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An efficient formic acid catalyzed one-pot synthesis of 4-arylquinoline 2-carboxylates in water via three-component coupling of arylamines, glyoxylates and phenylacetylenes has been described. 4-Arylquinoline 2-carboxylates 1o and 1q displayed significant antioxidant activity as indicated by their Fe-reducing power in the ferric reducing ability of plasma (FRAP) assay. The compounds were found to react directly with hydrogen peroxide, which might be one of the mechanisms of their antioxidant effect. Compounds 1o and 1q effectively quenched H2O2 and amyloid-β-generated reactive oxygen species (ROS) and also displayed significant protection against H2O2-induced neurotoxicity in human neuroblastoma SH-SY5Y cells. Additionally, all compounds exhibited promising P-glycoprotein induction activity in human adenocarcinoma LS-180 cells, indicating their potential to enhance amyloid-β clearance from Alzheimer's brains. Furthermore, all compounds were relatively non-toxic to SH-SY5Y and LS-180 cells (IC50 > 50 μM). The promising antioxidant, ROS quenching, neuroprotective and Pgp-induction activity of these compounds strongly indicate their potential as anti-Alzheimer's agents.

The design, optimization, and scale-up of a chromatographic process using immobilized metal ion affinity chromatography (IMAC) demands a thorough understanding of the fundamental factors governing the various interactions between immobilized metal ions and proteins. The results obtained and reported in this study emphasize the role of ionic strength and pH in governing the extent and mechanism of these interactions using WGA-TREN-Ni(II) as the model system. The adsorption data for various ionic strength (0–3 M NaCl) and pH (5–9) conditions have been analyzed, both qualitatively, in the light of the information available for other systems, and quantitatively, using four isotherm models viz. the general affinity interaction theory/the Langmuir model, the Freundlich model, the Temkin model, and the Langmuir–Freundlich model. This analysis indicates that only the combined Langmuir–Freundlich model is able to explain the adsorption behavior under the whole range of ionic strength and pH relevant for IMAC. The other three models are applicable to some extent only for low ionic strength (≤0.5 M NaCl) values. It is envisaged that this investigation would be useful in developing an improved quantitative understanding of the role of solution environment in governing IMA interactions and in designing and/or optimizing protein separations on preparatory scale.

Previous attempts to express the toxin complex genes of Photorhabdus luminescens W14 in Escherichia coli have failed to reconstitute their oral toxicity to the model insect Manduca sexta . Here we show that the combination of three genes, tcdA , tcdB , and tccC , is essential for oral toxicity to M . sexta when expression in E . coli is used. Further, when transcription from native toxin complex gene promoters is used, maximal toxicity in E . coli cultures is associated with the addition of mitomycin C to the growth medium. In contrast, the expression of tcdAB (or the homologous tcaABC operon) with no recombinant tccC homolog in a different P . luminescens strain, K122, is sufficient to confer oral toxicity on this strain, which is otherwise not orally toxic. We therefore infer that P . luminescens K122 carries a functional tccC -like homolog within its own genome, a hypothesis supported by Southern analysis. Recombinant toxins from both P . luminescens K122 and E . coli were purified as high-molecular-weight particulate preparations. Transmission electron micrograph (TEM) images of these particulate preparations showed that the expression of tcdAB (either with or without tccC ) in E . coli produces visible ∼25-nm-long complexes with a head and tail-like substructure. These data are consistent with a model whereby TcdAB constitutes the majority of the complex visible under TEM and TccC either is a toxin itself or is an activator of the complex. The implications for the potential mode of action of the toxin complex genes are discussed.

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the purification of natural and recombinant therapeutic products and is beginning to find industrial applications. The design, optimization, and scale-up of a chromatographic process using IMAC demands a thorough understanding to be developed regarding the fundamental factors governing the various interactions between immobilized metal ions and proteins. Consequently, there is an immediate need to find out a theory that is able to account for these interactions most efficiently in a qualitative as well as a quantitative manner. In view of this requirement, the interactions of several model proteins (lysozyme, ovalbumin, bovine serum albumin, conalbumin, and wheat germ agglutinin) with metal (Cu(II), Ni(II))-chelated IDA (iminodiacetate) and tris(2-aminoethyl)amine were investigated. The adsorption data were analyzed using four isotherm models, viz., the general affinity interaction theory/Langmuir model, the Freundlich model, the Temkin model, and the Langmuir–Freundlich model, and the sorption parameters were computed. Although the first three models were applicable to some protein–IMA–M(II) systems, the Langmuir–Freundlich model appeared to be the most efficient model for explaining the interactions of proteins with IMA–M(II) gels. Also, this model was able to explain cooperativity and binding heterogeneity in quantitative terms. It is envisaged that this analysis would be useful in developing an improved understanding of protein-immobilized metal ion interactions and providing guidelines for designing preparative-scale separations using IMAC.

Background & Aims: Glucagon-like peptide (GLP)-2, a product of the proglucagon gene, is expressed in enteroendocrine cells of the small and large intestine and is trophic to the gastrointestinal mucosa. GLP-2 also inhibits gastric acid secretion and emptying and upregulates intestinal hexose transport. GLP-2 acts via binding to a single G protein— coupled GLP-2 receptor (GLP-2R), but the cellular targets for the diverse actions of GLP-2 remain unknown. Methods: GLP-2R expression in rodent and human tissues was examined using a combination of Northern blotting, reverse-transcription polymerase chain reaction (RT-PCR), and immunocytochemistry. Results: A single major GLP-2R messenger RNA transcript was detected by Northern blot analysis in rodent stomach, duodenum, jejunum, ileum, and colon, but not in rodent esophagus. GLP-2R expression was also detected by RT-PCR in RNA from the hypothalamus, brain stem, and lung. Immunocytochemical localization of human GLP-2R expression using specific antisera detected GLP-2R immunopositivity in subsets of endocrine cell populations in the epithelium of the stomach and both the small and large bowel. Conclusions: These findings suggest that enteroendocrine-derived GLP-2 acts directly on endocrine cells to induce one or more downstream mediators of GLP-2 action in the gastrointestinal tract.

Leishmaniasis is a disease caused by the intracellular protozoan, Leishmania. A current treatment for cutaneous leishmaniasis involves the delivery of imidazoquinolines via a topical cream. However, there are no parenteral formulations of imidazoquinolines for the most deadly version of the disease, visceral leishmaniasis. This work investigates the use of electrospray to encapsulate the imidazoquinoline adjuvant resiquimod in acid sensitive microparticles composed of acetalated dextran (Ac-DEX) or Ac-DEX/Tween blends. The particles were characterized and tested both in vitro and in vivo. Solutions of Ac-DEX and resiquimod in ethanol were electrosprayed to generate approximately 2 μm Ac-DEX particles containing resiquimod with an encapsulation efficiency of 85%. To prevent particle aggregation, blends of Ac-DEX with Tween 20 and Tween 80 were investigated. Tween 80 was then blended with the Ac-DEX at ∼10% (w/w) of total polymer and particles containing resiquimod were formed via electrospray with encapsulation efficiencies between 40% and 60%. In vitro release profiles of resiquimod from Ac-DEX/Tween 80 particles exhibited the acid-sensitive nature of Ac-DEX, with 100% drug release after 8 h at pH 5 (phagosomal pH) and after 48 h at pH 7.4 (physiological pH). Treatment with Ac-DEX/Tween 80 particles elicited significantly greater immune response in RAW macrophages over free drug. When injected intravenously into mice inoculated with Leishmania, parasite load reduced significantly in the bone marrow compared to blank particles and phosphate-buffered saline controls. Overall, electrospray appears to offer an elegant, scalable way to encapsulate adjuvant into an acid sensitive delivery vehicle for use in treating visceral leishmaniasis.

The in vivo protective role of hydro-methanolic root extract of Withania somnifera (WS) was evaluated in alleviating lead nitrate (LN)-induced toxicity in male Swiss albino mice by measuring hematoserological profiles. The lead-treated (20 mg/kg body wt, p.o.) albino mice (25-30 g) concurrently received the root extract (200 and 500 mg/kg body wt, p.o.) once daily for the duration of six weeks. Animals exposed to LN showed significant (P0.001) decline in haemoglobin content, red blood cell count, white blood cell count, packed cell volume and insignificant decrease in mean corpuscular haemoglobin and mean corpuscular haemoglobin content, while mean corpuscular volume and platelet count were increased. A significant elevation (P0.001) in serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase, acid phosphatase and total cholesterol were also observed, when compared with control mice. Thus, the study demonstrated that the concurrent daily administration of root extract of WS protected the adverse effects of LN intoxication in mice.

Liquid−Liquid equilibria at 303.15 K were measured for water + 2,3-butanediol + 3-methyl-1-butanol with and without sodium sulfate and sodium carbonate. Complete phase diagrams were obtained by evaluating the solubility and tie-line results for ternary and quaternary systems. The consistency of the tie-line results was ascertained using Othmer−Tobias plots. The effect of salts on the distribution coefficient and selectivity of 2,3-butanediol was studied. Sodium carbonate was found to have more salting out power. Addition of salt was, therefore, found to improve not only the extracting power of the solvent but also the selectivity of the solvent for 2,3-butanediol.

Multiple gene transfections are often required to control the differentiation of embryonic stem cells. This is typically done by removing the cells from the culture substrate and conducting gene transfection via bulk electroporation (in suspension), which is then followed by further culture. Such repetitive processes could affect the growth and behavior of delicate/scarce adherent cells. We have developed a novel nanofiber-based sandwich electroporation device capable of in situ and in culture gene transfection. Electrospinning was used to deposit poly(ε-caprolactone)/gelatin nanofibers on the Al(2)O(3) nanoporous support membrane, on top of which a polystyrene microspacer was thermally bonded to control embryonic stem cell colony formation. The applicability of this system was demonstrated by culturing and transfecting mouse embryonic stem cells. Measurements of secreted alkaline phosphatase protein and metabolic activity showed higher transfection efficacy and cell viability compared to the conventional bulk electroporation approach.

A rapid immune response is required to prevent death from Anthrax, caused by Bacillus anthracis. We formulated a vaccine carrier comprised of acetalated dextran microparticles encapsulating recombinant protective antigen (rPA) and resiquimod (a toll-like receptor 7/8 agonist). We were able to protect against triplicate lethal challenge by vaccinating twice (Days 0, 7) and then aggressively challenging on Days 14, 21, 28. A significantly higher level of antibodies was generated by day 14 with the encapsulated group compared to the conventional rPA and alum group. Antibodies produced by the co-encapsulated group were only weakly-neutralizing in toxin neutralization; however, survival was not dependent on toxin neutralization, as all vaccine formulations survived all challenges except control groups. Post-mortem culture swabs taken from the hearts of vaccinated groups that did not produce significant neutralizing titers failed to grow B. anthracis. Results indicate that protective antibodies are not required for rapid protection; indeed, cytokine results indicate that T cell protection may play a role in protection from anthrax. We report the first instance of use of a particulate carrier to generate a rapid protective immunity against anthrax.

In the field of drug delivery, pH-sensitive polymeric microparticles can be used to release therapeutic payloads slowly in extracellular conditions (pH 7.4) and faster in more acidic areas in vivo, such as sites of inflammation, tumors, or intracellular conditions. Our group currently uses and is further developing the pH-sensitive polymer acetalated dextran (Ac-DEX), which is a biodegradable polymer with highly tunable degradation kinetics. Ac-DEX has displayed enhanced delivery of vaccine and drug components to immune and other cells, making it an extremely desirable polymer for immune applications. Currently, one of the degradation products of Ac-DEX is methanol, which may cause toxicity issues if applied at high concentrations with repeated doses. Therefore, in this manuscript we report the first synthesis and characterization of an Ac-DEX analog which, instead of a methanol degradation product, has a much safer ethanol degradation product. We abbreviate this ethoxy acetal derivatized acetalated dextran polymer as Ace-DEX, with the 'e' to indicate an ethanol degradation product. Like Ac-DEX, Ace-DEX microparticles have tunable degradation rates at pH 5 (intracellular). These rates range from hours to several days and are controlled simply by reaction time. Ace-DEX microparticles also show minimal cytotoxicity compared to commonly used poly(lactic-co-glycolic acid) (PLGA) microparticles when incubated with macrophages. This study aims to enhance the biocompatibility of acetalated dextran-type polymers to allow their use in high volume clinical applications such as multiple dosing and tissue engineering.

Insulin-expressing islet-like cell clusters derived from precursor cells have significant potential in the treatment of type-I diabetes. Given that cluster size and uniformity are known to influence islet cell behavior, the ability to effectively control these parameters could find applications in the development of anti-diabetic therapies. In this work, we combined micro and nanofabrication techniques to build a biodegradable platform capable of supporting the formation of islet-like structures from pancreatic precursors. Soft lithography and electrospinning were used to create arrays of microwells (150-500 μm diameter) structurally interfaced with a porous sheet of micro/nanoscale polyblend fibers (~0.5-10 μm in cross-sectional size), upon which human pancreatic ductal epithelial cells anchored and assembled into insulin-expressing 3D clusters. The microwells effectively regulated the spatial distribution of the cells on the platform, as well as cluster size, shape and homogeneity. Average cluster cross-sectional area (~14000-17500 μm(2)) varied in proportion to the microwell dimensions, and mean circularity values remained above 0.7 for all microwell sizes. In comparison, clustering on control surfaces (fibers without microwells or tissue culture plastic) resulted in irregularly shaped/sized cell aggregates. Immunoreactivity for insulin, C-peptide and glucagon was detected on both the platform and control surfaces; however, intracellular levels of C-peptide/cell were ~60 % higher on the platform.

2, 3-Butanediol, an important industrial chemical, is of interest because of its application as a solvent and liquid fuel additive. Liquid-liquid equilibria at 303.15 [+-] 0.5 K were measured for water + 2, 3-butanediol + butan-1-ol, + 3-methyl-1-butanol, + 4-methyl-2-pentanone, + tributyl phosphate, and + butyl acetate. Complete phase diagrams were obtained by evaluating the solubility and tie-line results for each ternary mixture. The consistency of the tie-line results was ascertained using an Othmer-Tobias plot. The distribution coefficient and separation factors were evaluated over the immiscibility region. Among the solvents studied, butan-1-ol is the most effective one though tributyl phosphate and 3-methyl-1-butanol may be preferred because of their low solubility and high selectivity.

The toxicity of Lead was recognized centuries ago, and it continued to pose serious threat to the health of children as well as adults. This review presents an overview of the current knowledge of toxic effects of Lead induced oxidative damage and also suggests some possible measures which could reduce the toxic effects of the metal. This paper examines the effect s of Lead in blood, soft tissues, haematopoietic system and the antioxidant defense system. On the other hand, data also indicated that lead is an essential element at low dietary intakes. Its deficiency was shown to depress growth, disturb iron metabolism, alter activities of some enzymes and disturb the metabolism of cholesterol, phospholipids and bile acids. It was found that lead toxicity is significant but a preventable health problem. Furthermore, work is needed to find the effective and safe intervention for lowering the lead exposure at the general population level. Key words: Lead toxicity, oxidative damage, haematological, antioxidant.

The present study was conducted to investigate the chemopreventive effects of hydro-ethanolic extract of Euphorbia neriifolia (EN) on N-nitrosodiethylamine (DENA) induced renal cancer in male Swiss albino mice. Animals were pretreated with EN extract (150 and 400 mg/kg body weight; p.o) and butylated hydroxyanisole (BHA) as a standard (0.5% and 1% BHA p.o) both for two week prior to the administration of single dose of DENA (50 mg/kg body weight; p.o). Various in vivo antioxidant biochemical parameters like lipid peroxidation (LPO), superoxide dismutase (SOD), and catalase (CAT) were evaluated to determine the reno-protective and antioxidant activity of EN. DENA increased oxidative stress through increase in LPO and decrease in antioxidant enzymes (SOD, and CAT). The EN extract significantly restored the antioxidant enzyme level in the kidney and exhibited significant dose dependant protective effect against DENA induced nephrotoxicity, which can be mainly attributed to the antioxidant property of the extract. This study rationalized the ethno-medicinal use of EN for protection against renal cancer.

Histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, when incubated at elevated temperatures forms many species of protein. The two major species are HPr-1 and HPr-2, which have been shown to lack one or two amides, respectively (Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 7023-7033). The formation of HPr-1 and HPr-2 is shown to be pH-dependent and does not occur readily below pH 6. Investigation of the identities and properties of the two residues that deamidate involved creation of site-directed mutants at the 6 glutamine and 2 asparagine residues of HPr; description of their deamidation species by isoelectric focusing; determination of their relative antibody binding properties; assay of their phosphoacceptor and phosphodonor activities; characterization of tryptic and V8-protease peptides; obtaining two-dimensional nuclear magnetic resonance spectra of HPr, HPr-1, and several mutants. It was determined that the sequential deamidation of Asn-38 and Asn-12 yields HPr-1 and HPr-2. Both residues exist as Asn-Gly pairs, and both deamidations probably form isoaspartyl acid. HPr from Bacillus subtilis and Staphylococcus carnosus which also have Asn-Gly at residues 38 and 39 form HPr-1 species presumably by deamidation. HPr from Streptococcus faecalis which does not have Asn-38 does not form a HPr-1 species. The E. coli mutant HPrs, N12D and Q51E, residues that may be involved in the active site, had impaired phosphohydrolysis properties and decreased phosphoenolpyruvate:sugar phosphotransferase system activity.

The location and description of epitopes on proteins describe the basis of immunological specificity. The 2.8-A structure of the phosphocarrier protein, HPr from Escherichia coli, complexed to the Fab fragment of the monoclonal antibody, Jel42, has been determined. This allows the first comparison of epitope predictions from extensive site-directed mutagenesis experiments, coupled with biological activity studies (Sharma, S., Georges, F., Klevit, R. E., Delbaere, L. T. J., Lee, J. S., and Waygood, E. B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4877-4881), with those from x-ray analysis. There are 14 amino acid residues of E. coli HPr that interact with the Jel42 antigen-binding site. Nine of these were correctly assigned by the mutagenesis studies. Of the 5 remaining residues, Met-1 could not be altered; two others appear to have critical roles in determining protein conformation; the other 2 residues have a minimal effect on antibody binding since they are located on the periphery of the epitope with one face of their side chains in van der Waals contact with the antibody and the other face in contact with solvent. Four residues were incorrectly assigned to the epitope. These residues were located adjacent to epitope residues that were likely perturbed by these mutations. This study demonstrates that mutations which caused greater than 10-fold changes in antibody binding affinity were correctly assigned to the epitope by the mutagenesis experiments. Guidelines are also presented in order to minimize incorrect assignments.

Phaseolus mungo lectin in pure form was isolated from seeds by affinity chromatography on a galactosyl Sepharose 6B column. The lectin contained 8.3% neutral carbohydrate. It absorbed maximally near 278 nm, and the corresponding maxima in fluorescence excitation and emission spectra occurred, respectively, at 278 and 337 nm. Its elution profile from an analytical Sephadex G-150 column was consistent with a molecular weight of 137 000 and a Stokes radius of 4.3 nm; the frictional ratio, f/f0, of the lectin was calculated to be 1.26. The lectin moved as a single protein band of Mr 66 000 in SDS-PAGE under both reducing and nonreducing conditions. The results suggested that the lectin is a dimer whose overall native conformation is nearly globular. From the effect of 10 saccharides on the hemagglutinating activity of lectin against trypsinized rabbit erythrocytes, it was concluded that the lectin was galactose/N-acetylgalactosamine-specific, having significantly reduced affinity for N-acetylgalactosamine. The lectin showed preference for alpha-glycoside and probably does not contain a hydrophobic binding site.

Brain is a target of stress along with the immune, metabolic, and cardiovascular systems of the body. In the present work, the preventive roles of a multivitamin–mineral supplement and vitamins (E + C) in chronic unpredictable stress (CUS)-induced oxidative damage were studied in the brain and heart of Swiss albino mice. Thirty-two mice were randomized to one of the following groups: control + vehicle, CUS + vehicle, CUS + multivitamin–mineral, and CUS + vitamins (E + C). CUS was applied for 4 weeks, and multivitamin–mineral and vitamins (E + C) were administered orally for the same period. CUS led to a negative impact on all the biochemical parameters analyzed. Elevation in malondialdehyde and reduction in glutathione levels were found. The activities of superoxide dismutase, catalase, glutathione S-transferase, and glutathione reductase were decreased. Treatment with multivitamin–mineral and vitamins (E + C) brought these parameters to near normal levels. Multivitamin–mineral was found more restitutive than combined vitamins (E + C) doses. The present study hypothesizes that supplementation with a multivitamin–mineral may prove more effective than vitamin treatment alone in the alleviation of oxidative damage in brain and heart during periods of chronic stress.

Guided assembly of microscale tissue subunits (i.e. 3D cell clusters/aggregates) has found applications in cell therapy/tissue engineering, cell and developmental biology, and drug discovery. As cluster size and geometry are known to influence cellular responses, the ability to spatially control cluster formation in a high throughput manner could be advantageous for many biomedical applications. In this work, a micro- and nanofabricated platform was developed for this purpose, consisting of a soft-lithographically fabricated array of through-thickness microwells structurally bonded to a sheet of electrospun fibers. The microwells and fibers were manufactured from several polymers of biomedical interest. Human hepatocytes were used as model cells to demonstrate the ability of the platform to allow controlled cluster formation. In addition, the ability of the device to support studies on semi-controlled heterotypic interactions was demonstrated by co-culturing hepatocytes and fibroblasts. Preliminary experiments with other cells of interest (pancreatic cells, embryonic stem cells, and cardiomyocytes) were also conducted. Our platform possesses several advantages over previously developed microwell arrays: a more in vivo-like topographical stimulation of cells; better nutrient/waste exchange through the underlying nanofiber mat; and easy integration into standard two-chamber cell culture well systems.

Thirty-four of the 85 residues of the histidine-containing protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system have been changed by site-directed mutagenesis. Many of the mutations have wild-type activity suggesting an unaltered tertiary structure but have altered binding to three monoclonal antibodies: Jel42, Jel44, and Jel323. This altered binding defines the residues that are involved in the epitopes of HPr. At present, two different three-dimensional structures have been determined for HPr, one from two-dimensional nuclear magnetic resonance spectra and the other from x-ray diffraction of HPr crystals. The epitope mapping for Jel42 does not distinguish between the tertiary structures. However, only the HPr structure derived from two-dimensional nuclear magnetic resonance spectra is consistent with a contiguous surface binding site that can be defined as the epitope for Jel44. Thus the x-ray structure may represent a partially unfolded HPr.

Over the last three decades considerable advances have marked the field of drug delivery technology, resulting in many breakthroughs in clinical medicine. However, major unmet needs remain. Among these are broad categories of: 1) Continuous release of therapeutic agents over extended time periods and in accordance to a pre-determined temporal profile [38, 60]; 2) Local delivery at a constant rate to the tumor microenvironment, to overcome much of the systemic toxicity and improve anti-tumor efficacy; 3) Improved ease of administration, increasing patient compliance, while minimizing the needed intervention of healthcare personnel and decreasing the length of hospital stays [6, 46]. Success in addressing some or all of these challenges would potentially lead to improvements in efficacy and patient compliance, as well as minimization of side effects [66].

The creation of antifouling surfaces is one of the major prerequisites for silicon-based micro-electrical-mechanical systems for biomedical and analytical applications (known as BioMEMS). Poly(ethylene glycol) (PEG), a water-soluble, nontoxic, and nonimmunogenic polymer has the unique ability to reduce nonspecific protein adsorption and cell adhesion and, therefore, is generally coupled with a wide variety of surfaces to improve their biocompatibility. To this end, we have analyzed PEG thin films of various grafting densities (i.e., number of PEG chains per unit area) coupled to silicon using a single-step PEG-silane coupling reaction scheme using variable-angle ellipsometry. Initial PEG concentration and coupling time were varied to attain different grafting densities. These data were theoretically analyzed to understand the phenomenon of PEG film formation. Furthermore, all the PEG films were evaluated for their ability to control biofouling using albumin and fibrinogen as the model proteins. PEG thin films formed by using higher PEG concentrations ( > or = 10 mM PEG) or coupling time ( > or = 1 h) demonstrated enhanced protein fouling resistance behavior. This analysis is expected to be useful to form PEG films of desired grafting density on silicon substrates for appropriate application.

The creation of nonfouling surfaces is one of the major prerequisites for microdevices for biomedical and analytical applications. Poly(ethylene glycol) (PEG), a water soluble, nontoxic, and nonimmunogenic polymer has the unique ability of reducing nonspecific protein adsorption and cell adhesion and, therefore, is generally coupled with a wide variety of surfaces to improve their biocompatibility. The performance of these modified surfaces for long-term biomedical applications largely depends on the stability of these PEG films. To this end, we have investigated the stability of covalently coupled ultrathin PEG films on silicon in aqueous in vivo like conditions for a period of 4 weeks. The PEG-modified silicon substrates were incubated in PBS (37 degrees C, pH 7.4, 5% CO2) for different periods of time and then characterized using the techniques of ellipsometry, contact angle measurement, X-ray photoelectron spectroscopy, and atomic force microscopy. The ability of the PEG-modified surfaces to control protein fouling was examined by protein adsorption studies using fluorescein isothiocyanate labeled bovine serum albumin and ellipsometry. Furthermore, the ability of these films to control fibroblast adhesion was examined. Studies suggest that the PEG-modified surfaces retain their protein and cell repulsive nature even though the PEG film thickness decreases for the period of investigation.

In the past two decades, the biological and medical fields have seen great advances in the development of biosensors capable of quantifying biomolecules. Many of these biosensors have micro- and nano-scale features, are fabricated using biochip technology, and use silicon as a base material. The creation of antifouling sensor interfaces is critical to avoid serious consequences that arise due to their contact with biological fluids. To this end, we have created thin PEG interfaces of various grafting densities on silicon using a single-step PEG-silane coupling reaction scheme. Initial PEG concentration (5-50 mM) and coupling time (0.5-24 h) were varied to attain different grafting densities, and different PEG interfaces so created were analyzed using XPS and AFM. Furthermore, all the PEG interfaces were evaluated using XPS and AFM for their antifouling abilities using fibrinogen as the model protein. Results indicated that PEG interfaces created in this investigation are appropriate for biosensors with micro- and nano-scale features, and are efficient in controlling protein fouling.

Bacteriocins are proteins produced by bacteria to destroy other bacteria occupying their ecological niche. Photorhabdus luminescens is an insect pathogenic bacterium carried by an entomopathogenic nematode and occupies several different niches in its life cycle. The nematode enters the insect and releases a single strain of P. luminescens. The bacteria then kill the host and the bacteria and nematodes replicate within the cadaver. Strikingly, at the end of the infection the cadaver is still occupied by a single strain of bacterium, suggesting that P. luminescens can destroy other bacteria entering, or present within, the insect. Here we describe four loci encoding 'lumicins' in P. luminescens subsp. akhurstii strain W14. The lumicins are novel bacteriocins capable of killing other strains of Photorhabdus and Escherichia coli. These loci predict killer proteins and multiple dual type immunity proteins with domains similar to pyocins and colicins. The killer proteins are chimeric in nature with multiple domains, one of which is similar to the uropathogenic-specific protein (USP) described from uropathogenic E. coli. The implications of these novel bacteriocins for the lifestyle of Photorhabdus and the potential role of USP as a bacteriocin in E. coli are discussed.

Summary Invertebrates, including insects, are being developed as model systems for the study of bacterial virulence. However, we understand little of the interaction between bacteria and specific invertebrate tissues or the immune system. To establish an infection model for Photorhabdus, which is released directly into the insect blood system by its nematode symbiont, we document the number and location of recoverable bacteria found during infection of Manduca sexta. After injection into the insect larva, P. luminescens multiplies in both the midgut and haemolymph, only later colonizing the fat body and the remaining tissues of the cadaver. Bacteria persist by suppressing haemocyte-mediated phagocytosis and culture supernatants grown in vitro, as well as plasma from infected insects, suppress phagocytosis of P. luminescens. Using GFP-labelled bacteria, we show that colonization of the gut begins at the anterior of the midgut and proceeds posteriorly. Within the midgut, P. luminescens occupies a specific niche between the extracellular matrix and basal membrane (lamina) of the folded midgut epithelium. Here, the bacteria express the gut-active Toxin complex A (Tca) and an RTX-like metalloprotease PrtA. This close association of the bacteria with the gut, and the production of toxins and protease, triggers a massive programmed cell death of the midgut epithelium.

Increasing demands for more sophisticated and more biocompatible artificial medical devices in the recent years have stimulated researchers to either develop new technologies or adopt the established technologies from other scientific fields. Microfabrication technology (MEMS or micro-electrical-mechanical systems), mainly used in IC and microelectronics industry, has elicited increased interest among researchers due to the ability to control microstructure, topography, and feature size on the micro- and nano- scale; and control of surface chemistry in a precise manner through biochemical coupling and photopatterning processes. However, the applications of this technology are limited by the restricted choice of the materials (e.g. insulators, semiconductors and some metals) that can be used for micromachining. The most common materials used for biomedical applications are silicon and silicon dioxide. Though these materials are reasonably inert and biostable, several studies1–6 on silicon implants have shown fibrosis and scar tissue formation, thus limiting the long term functioning of the microdevice. Hence, in order to develop efficient implantable microdevices with minimal chances of immunorejection, it is extremely desirable to develop surface modification strategies to augment the biocompatibility of silicon before using it for biomedical applications.

Multidrug-resistant tumor cells overexpress P-glycoprotein (170 kDa), a member of the ABC (ATP Binding Cassette)-transporter superfamily. P-glycoprotein has been implicated in transport of a broad range of amphiphilic, hydrophobic drugs from tumor cells. The sequence and structural organization of P-glycoprotein, which consists of 12 transmembrane helices and two cytoplasmic nucleotide binding domains, is similar to other ABC-transporters. It is believed that the nucleotide binding domains of various ABC transporters, which have 30-50% sequence identity, play an important role in coupling ATP hydrolysis to the transport process. To allow structure-function studies of the nucleotide binding domains, the carboxyl-terminal nucleotide binding domain (NBD) of Chinese hamster P-glycoprotein has been cloned, overexpressed, and purified both by itself and as a fusion with maltose-binding protein. It has been demonstrated that the carboxyl-terminal NBD, when overexpressed in Escherichia coli in the absence of transmembrane helices, has very low ATPase activity. This suggests that the amino-terminal nucleotide binding domain and possibly interaction with the transmembrane domains may be required for full ATPase activity. It is also consistent with the idea that the ATPase activity of P-glycoprotein is stimulated in the presence of drugs. Circular dichroism spectral analysis and the ability of carboxyl-terminal NBD, both by itself and as a fusion with maltose-binding protein, to bind ATP-agarose beads and P-glycoprotein specific monoclonal antibodies suggests that the polypeptide folds into a functional domain. Gel filtration chromatography and cross-linking studies indicate that the carboxyl-terminal NBD has a tendency to self-associate to form oligomers. It is speculated that the carboxyl-terminal NBD may play a role in self-association of P-glycoprotein molecules in the plasma membrane.

Controlling the microscale environment in three-dimensional (3D) matrices for tissue engineering applications is a challenging but necessary goal. In this work, the effect of discrete microscale structures (microrods) on cell proliferation was assessed in 3D gels. Microrods were fabricated out of SU-8 with dimensions of 100 x 15 x 15 microm (L x H x W) and incorporated into Matrigel seeded with fibroblasts. The 3D microrod-Matrigel composite system inhibited proliferation of both primary and cell-line fibroblasts compared to cells seeded in Matrigel alone. To rule out bulk mechanical effects, the bulk shear modulus (G') and loss modulus (G") were assessed between 0.1 and 5 Hz for both Matrigel and microrod-Matrigel composites. The incorporation of microrods did not change the bulk stiffness of the gel. Moreover, it was determined that the chemistry of the microrod material itself did not inhibit cell proliferation. Therefore, results indicate that the presence of suspended microscale structures in three dimensions can regulate cell proliferation in a dose-dependent manner. This system provides a biocompatible, long-term way to modulate cell growth in 3D cultures and is amenable to in vivo applications.

The effect of stretching condition on stress-induced crystallization of amorphous polyethylene terephthalate (PET) film has been studied. Cast PET film was stretched at 85°C with stretching rates ranging from 100% per minute to 600% per minute for various stretching ratios. The stretched film samples were annealed at 140°C both under tension as well as under relaxed conditions. Tensile properties were determined using Instron Tensile Tester and results were compared. Tenacity, yield stress, and initial modulus were found to increase while elongation at break decreased with the increase in stretching rate. The degree of crystallinity was measured by x-ray diffraction as well as density methods. Birefringence and crystalline orientation factor values were determined experimentally and using these, amorphous orientation factor values were calculated. Birefringence, crystalline orientation and amorphous orientation values were found to increase with the increase in stretching rate. These studies showed that at low stretching rates a significant amount of relaxation takes place, resulting in lower orientation and strength. On the other hand, at higher stretching rates relaxation is minimized thus providing high orientation and strength. Upon annealing the crystallinity values increased as expected, however the trend of orientation and mechanical properties with respect to stretching rate was similar to those of unannealed films.

Triple differential cross-sections for the ionization of helium by fast positrons are calculated in a ‘correlated’ first Born approximation supplemented by the inclusion of post-collision interaction effects. The results are analysed with respect to electron-helium experimental data of Jung and coworkers in coplanar asymmetric geometry.

The triple-differential cross sections for the electron-impact ionization of helium in coplanar asymmetric geometry have been calculated by use of a second Born approximation which employs an improved choice for the target continuum-state wave function. The results are compared with the absolute experimental data of K. Jung et al. (J. Phys. B 18, 2955 (1985)) at an incident energy of 600 eV and are found to be in very good agreement with them. The corresponding results for the positron-impact case are also presented.

The recent absolute data of Jung et al. for the ionization of helium in a coplanar asymmetric geometry at an incident electron energy of 600 eV are analyzed by using a product of two Coulomb wave functions in the final state. This model leads to an improvement in the binary-peak to recoil-peak ratio and in the location of the binary peak compared with the first Born approximation.

The triple-differential cross section for the single ionization of helium in the coplanar asymmetric geometry has been calculated by using a second Born approximation which employs an improved choice of the target continuum-state wave function. The contribution of still higher-order terms of the scattering amplitude is also considered via the usual Glauber approximation. The results are compared with the recent data of Jung et al. at an incident energy of 600 eV and are found to be in very good agreement with them.

Keywords: Phosphoryl transfer; Epitopes; Two-dimensional NMR; Protein structure; X-ray diffraction; Sugar transport

A hybrid second-order model, in which the first-Born-approximation amplitude is evaluated by using an improved choice of the target continuum-state wave function and the second-Born-approximation amplitude in the usual way, is used to obtain the triple-differential cross sections for the ionization of helium. The results are compared with the recent data of Jung et al. in the coplanar asymmetric geometry at an incident electron energy of 600 eV and are found to be in very good agreement.