonly studied as either anticancer⁶ or immunological treatments. There are currently nine anticancer and ten immunological mAbs approved in the US. These products have taken approximately the same length of time for clinical development (6.5 years). The length of the FDA review period was found to vary depending on whether the product was given priority or standard review (average 6.9 or 20.4 months, respectively).Looking forward, Dr. Reichert suggested that antibody fragments and modified versions (pegylated, alternate glycosylation, Fc engineered) are likely to enter the clinical pipeline in increasing numbers. The focus is likely to remain on human IgG, but designed protein scaffolds and domain antibodies will also be included in company pipelines.The meeting then turned to discussion of potentially disruptive science and technologies. Stefan Wildt (Merck) reviewed the development of glycoengineered yeast, which he described as a versatile glycoprotein expression platform. He first emphasized the importance of glycosylation, which affects circulating half life, tissue distribution, potency and immunogenicity of therapeutic proteins. Any new bioprocesses need to be scalable, portable, and provide analytically comparable protein at all scales. The primary biomanufacturing platforms are bacterial (e.g., E. coli), fungal (e.g., Pichia pastoris), and mammalian cell culture (e.g., CHO cells). Fungal platforms are currently used for production of common industrial enzymes, but have not been used extensively for production of therapeutics because the yeast glycosylation pathway yields products that are potentially immunogenic in humans. GlycoFi Inc., a wholly owned subsidiary of Merck & Co., Inc. has developed Pichia with humanized glycosylation to circumvent this problem.In yeast, the carbohydrate processing occurs sequentially in the secretory pathway, like an assembly line. The enzymes act one after another and their actions are separated in time and space. As a consequence, humanized yeast produce proteins with human glycans that are highly uniform. In contrast, traditional mammalian cell production systems produce functional glycoproteins that are heterogeneous and contain non-human glycoforms. As reported by Hamilton et al.,⁷ GlycoFi eliminated yeast-specific glycosylation in Pichia pastoris and introduced 14 heterologous genes; this process yielded yeast capable of producing complex glycoproteins with greater than 90% terminal sialylation. Candidate protein can be produced in a bioreactor process that takes three to seven days, which is somewhat shorter than the time for production in mammalian cells.For example, Dr. Wildt discussed MK2578, which is a pegylated erythropoietin that is terminally sialylated with N-glycans. The candidate is currently in Phase 1 studies as a potential treatment for anemia. The glycosylation fidelity from the Pichia platform is retained when protein is produced at laboratory scale to up to 2,000 L. The yeast can also be used to produce antibody as IgG1. Compared to CHO cell produced IgG1, candidates produced in yeast were found to be more potent in inducing ADCC and could bind antigen as well. In preclinical studies, yeast-produced mAb glycovariants demonstrated good results in PK studies in Rhesus monkeys and C57BL mice. In conclusion, Dr. Wildt remarked that the selection process, which includes screening for titer, fermentability, glycosylation and protein quality, can result directly in production strains. Yield is approximately 1.4 grams per liter for antibody candidates, but yields up to 2 grams per liter can be achieved.Annie De Groot (EpiVax) presented information on methods to reduce protein immunogenicity by design through deimmunization and tolerance induction. She noted the parallels between vaccine use, when an immune response is desired, and immunogenic therapeutic proteins, which elicit an immune response that is not desired. In both cases, a payload coupled with a delivery vehicle and an adjuvant determine immunogenic potential. T-cell epitopes are a key contributing factor. Like proteins, antibodies are processed by antigen presenting cells. The activated T-cells in turn activate B-cells; in the absence of T-cells, no antibody formation is observed.EpiVax has developed an array of in silico tools and techniques to predict whether proteins will be immunogenic. These include EpiMatrix, in which overlapping 9-mer peptide frames are evaluated for binding potential to eight common class II HLA alleles. The ClustiMer algorithm can be used to find regions of high immunogenicity. Using these methods, an overall immunogenicity score can be estimated. These in silico results can be validated in vitro and in vivo (e.g., HLA transgenic mice).The approach has been clinically validated. Koren et al.⁸ reported on use of EpiMatrix analysis of a recombinant fusion protein that predicted promiscuous T-cell epitopes in the C-terminal region. In a phase 1 study of 76 subjects, 37% developed antibodies after one injection of the protein candidate. EpiMatrix correctly predicted the immunogenic region and the likelihood that the protein would be immunogenic in the clinic.These results suggest that the technology might be useful as part of an overall strategy for assessing antibody responses in non-clinical and clinical settings.^{9, 10} In addition, rational modification of epitopes identified using the technology could effectively ‘deimmunize’ protein candidates. Dr. De Groot also discussed the discovery of ‘Tregitopes’ (highly conserved regulatory T-cell epitopes) that are promiscuous, high affinity HLA binders found in IgG. She noted that there is a correlation of antibody immunogenicity with the presence of Tregitopes. Dr. De Groot and co-workers have demonstrated that co-incubation of peripheral blood mononuclear cells (PBMCs) with the Tregitopes can lead to suppression of immune response to other antigens. This suggests that the engineering of Tregitopes into antibodies or other proteins might lead to the development of less immunogenic candidates.Modular IMmune In vitro Constructs (MIMIC), which is an in vitro biomimetic human immune system developed to accurately model the immunotoxicity and immunogenicity of drug candidates, was reviewed by William Warren (Vaxdesign). The MIMIC system is designed to serve as a ‘clinical trial in a well’ by providing predictive HTP in vitro immunology assessment of drug candidates. Primary human donor cells are used to simulate human responses to agents such as vaccines and drugs. The system consists of three modules: (1) Simulation of innate immunity with a peripheral tissue equivalent (PTE) module; (2) Simulation of adaptive immunity with the lymphoid tissue equivalent (LTE); and (3) a functional assay or disease model. The PTE module comprises one monolayer of endothelial cells grown over a 3D collagen matrix. Human PBMCs from donors are used to seed the module; monocytes extravasate through the endothelial cells and differentiate into antigen presenting cells. The PTE module can be used to assess reactogenicity and immunotoxicity responses. The LTE module functionally reproduces the environment of a human lymph node. Within the module, T-cells, B-cells, antigen-presenting cells or follicular dendritic cells interact, leading to immune stimulation that results in activation of lymphocytes, cytokine generation and antibody production. The activated lymphocytes, cytokine profiles and antibodies are then characterized using various methods.Dr. Warren discussed use of the modules to measure the magnitude and quality of T-cell response to vaccines. He noted that primary CD8 T-cell response, in vitro humoral and B-cell response, antibody titer, and microneutralization can be assessed. A correlation analysis of MIMIC response versus serum titer in hepatitis B and influenza vaccination has been performed. Results suggest that the MIMIC system can be used to predict whether a vaccine would be efficacious before going to the clinic. In addition to immunogenicity, the system acts as a biomimetic for evaluation of immunotoxicity and can be used as an inflammation model or in vitro infectious disease model. Use of the system has the potential to accelerate the entire drug development timeline, and decrease failures by providing better data for evaluation of preclinical candidates.Karyn O''Neil (Centyrex, a Johnson & Johnson Internal Venture) described alternative scaffolds that are being used as new biotherapeutic platforms by Johnson & Johnson. She started by wondering whether mAbs are always the best choice since there are reasons to develop alternatives. For example, desirable epitopes might be immunologically silent, alternatives to injection delivery are a challenge, full-size antibodies penetrate tissue and tumors poorly, and royalties might be due on numerous phases of the mAb discovery, screening, development and production process. However, requirements for a next-generation platform, which include the expansion of the range and possibility of targets, lower cost of development and manufacturing complexity, novel delivery, elimination of cold storage, clear freedom to operate and no intellectual property issues, are difficult to meet. Alternative scaffolds do meet many of the aforementioned requirements, and these molecules have favorable biophysical characteristics. Alternative scaffolds can be readily formatted into multi-specific binders with relevant biological activity. For example, Lu et al.¹¹ combined variable regions of two antagonistic antibodies to produce a human IgG-like bispecific antibody that could strongly inhibit the growth of two different human tumors in HT29 xenografts in vivo.Johnson & Johnson''s strategy toward the use of alternative scaffolds involves development of both Centyrins^™ and DARPins^™, which are viewed as complimentary molecule types. Both are small (10 to 18 kDa) single domain proteins that have high affinity (low picomolar to femtomolar range) and high selectivity for their targets. They are compatible with technologies that improve serum half-lives and seem to have low immunogenicity and low toxicity. They are also very stable and can be expressed at high levels in soluble form. DARPins^™ have flat wide surfaces that are better suited for disrupting protein-protein interactions,¹² whereas Centyrins^™ have extended loops that can interact in protein clefts, enzyme active sites and protein channels.¹³DARPins^™, which are being developed as part of a collaboration between Molecular Partners and Johnson & Johnson, are selected from in vitro display of very large (10¹²) libraries. The method uses PCR and affinity maturation, and candidates with slow off-rates can be selected. In this way, high affinity, neutralizing DARPins^™ can be selected within weeks. Melting properties can be used for selection, resulting in DARPins^™ candidates with good biophysical properties. Small scale (2 mL) expression of DARPins^™ can yield approximately 1 mg each for additional characterization.The Centyrins^™ scaffolds have loops that are analogous to the CDRs of antibodies. The molecules have excellent biophysical properties (>100mg/mL expression, >170mg/mL solubility, >82°C melting temperature, low predicted immunogenicity, stable in serum for more than one month), and can be engineered for improved stability. An in vitro display system licensed from Isogenica utilizes CIS-display technology for Centyrins^™ selection. This is proven technology for peptide display. Libraries are potentially quite large (10¹³). The CIS-display allows rapid panning and selection of binders with a PCR step that allows for in vitro evolution of binders. Rational design of library diversity can improve scaffold properties.¹⁴ A green fluorescent protein solubility/folding reporter assay¹⁵ is used to assess library quality. The unique properties of alternative scaffolds can be exploited in numerous areas, such as bispecific molecules, medical device, encapsulation, novel formulation and delivery, drug/toxin or radionuclide conjugation, imaging, biosensor, purification technologies and intracellular expression.Cutting-edge analytical technologies became the focus of the meeting in the afternoon session. Steve Cohen (Waters Corporation) discussed chromatographic analysis for biopharmaceuticals with an emphasis on current trends and future prospects. He first discussed ultra performance liquid chromatography (UPLC) utilizing sub-2 micron particle packing (1.7 µmeter with either 130 angstrom or 300 angstrom pores). Compared to HPLC with 3.5 µmeter particle packing, UPLC gives sharper peaks, and can improve resolution of samples in the same run time or achieve comparable resolution and selectivity in a reduced run time (80 versus 120 minutes). Dr. Cohen then presented results of LC/ultraviolet (UV) analysis of a reduced monoclonal antibody, and a murine monoclonal IgG reduced and alkylated standard run at elevated temperature. He noted that the high temperature (80–90°C) is absolutely required for reasonable chromatography. Analytical methods for monitoring glycosylation of mAbs are important because bioprocess conditions can cause variation in high mannose type, truncated forms, reduction of tetra-antennary and increase in tri- and biantennary structures, less sialyated glycans and less glycosylation.Dr. Cohen also reviewed new separations technologies such as monolithic materials and chip based nanoscale separations. He presented an example of the use of a ceramic microfluidic UPLC system and the software tool BiopharmaLynx 1.2 to perform humanized peptide mapping. Three dimentional structure analysis using amide hydrogen exchange was also discussed. In a continuous labeling experiment, labeling occurs at 25°C, pH 7, and aliquots are removed and quenched at 0°C, pH 2.5. The protein sample can be subjected to HPLC/UPCL directly, or subjected to online digestion and then HPLC/UPCL. Electrospray mass spectrometry then provides information about isotope pattern and deuterium content that can be used to determine exchange rates. Use of UPLC will provide sharper peaks and improved spectral quality.¹⁶ The technique can be used for quality control or comparability of samples, e.g., differentiation of correctly folded protein from incorrectly folded protein.Tom Laue (University of New Hampshire) discussed advances in analytical ultracentrifugation (AUC) and analytical electrophoresis (AE). Dr. Laue remarked that AUC provides a framework for thinking about concentrated solutions and proximity energies. AUC can be used to characterize proteins in high concentration formulations. Proximity energies at high concentrations may be positive or negative, and are dependent on such factors as distance, orientation, solvent and time. Potential energy is dependent on forces such as charge-charge, charge-dipole, dipole-dipole, hydrogen bonding, dispersion, dipole induced dipole, charge induced dipole and van der Waals interactions. AUC with fluorescence detection can be used to characterize labeled proteins in concentrated samples such as plasma. For example, mAb interactions in plasma can be observed using fluorescence detected sedimentation. Weak electrostatic interactions will dominate molecular behavior in concentrated solutions.Dr. Laue then discussed AE as a technique to determine accurate values of protein charge. Interestingly, monoclonal IgGs have an actual charge that is aberrantly low compared to the calculated value (e.g., 2 versus 24). The low charge leads to problems with poor solubility and high viscosity. Dr Laue speculated that the mAb charge suppression may have some housekeeping function such as weakening charge interactions with anionic plasma proteins, or altering co-operativity for Fc FcR binding or other functions. The low charge may be due to a combination of pKa shifts, anion binding (territorial or site), and carbohydrate involvement. Many of the viscosity and solubility problems encountered during processing may be traced to the low charge on IgGs. He urged attendees to measure the charge and not to rely on calculated charge estimates (e.g., from isoelectric point measurements).Kermit Murray (Louisiana State University) discussed coupling microfluidic chips to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The chips can speed proteomics by serving as a single platform for automated cell culturing, digestion, separation and sample deposition. The system is based on synthetic polymer microfluidic devices, with chip components fabricated on a poly(methyl) methacrylate plate using the hot embossing method and off-line MALDI analysis. A key component of the system is a trypsin microreactor. Assembled chips are processed using a pressure-driven or electrokinetic flow; the system utilizes a Dionex LC and a Probot MALDI plate spotter for the former. Digested peptides are coaxially mixed with a MALDI matrix solution and deposited on a MALDI target. Chips are stable for about one month.Dr. Murray presented experimental results from analyses of cytochrome c under various flow rates. A flow rate of 1 µL/min, with a residence time of approximately 24 seconds within the reaction bed, provided 67% sequence coverage, which increased to 72% when residence time was increased to 48 seconds. Use of the 1 µL/min flow rate resulted in sequence coverages of 35%, 58%, and 47% for 10 µM samples of bovine serum albumin, myoglobin and phosphorylase b, respectively. The digestion efficiency was improved using an electrokinetically driven microreactor using a micro-post structured chip. Using the micro-post system, sequence coverage of 10 µM cytochrome c was 89%; sequence coverage decreased when protein concentration of the sample was lower. Whole bacterial cells can be analyzed using the system. Digestion and deposition of E. coli resulted in identification of the aminoglycoside 3′-phosphotransferase type 1, with 57% sequence coverage.A two-chamber chip developed by Dr. Murray and colleagues can also be used to provide MALDI MS results for bacteria. It has applications for analysis of sepsis, pneumonia, tuberculosis, blood supply QA/QC, and environmental pathogen samples. The cell culture chamber has sample and media inlet, as well as outlet, channels; the culture chamber itself has a 3 mm diameter and 300 µm depth. The system uses a PMMA chip and PDMS cover. The channel surfaces are sterilized with UV. After assembly, the chamber is filled with nutrient broth, approximately 4,000 E. coli cells are added and the reservoirs are closed. The bacteria are cultured for 24 hours at 37°C. One µL of E. coli is then deposited on the MALDI target plate. In an experiment using ATCC#9637, #11303, or #11775, some cellular protein peaks were found. The results suggest that such on-chip culturing could be used for fingerprint analysis. Finally, Dr. Murray discussed preliminary work on a temperature regulated chip with heating and cooling elements.The topic of mass spectrometry (MS)-based strategies to study protein architecture, dynamics and binding was reviewed by Igor Kaltashov (University of Massachusetts, Amherst). He first noted that biopharmaceuticals have higher order structure and conformational heterogeneity, and various perturbations or changes in the production process can result in alterations of the primary or higher order structure that can have deleterious effects on the efficacy, immunogenicity or stability of the protein. MS has been applied to the structural characterization of recombinant protein pharmaceuticals¹⁷ and specifically therapeutic antibodies.¹⁸ The tertiary and quaternary protein structure can be characterized directly in solution by electrospray ionization (ESI) MS.As an example of the use of MS in structure characterization, Dr. Kaltashov discussed studies done on alkylated interferon β-1a (IFN-β-1a). Alkylation at Cys-17 of the protein results in 50–90% reduction of the antiviral activity. He remarked that ‘classical’ biophysical techniques such as size exclusion chromatography, fluorescence, far-UV circular dichroism (CD) and near-UV CD were not very informative regarding conformational changes between the alkylated and unmodified forms. Two complementary MS-based techniques, analysis of ionic charge state distribution and hydrogen/deuterium exchange (HDX), were used to monitor conformational changes.¹⁹ The analysis of the ionic charge distributions indicated a decrease in conformational stability in the alkylated form; the partial unfolding was revealed by the presence of protein ions in the ESI mass spectra with significantly higher charge density compared to the unmodified version. In HDX MS, measurements can be carried out under conditions closely mimicking the formulation buffer, and thermodynamic information is derived from biophysical measurements. Global HDX MS revealed higher flexibility of alkylated IFN. Backbone flexibility was observed to be distributed unevenly across the polypeptide sequence. Structural studies suggest that the loss of antiviral activity of the alkylated form is due to destabilization of a region of IFN that binds with its low affinity receptor (IFNAR1), and disruption of ternary complex formation.Dr. Kaltashov concluded by suggesting that ESI MS can be used to characterize highly heterogeneous systems and presented findings from a study of heparin, which is very heterogeneous, and difficult to characterize by MS. With colleagues, he has developing a mass spectrometry-based strategy for characterization of anti-thrombin interaction with low molecular weight heparin and heparin oligomers.²⁰Genentech''s use of high throughput (HTP) methods in bioprocess development was discussed by Judy Chou (Genentech). She described analytical methods as the ears and eyes of the production process. Use of a high throughput platform is directly related to the need for rapid analysis of bioprocess samples. The need for speedy analysis, which enables new products to get to patients in a timely fashion, has to be balanced with the need for extensive sample characterization that might be time-consuming. The aim is to leverage new technology, especially in HTP purification and automation to increase the number of experiments while reducing resources required and shortening timelines. The HTP approach involves scaling down cell culture, use of protein A well-plate purification (PAWP), purification in plate (PiP), HTP impurity analysis and an at-line reverse phase high performance liquid chromatography (RP-HPLC) method.In the PAWP method, 24- or 96-well plates with protein A are used. The plates can be subjected to centrifugation or vacuum procedures, then samples are directly analyzed by HPLC, liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis (CE), or image capillary isoelectric focusing (icIEF) techniques. The method allows product quality tests to be expedited, and allows the company to address product quality issues early on and reduce resource and time cost later. Use of the PAWP method was directly compared with use of a standard purification procedure (protein A column). Samples subjected to both methods gave similar results in an array of tests (SEC IEC, CZE, icIEF analysis, CE-Glycan assay, peptide mapping).RP-HPLC rapid monitoring can be used as a fast method to monitor mAb fragments in both the purified samples and the cell culture fluids without any sample preparation procedure. It provides a powerful tool to look into antibody reduction issues and helps to monitor as well as to develop bioprocess to mitigate the risk of losing product quantity and quality. Furthermore, the on-line RP-HPLC-Mass Spectroscopy (MS) enabled the understanding of the new peaks identified in the cell culture fluids and increased the process knowledge during the development and operation phases.HTP impurity assays were also developed as a tool to quickly narrow down purification conditions. A CHO protein Meso Scale Discovery (MDS) impurity assay that utilizes electrochemiluminescence was incorporated in an HTP process. Plates can be prepared and stored for up to nine months. The MSD assay was compared to ELISA at various purification steps and the difference was found to be less than 15%, whereas only 10% of resources are used to perform assay and the total assay time was only 2.5 hours for ∼400 samples. In addition, a leached Protein A HTP assay based on MSD technology was also developed. In order to prevent the signal masking introduced by the products, a novel approach of acidification combined with effective blockers was implemented. The new method is generic to all the molecules tested so far and only takes three hours for ∼400 samples with limited amount of resources needed.A novel TEACAN system that puts all the relevant analytical assays as well as the PiP and High throughput Formulation development in an assembly line is currently being used. Dr. Chou mentioned that the details of the methods she described will be published soon.     

Effects of cultivation practice on floristic and flowering diversity of spontaneously growing plant species on arable fields

In the past, the floristic diversity of arable fields has been described in terms of species diversity (SD) and their degree of coverage (C), but never in combination with the recording of the actually flowered species (FS) and their flowering intensity (FI) to striking differences in the cultivation methods on arable land. In relation to SD and C, however, FS and FI may provide important additional information on the functional biodiversity of fields. The aim was therefore to investigate the effects of (a) conventional, (b) organic, and (c) smallholder (never application of herbicides) on the floristic diversity. Using a region in Germany, we investigated SD, C, FS, and FI synchronously in (a), (b), and (c), by 356 vegetation surveys (5 × 5 m plots) conducted in spring and summer in 2019 in winter cereals. Statistical tests were used to analyze the differences between (a), (b), and (c). The medians were used to compare the floristic diversity of (a), (b), and (c) and finally relationships of FS and FI to SD were analyzed in relation to the cultivation methods. Significant differences in SD, C, FS, and FI were found between the (a), (b), and (c) in spring and summer characterized by sharp declines from (c) to (b) to (a). A drastic reduction in floristic diversity from (c) 100 to (b) 52 to (a) 3 was determined. Plants in flower (FS, FI) were very poorly in (a), moderately well to well in (b), and well to very well represented in (c). (C) to (a) was characterized by a sharp decline and from (a) to (b) by sharp increase in floristic diversity. With current acreage proportions of (a) in mind, this would affect, about one third of land area in Germany, associated with a drastic reduction in functional biodiversity for insects.     

Insulin-like growth factor I increases alpha Vbeta 3 affinity by increasing the amount of integrin-associated protein that is associated with non-raft domains of the cellular membrane.

Insulin-like growth factor I (IGF-I) stimulates an increase in alpha(V)beta(3) ligand binding. Stimulation of smooth muscle cells by IGF-I requires alpha(V)beta(3) ligand occupancy, and enhanced alpha(V)beta(3) ligand occupancy augments IGF-I actions. Therefore, IGF-I-induced changes in alpha(V)beta(3) ligand binding may act to further enhance IGF-I actions. Integrin-associated protein (IAP) has been shown to be associated with alpha(V)beta(3) and is required for the binding of alpha(V)beta(3) to vitronectin-coated beads. We therefore investigated whether IGF-I could stimulate IAP-alpha(V)beta(3) association resulting in enhanced ligand binding. IGF-I stimulated an increase in IAP-alpha(V)beta(3) association. This was due, at least in part, to an IGF-I-stimulated redistribution of IAP from the Triton-insoluble fraction of the cell to the Triton-soluble fraction of the cell, where most of the alpha(V)beta(3) was located. Inhibition of the phosphatidylinositol 3-kinase pathway blocked both the redistribution of IAP and the increase in IAP-alpha(V)beta(3) association, providing further evidence that the redistribution of IAP is essential for the increase in association. An anti-IAP monoclonal antibody, blocked both the IGF-I-stimulated increase in IAP-alpha(V)beta(3) complex formation and cell migration. IGF-I-stimulated translocation of IAP and increase in IAP-alpha(V)beta(3) association represent an important process by which IGF-I modulates alpha(V)beta(3) ligand binding and cellular responses.     

Introgression of chromosome 13 in Dahl salt-sensitive genetic background restores cerebral vascular relaxation

To evaluate the potential role of impaired renin-angiotensin system (RAS) function in contributing to reduced vascular relaxation in Dahl salt-sensitive (S) rats, responses to ACh (10(-6) mol/l) and hypoxia (Po(2) reduction to 40-45 mmHg) were determined in isolated middle cerebral arteries of Dahl S rats, Brown Norway (BN) rats, and consomic rats having chromosome 13 (containing the renin gene) or chromosome 16 of the BN rat substituted into the Dahl S genetic background (SS-13(BN) and SS-16(BN), respectively). Arteries of BN rats on a low-salt (LS) diet (0.4% NaCl) dilated in response to ACh and hypoxia, whereas dilation in response to these stimuli was absent in Dahl S rats on LS diet. Vasodilation to ACh and hypoxia was restored in SS-13(BN) rats on an LS diet but not in SS-16(BN) rats. High-salt diet (4% NaCl), to suppress ANG II, eliminated vasodilation to hypoxia and ACh in BN and in SS-13(BN) rats. Treatment of SS-13(BN) rats with the AT(1) receptor antagonist losartan also eliminated the restored vasodilation in response to ACh and hypoxia. These studies suggest that restoration of normal RAS regulation in SS-13(BN) consomic rats restores vascular relaxation mechanisms that are impaired in Dahl S rats.     

G protein beta 5 subunit interactions with alpha subunits and effectors

When the beta(5) (short form) and gamma(2) subunits of heterotrimeric G proteins were expressed with hexahistidine-tagged alpha(i) in insect cells, a heterotrimeric complex was formed that bound to a Ni-NTA-agarose affinity matrix. Binding to the Ni-NTA-agarose column was dependent on expression of hexahistidine-tagged alpha(i) and resulted in purification of beta(5)gamma(2) to near homogeneity. Subsequent anion-exchange chromatography of beta(5)gamma(2) resulted in resolution of beta(5) from gamma(2) and further purification of beta(5). The purified beta(5) eluted as a monomer from a size-exclusion column and was resistant to trypsin digestion suggesting that it was stably folded in the absence of gamma. beta(5) monomer could be assembled with partially purified hexahistidine-tagged gamma(2) in vitro to form a functional dimer that could selectively activate PLC beta2 but not PLC beta3. alpha(o)-GDP inhibited activation of PLC beta2 by beta(5)gamma(2) supporting the idea that beta(5)gamma(2) can bind to alpha(o). beta(5) monomer and beta(5)gamma(2) only supported a small degree of ADP ribosylation of alpha(i) by pertussis toxin (PTX), but beta(5) monomer was able to compete for beta(1)gamma(2) binding to alpha(i) and alpha(o) to inhibit PTX-catalyzed ADP ribosylation. These data indicate that beta(5) functionally interacts with PTX-sensitive GDP alpha subunits and that beta(5) subunits can be assembled with gamma subunits in vitro to reconstitute activity and also support the idea that there are determinants on beta subunits that are selective for even very closely related effectors.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Definition of the structural elements in plasminogen required for high-affinity binding to apolipoprotein(a): a study utilizing surface plasmon resonance

Lipoprotein(a) [Lp(a)] is suggested to link atherosclerosis and thrombosis owing to the similarity between the apolipoprotein(a) [apo(a)] moiety of Lp(a) and plasminogen. Lp(a) may interfere with tPA-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoaguable state in vivo. The present study employed surface plasmon resonance (SPR) to examine the binding interaction between plasminogen and a physiologically relevant, 17-kringle recombinant apo(a) species [17K r-apo(a)] in real time. Native, intact Glu(1)-plasminogen bound to apo(a) with substantially higher affinity (K(D) approximately 0.3 microM) compared to a series of plasminogen fragments (K1-5, K1-3, K4, K5P, and tail domain) that interacted weakly with apo(a) (K(D) > 50 microM). Treatment of Glu(1)-plasminogen with citraconic anhydride (a lysine modification reagent) completely abolished binding to wild-type 17K r-apo(a), whereas citraconylated 17K r-apo(a) decreased binding to wild-type Glu(1)-plasminogen by approximately 50%; inhibition of binding was also observed using the lysine analogue epsilon-aminocaproic acid. Whereas native Glu(1)-plasminogen exhibited monophasic binding to 17K r-apo(a), truncated Lys(78)-plasminogen exhibited biphasic binding. Altering Glu(1)-plasminogen from its native, closed conformation (in chloride buffer) to an open conformation (in acetate buffer) also yielded biphasic isotherms. These SPR data are consistent with a two-state kinetic model in which a conformational change in the plasminogen-apo(a) complex may occur following the initial binding event. Differential binding kinetics between Glu(1)-/Lys(78)-plasminogen and apo(a) may explain why Lp(a) is a stronger inhibitor of tPA-mediated Glu(1)-plasminogen activation compared to Lys(78)-plasminogen activation.     

Highly efficient technetium-99m labeling procedure based on the conjugation of N-[N-(3-diphenylphosphinopropionyl)glycyl]cysteine ligand with poly(ethylene glycol)

The PN(2)S N-(N-(3-diphenylphosphinopropionyl)glycyl)cysteine ligand was conjugated to methoxy-poly(ethylene glycol)-amino (mPEG-NH(2)) 5 and 20 kDa to yield PN(2)S(Trt)-PEG(5000) 1 and PN(2)S(Trt)-PEG(20000) 2, and then detritylated to PN(2)S-PEG(5000) 4 and PN(2)S-PEG(20000) 5. When an acidic solution of (99m)TcO(4)(-) is added to 4 or 5 in solid form, a quantitative yield in a single labeled species, (99m)Tc-labeled PN(2)S-PEG(5000) 9 and (99m)Tc-labeled PN(2)S-PEG(20000) 10, respectively, is obtained. The reaction occurs in less than 15 min at room temperature for 4 and 35 degrees C for 5. This labeling procedure avoids the use of an external reducing agent, and it is based on the amphiphilic properties of PN(2)S-PEGs. Once in water, 4 and 5 self-assemble in micelles, which catalyze the metal reduction by means of an electron pair transfer from the phosphorus to technetium. The [(99m)TcO](3+) species is then coordinated, and at micelle level, both the (P)ON(2)S and the PN(2)S coordinations are possible, as demonstrated by reacting (99m)Tc-gluconate and ReOCl(3)(PPh(3))(2) with 4 and 5 and with the oxidized analogous (P)ON(2)S-PEG(5000) 6. Compounds 9 and 10 exhibited a high stability both in vitro and in vivo. Biodistribution studies in mice also indicated that PN(2)S linking and (99m)Tc labeling do not modify PEG behavior in water and in vivo since the polymer dictates the fate of the conjugate.     

ATP-binding modes and functionally important interdomain bonds of sarcoplasmic reticulum Ca2+-ATPase revealed by mutation of glycine 438, glutamate 439, and arginine 678

ATP binds to sarcoplasmic reticulum Ca(2+)-ATPase both in a phosphorylating (catalytic) mode and in a nonphosphorylating (modulatory) mode, the latter leading to acceleration of phosphoenzyme turnover (Ca(2)E(1)P --> E(2)P and E(2)P --> E(2) reactions) and Ca(2+) binding (E(2) --> Ca(2)E(1)). In some of the Ca(2+)-ATPase crystal structures, Arg(678) and Glu(439) seem to be involved in the binding of nucleotide or an associated Mg(2+) ion. We have replaced Arg(678), Glu(439), and Gly(438) with alanine to examine their importance for the enzyme cycle and the modulatory effects of ATP and MgATP. The results point to the key role of Arg(678) in nucleotide binding and to the importance of interdomain bonds Glu(439)-Ser(186) and Arg(678)-Asp(203) in stabilizing the E(2)P and E(2) intermediates, respectively. Mutation of Arg(678) had conspicuous effects on ATP/MgATP binding to the E(1) form and ADP binding to Ca(2)E(1)P, as well as ATP/MgATP binding in modulatory modes to E(2)P and E(2), whereas the effects on ATP/MgATP acceleration of the Ca(2)E(1)P --> E(2)P transition were small, suggesting that the nucleotide that accelerates Ca(2)E(1)P --> E(2)P binds differently from that modulating the E(2)P --> E(2) and E(2) --> Ca(2)E(1) reactions. Mutation of Glu(439) hardly affected nucleotide binding to E(1), Ca(2)E(1)P, and E(2), but it led to disruption of the modulatory effect of ATP on E(2)P --> E(2) and acceleration of the latter reaction, indicating that ATP normally modulates E(2)P --> E(2) by interfering with the interaction between Glu(439) and Ser(186). Gly(438) seems to be important for this interaction as well as for nucleotide binding, probably because of its role in formation of the helix containing Glu(439) and Thr(441).     

Pseudomonas pseudoalcaligenes KF707 upon biofilm formation on a polystyrene surface acquire a strong antibiotic resistance with minor changes in their tolerance to metal cations and metalloid oxyanions

The susceptibility to various biocides was examined in planktonic cells and biofilms of the obligate aerobe, PCBs degrader, Pseudomonas pseudoalcaligenes KF707. The toxicity of two antibiotics, amikacin and rifampicin, three metalloid oxyanions (AsO(2) (-), SeO(3) (2-), TeO(3) (2-)) and three metal cations (Cd(2+), Ni(2+), Al(3+)) was tested at two stages of the biofilm-development (4 and 24 h) and compared to planktonic cells susceptibility. Mature biofilms formed in rich (LB, Luria-Bertani) medium were thicker (23 mum) than biofilms grown in minimal (SA saccarose-arginine) medium (13 mum). Early grown (4 h) SA-biofilms, which consisted of a few sparse/attached cells, were 50-100 times more resistant to antibiotics than planktonic cells. Conversely, minor changes in tolerance to metal(loid)s were seen in both SA- and LB-grown biofilms. In contrast to planktonic cells, no reduction of TeO(3) (2-) to elemental Te(0) or SeO(3) (2-) to elemental Se(0) was seen in KF707 biofilms. The data indicate that: (a) metal tolerance in KF707 biofilms, under the growth and exposure conditions described here, is different than antibiotic tolerance; (b) KF707 planktonic cells and biofilms, are almost equally susceptible to killing by metal cations and oxyanions, and (c) biofilm-tolerance to TeO(3) (2-) and SeO(3) (2-) is not linked to metalloid reduction; this means that KF707 planktonic cells and biofilms differ in their physiology and strategy to counteract metalloid toxicity.     

Vaccination of lactating dairy cows for the prevention of aflatoxin B1 carry over in milk

The potential of anaflatoxin B(1) (AnAFB(1)) conjugated to keyhole limpet hemocyanin (KLH) as a vaccine (AnAFB(1)-KLH) in controlling the carry over of the aflatoxin B(1) (AFB(1)) metabolite aflatoxin M(1) (AFM(1)) in cow milk is reported. AFB(1) is the most carcinogenic compound in food and foodstuffs amongst aflatoxins (AFs). AnAFB(1) is AFB(1) chemically modified as AFB(1)-1(O-carboxymethyl) oxime. In comparison to AFB(1), AnAFB(1) has proven to be non-toxic in vitro to human hepatocarcinoma cells and non mutagenic to Salmonella typhimurium strains. AnAFB(1)-KLH was used for immunization of cows proving to induce a long lasting titer of anti-AFB(1) IgG antibodies (Abs) which were cross reactive with AFB(1), AFG(1), and AFG(2). The elicited anti-AFB(1) Abs were able to hinder the secretion of AFM(1) into the milk of cows continuously fed with AFB(1). Vaccination of lactating animals with conjugated AnAFB(1) may represent a solution to the public hazard constituted by milk and cheese contaminated with AFs.     

δ(13) C of leaf-respired CO(2) reflects intrinsic water-use efficiency in barley

Leaf intrinsic water-use efficiency (WUE), the ratio of photosynthetic rate to stomatal conductance (A/g(s) ), is a key plant trait linking terrestrial carbon and water cycles. A rapid, integrative proxy for A/g(s) is of benefit to crop breeding programmes aiming to improve WUE, but also for ecologists interested in plant carbon-water balance in natural systems. We hypothesize that the carbon isotope composition of leaf-respired CO(2) (δ(13) C(Rl) ), two hours after leaves are transferred to the dark, records photosynthetic carbon isotope discrimination and so provides a proxy for A/g(s) . To test this hypothesis, δ(13) C(Rl) was measured in four barley cultivars grown in the field at two levels of water availability and compared to leaf-level gas exchange (the ratio of leaf intercellular to ambient CO(2) partial pressure, C(i) /C(a) , and A/g(s) ). Leaf-respired CO(2) was more (13) C-depleted in plants grown at higher water availability, varied between days as environmental conditions changed, and was significantly different between cultivars. A strong relationship between δ(13) C(Rl) and δ(13) C of sucrose was observed. δ(13) C(Rl) was converted into apparent photosynthetic discrimination (Δ(13) C(Rl) ) revealing strong relationships between Δ(13) C(Rl) and C(i) /C(a) and A/g(s) during the vegetative stage of growth. We therefore conclude that δ(13) C(Rl) may provide a rapid, integrative proxy for A/g(s) in barley.     

Differential behavior of sPLA2-V and sPLA2-X in human neutrophils

Neutrophils and differentiated PLB-985 cells contain various types of PLA(2)s including the 85 kDa cytosolic PLA(2) (cPLA(2)), Ca(2+)-independent PLA(2) (iPLA(2)) and secreted PLA(2)s (sPLA(2)s). The present study focuses on the behavior of sPLA(2)s in neutrophils and PLB cells and their relationship to cPLA(2)alpha. The results of the present research show that the two types of sPLA(2) present in neutrophils, sPLA(2)-V and sPLA(2)-X, which are located in the azurophil granules, are differentially affected by physiological stimuli. While sPLA(2)-V is secreted to the extacellular milieu, sPLA(2)-X is detected on the plasma membranes after stimulation. Stimulation of neutrophils with formyl-Met-Leu-Phe (fMLP), opsonized zymosan (OZ) or A23187 resulted in a different kinetics of sPLA(2) secretion as detected by its activity in the neutrophil supernatants. Neutrophil priming by inflammatory cytokines or LPS enhanced sPLA(2) activity detected in the supernatant after stimulation by fMLP. This increased activity was due to increased secretion of sPLA(2)-V to the supernatant and not to release of sPLA(2)-X. sPLA(2) in granulocyte-like PLB cells exhibit identical characteristics to neutrophil sPLA(2), with similar activity and optimal pH of 7.5. Granulocyte-like cPLA(2)alpha-deficient PLB cells serve as a good model to study whether sPLA(2) activity is regulated by cPLA(2)alpha. Secretion and activity of sPLA(2) were found to be similar in granulocyte-like PLB cells expressing or lacking cPLA(2)alpha, indicating that they are not under cPLA(2)alpha regulation.     

Interaction of lecithin:cholesterol acyltransferase (LCAT).alpha 2-macroglobulin complex with low density lipoprotein receptor-related protein (LRP). Evidence for an alpha 2-macroglobulin/LRP receptor-mediated system participating in LCAT clearance.

The reaction of lecithin:cholesterol acyltransferase (LCAT) with high density lipoproteins (HDL) is of critical importance in reverse cholesterol transport, but the structural and functional pathways involved in the regulation of LCAT have not been established. We present evidence for the direct binding of LCAT to alpha(2)-macroglobulin (alpha(2)M) in human plasma to form a complex 18.5 nm in diameter. Forty percent of plasma LCAT-HDL was associated with alpha(2)M; moreover, most of the LCAT in cerebrospinal fluid and in the medium of cultured human hepatoma cell line was associated with alpha(2)M. Purified recombinant human LCAT (rLCAT) labeled with (125)I bound to native and methylamine-activated alpha(2)M (alpha(2)M-MA) in vitro in a time- and concentration-dependent manner, and this binding did not depend on the presence of lipid. rLCAT bound to alpha(2)M-MA with greater affinity than to alpha(2)M. Furthermore, rLCAT did not activate alpha(2)M as phosphatidylcholine-specific phospholipase C does. Reconstituted HDL particles (LpA-I) inhibited the binding of rLCAT to alpha(2)M more efficiently than native HDL(3) did. LCAT associated with alpha(2)M was enzymatically inactive under both endogenous and exogenous assay conditions. Purified rLCAT alone did not bind to low density lipoprotein receptor-related protein (LRP) as lipoprotein lipase (LPL) does; however, when rLCAT was combined with alpha(2)M-MA to form a complex, binding, internalization, and degradation of rLCAT took place in LRP-expressing cells (LRP (+/+)) but not in cells deficient in LRP (LRP (-/-)). It is concluded that the binding of LCAT to alpha(2)M inhibits its enzymatic activity. Furthermore, the finding supports the possibility that the LRP receptor can act in vivo to mediate clearance of the LCAT-alpha(2)M complex and may significantly influence the bioavailability of LCAT.     

Antioxidant properties of cystic fibrosis sputum

Oxidative stress is a likely contributor to the pathogenesis of cystic fibrosis (CF) lung disease. However, hydrogen peroxide (H(2)O(2)), a physiological oxidant, is not elevated in CF exhalates. H(2)O(2) may be neutralized by antioxidants in CF airway secretions. The H(2)O(2)-detoxifying capacity of CF airway secretions, obtained via sputum induction, was studied in an in vitro H(2)O(2) cytotoxicity model. 16HBE14o- cells were exposed to H(2)O(2) in culture medium containing either 0 or 10% fetal bovine serum (FBS) or 10% CF sputum supernatant (extracted without use of dithiothreitol). The efficiency of H(2)O(2) neutralization was estimated by measuring intracellular oxidant levels (dihydrorhodamine 123) after 2 h and cell viability (propidium iodide) after 24 h of H(2)O(2) exposure. Furthermore, the presence of reduced thiols (DTNB assay) and reduced glutathione (recycling assay) in CF sputum samples was evaluated. CF sputum extracts completely prevented intracellular oxidant accumulation seen in cells incubated with H(2)O(2) in both control media (i.e., 0 or 10% FBS). Furthermore, CF sputum abolished cell death in 16HBE14o- cells exposed to up to 1 mM H(2)O(2). In contrast, there was 100% cytotoxicity in cells exposed to 600 microM H(2)O(2) in both control media. The H(2)O(2)-detoxifying potential of CF sputum was sustained after catalase and heme peroxidases were inactivated by sodium azide, which does not affect glutathione peroxidase. In addition, reduced protein thiols were found in abundance in CF sputum. In conclusion, CF sputum is capable to neutralize H(2)O(2) and abundant reduced thiols and/or glutathione peroxidase are fully sufficient to detoxify H(2)O(2).     

Functional effect of longitudinal heterogeneity in constricted airways before and after lung expansion

Heterogeneity in narrowing among individual airways is an important contributor to airway hyperresponsiveness. This paper investigates the contribution of longitudinal heterogeneity (the variability along the airway in cross-sectional area and shape) to airway resistance (R(aw)). We analyzed chest high-resolution computed tomography scans of 8 asthmatic (AS) and 9 nonasthmatic (NA) subjects before and after methacholine (MCh) challenge, and after lung expansion to total lung capacity. In each subject, R(aw) was calculated for 35 defined central airways with >2 mm diameter. Ignoring the area variability and noncircular shape results in an underestimation of R(aw) (%U(total)) that was substantial in some airways (～50%) but generally small (median <6%). The average contribution of the underestimation of R(aw) caused by longitudinal heterogeneity in the area (%U(area)) to %U(total) was 36%, while the rest was due to the noncircularity of the shape (%U(shape)). After MCh challenge, %U(area) increased in AS and NA (P < 0.05). A lung volume increase to TLC reduced %U(total) and %U(area) in both AS and NA (P < 0.0001, except for %U(total) in AS with P < 0.01). Only in NA, %U(shape) had a significant reduction after increasing lung volume to TLC (P < 0.005). %U(area) was highly correlated, but not identical to the mean-normalized longitudinal heterogeneity in the cross-sectional area [CV(2)(A)] and %U(shape) to the average eccentricity of the elliptical shape. This study demonstrates that R(aw) calculated assuming a cylindrical shape and derived from an average area along its length may, in some airways, substantially underestimate R(aw). The observed changes in underestimations of R(aw) with the increase in lung volume to total lung capacity may be consistent with, and contribute in part to, the differences in effects of deep inhalations in airway function between AS and NA subjects.     

Up-regulation of alphavbeta3 integrin expression is a novel molecular response to chemotherapy-induced cell damage in a heregulin-dependent manner

alpha(v)beta(3) integrin has a dual role in apoptosis. Whereas ligated alpha(v)beta(3) activates cell survival pathways and suppresses pro-apoptotic signals, unligated alpha(v)beta(3) or integrins bound to soluble ligands promote apoptosis. In this study, we assessed the role of alpha(v)beta(3) in chemosensitivity of breast cancer cells expressing different levels of heregulin (HRG). Expression levels of the RGD-binding integrins alpha(v)beta(3) were measured in MDA-MB-231 human breast cancer cells and its low HRG-expressing derivative (MDA-MB-231/AS31) treated with the microtubule-interfering agents (MIAs) paclitaxel and vincristine. Following treatment, only alpha(v)beta(3) levels were significantly increased in MDA-MB-231 cells. Interestingly, alpha(v)beta(3) expression was more significantly up-regulated in the MDA-MB-231/AS31 cells than in the parental cells. This MIA-induced increase of alpha(v)beta(3) expression was correlated with a decrease in cell viability and an increase in apoptosis in MDA-MB-231/AS31 cells, indicating that overexpression of alpha(v)beta(3) is linked to chemotherapy-induced cell death in low HRG-expressing breast cancer models. Moreover, a paclitaxel-induced increase of alpha(v)beta(3) was also observed in MCF-7 cells but not in an doxorubicin-resistant derivative that shows cross-resistance to paclitaxel, further providing evidence that the extent of alpha(v)beta(3) up-regulation is related to cell damage. These results indicate that alpha(v)beta(3) integrin is dramatically up-regulated in low HRG-expressing breast cancer models that are highly responsive to MIAs, thus providing a novel molecular marker of chemosensitivity influenced by HRG levels in breast cancer cells.     

Studies of the receptor-bound conformation of alphaIIbbeta3 antagonists by 15N-edited NMR spectroscopy

We report the results of NMR studies and computer simulations of potent antagonists reflective of the alpha(IIb)beta(3) receptor-bound conformations. The peptides c[Mpa-(15)N-Arg(1)-(15)N-Gly(2)-(15)N-Asp(3)-(15)N-Phe(4)-(15)N-Arg(5)-Cys]-NH(2) (Phe-Arg analog) (Mpa: 3-mercaptopropionic acid) and c[Mpa-(15)N-Arg(1)-(15)N-Gly(2)-(15)N-Asp(3)-(15)N-Asp(4)-(15)N-Val(5)-Cys]-NH(2) (Asp-Val analog) were subjected to (15)N-edited NMR experiments to study the conformations of these peptides in the absence and in the presence of alpha(IIb)beta(3) receptor. The NMR studies of the Phe-Arg analog, a selective alpha(IIb)beta(3) antagonist, resulted in distinctly different experimental data in the presence and absence of the receptor. The computer simulations for this peptide resulted in one large family of structures consistent with the experimental data. This conformation suggests a type I beta-turn spanning residues Arg(1) and Gly(2) when bound to the receptor and we were able to establish a model for the three dimensional arrangement of the pharmacophores. The studies on the Asp-Val analog, an alpha(v)beta(3) antagonist that binds to the alpha(IIb)beta(3) with moderate affinity, resulted in conformations that are not as well defined as those for the Phe-Arg analog but are consistent with the model established for this analog. These results are important for the design of novel alpha(IIb)beta(3) antagonists.