Tris-NaCl Buffer Research Articles

Medicinal potential of antimicrobial peptides from two plants against <i>Bacillus cereus</i> and <i>Staphylococcus aureus</i>

Bacillus cereus and Staphylococcus aureus are the most important bacteria that cause nosocomial infection and are resistant to antibiotics. Crude proteins from Cassia fistula and Ricinus communis were isolated to study their medicinal potential against Bacillus cereus, and Staphylococcus aureus. Extraction of the crude proteins from plants was done by phosphate buffer saline (PBS) and Tris NaCl buffer by using the roots and seeds of both plants. Antimicrobial activity was checked against bacterial strains by using agar disc diffusion and agar well diffusion methods. Zones of inhibitions were measured. On well diffusion method, PBS buffer protein extract of C. fistula roots showed a maximum zone of inhibition of 25 mm against B. cereus. Tris NaCl buffer extracts of C. fistula roots and seeds showed zones of inhibition of 12mm and 5mm respectively against S. aureus while Ricinus communis roots showed a zone of 12mm against B. cereus. Because the protein of the plants showed good antimicrobial activity, we can use these plants against various diseases caused by Bacillus cereus and Staphylococcus aureus.

Extraction and quantification of antimicrobial peptides from medicinal plants through TrisNaCl and PBS buffer

Nature has provided plants with their own specific defence system that protect the plant from several traumatic conditions. These include environmental conditions like drought, harsh climate changes, wounding, pathogen attack and other biological as well as a biological stresses. In order to deal with all these harmful occurrences, plants synthesize a wide range of defence factors that include both primary as well as secondary metabolites. Out of these the most popular are the defence proteins which are known as antimicrobial peptides (AMP). These AMPs are actually the pathogenesis-related (PR) defence proteins.  These proteins are activated under the control of defence system of plant whenever triggered by the alarming situation. In the current study Crude protein extraction of four medicinally important plants named as Cassia fistula, Albizia lebbeck, Saccharum officinarum & Cymbopogon citratus was performen. Extraction was done in TrisNaCl and PBS buffer. Quantification of the protein content in the extract was done by Bradford assay. Concentration of protein from TrisNaCl buffer extracts was high as compared to the extracts from PBS buffer. As these proteins play their protective role in defence of the plants against pathogen attack so these extracts can better be used to check the antimicrobial activity of these plants in future to treat several infectious diseases in humans.

Analytical strategy for studying the formation and stability of multilayered films containing gold nanoparticles.

The design of layer-by-layer (LbL) polyelectrolyte films including nanoparticles is a growing field of innovation in a wide range of biomedical applications. Gold nanoparticles (AuNPs) are very attractive for further biomolecule coupling to induce a pharmacological effect. Nanostructured LbL films coupled with such metallic species show properties that depend on the conditions of construction, i.e. the polymer nature and dissolution buffer. Tripartite LbL films (polycation, AuNP, and polyanion) were evaluated using two different polycationic polymers (poly(allylamine hydrochloride) (PAH), poly(ethylene imine) (PEI)) and various medium conditions (salts, i.e. phosphate, Tris or Tris-NaCl buffers, and concentration). AuNP incorporation and film stability were analysed by visible spectrophotometry, capillary zone electrophoresis, a quartz crystal microbalance, and high-performance liquid chromatography. The ideal compromise between AuNP loading and film stability was obtained using PAH prepared in Tris-NaCl buffer (0.01-0.15M). This condition allowed the formation of a LbL film that was more stable than the film with PEI and provided an AuNP quantity that was 4.8 times greater than that of the PAH-PBS-built film. In conclusion, this work presents an analytical strategy for the characterization of nanostructured multilayer films and optimization of LbL films enriched with AuNPs to design biomedical device coatings.

Antibacterial Activity of Tris NaCl and PBS Buffer Protein Extract of Cassia fistula, Saccharum officinarum, Albizia lebbeck and Cymbopogon citrates Against Bacterial Strains.

Medicinal plants are gaining popularity over synthetic medicines because antibiotic resistance demands the alternative source of medication. In the present research, the crude protein extraction of 4 medicinal plants Cassia fistula, Saccharum officinarum, Albizia lebbeck and Cymbopogon citrates was carried out. Crude protein extraction was done by 2 different buffers i.e. Tris NaCl buffer and PBS buffer. Protein confirmation was done by Bradford assay in the spectrophotometer. Antibacterial potential was checked and compared against Escherichia coli, Bacillus subtilis, Neisseria gonorrhoea, Bacillus cereus and Proteus mirabilis. Antibacterial assay was performed by disc diffusion method, agar well method and zones of inhibition were calculated. The study results indicated that Tris NaCl extracts’ antimicrobial potential is higher than that of the PBS buffer. On disc diffusion method the Tris NaCl buffer extracts of Cymbopogon citrates showed maximum zone of inhibition 11 mm and 9 mm against Bacillus subtilis and Bacillus cereus respectively and control chloramphenicol showed maximum zone of inhibition 26 mm against Bacillus subtilis. Cassia fistula showed maximum zone of inhibition of 7 mm against Bacillus cereus while Saccharum officinarum and Albizia lebbeck didn’t show the any antibacterial activity. On the other hand, Protein extracts from PBS buffer didn’t show zone of inhibition against any bacteria. Only Albizia lebbeck showed minute zone of inhibition against Neisseria gonorrhea. On well diffusion method, Cassia fistula Tris NaCl protein extract showed the maximum zone of inhibition 20 mm and 18 mm against Proteus mirabilis and Bacillus subtilis respectively. While Albizia lebbeck PBS protein extract showed the maximum zone of inhibition 19 mm and 17 mm against Bacillus subtilis and Bacillus cereus. The results revealed that the protein extract of Albizia lebbeck, Cymbopogon citrates and Cassia fistula can be used tosynthesize antimicrobial drugs to treat the bacterial infections.

DNA binding and cleavage studies of novel Betti base substituted quaternary Cu(II) and Zn(II) phthalocyanines

Betti base substituted zinc and copper phthalocyanines (ZnBBPc and CuBBPc) and their quaternized derivatives qZnBBPc and qCuBBPc were synthesized and characterized for the first time. The photophysical quantum yield (ΦF) and singlet oxygen quantum yield (ΦΔ) of ZnBBPc and qZnBBPc were measured using comparative and 1,3-diphenylisobenzofuran (DPBF) absorption titration methods respectively. The DNA binding activities of qZnBBPc and qCuBBPc against Calf-Thymus DNA (CT-DNA) were investigated using UV–visible absorption titrations, fluorescent displacement assays with classical intercalator ethidium bromide (EB) and minor groove binder Hoechst dye (HD), as well as by carrying out viscosity measurements in Tris-NaCl buffer (pH 7.4). The results indicated that qCuBBPc and qZnBBPc phthalocyanines showed strong affinities for CT-DNA through intercalation with binding constants (Kb) of the order of 105 M−1. The cleavage activities of compounds on supercoiled pBR322 plasmid DNA were determined by agarose gel electrophoresis in the presence and absence of light and additives. The compound qCuBBPc showed hydroxyl radical mediated oxidative cleavage, while the complex qZnBBPc exhibited singlet oxygen mediated photocleavage.

Protocols for Studying Formation and Mineralization of Dental Tissues In Vivo: Extraction Protocol for Isolating Dentin Matrix Proteins from Developing Teeth.

The organic material in developing dentin is 90% type I collagen and 10% non-collagenous proteins. The key to understanding dentin biomineralization is to study how these proteins collectively precipitate and organize hydroxyapatite crystals. The first step in characterizing the proteins within a mineralizing matrix is to efficiently extract and isolate the essential molecular participants and elucidate their structural and biochemical properties. In this study, we expanded previous approaches to develop an improved strategy for the extraction of extracellular matrix proteins from the dentin of developing teeth. Proteins in dentin powder were sequentially extracted in the order Tris-guanidine buffer, HCl-formic acid solution, acetic acid-NaCl solution, Tris-NaCl buffer, and a second Tris-guanidine buffer. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by gelatin or casein zymography, and by Western blot analysis using dentin sialoprotein (DSP)- or dentin glycoprotein (DGP)-specific antibodies. This approach was used to purify assorted porcine dentin non-collagenous proteins.

Human Placenta Laminin-111 as a Multifunctional Protein for Tissue Engineering and Regenerative Medicine.

Laminins are major components of all basement membranes surrounding nerve or vascular tissues. In particular laminin-111, the prototype of the family, facilitates a large spectrum of fundamental cellular responses in all eukaryotic cells. Laminin-111 is a biomaterial frequently used in research, however it is primarily isolated from non-human origin or produced with time-intensive recombinant techniques at low yield.Here, we describe an effective method for isolating laminin-111 from human placenta, a clinical waste material, for various tissue engineering applications. By extraction with Tris-NaCl buffer combined with non-protein-denaturation ammonium sulfate precipitation and rapid tangential flow filtration steps, we could effectively isolate native laminin-111 within only 4 days. The resulting material was biochemically characterized using a combination of dot blot, SDS-PAGE, Western blot and HPLC-based amino acid analysis. Cytocompatibility studies demonstrated that the isolated laminin-111 promotes rapid and efficient adhesion of primary Schwann cells. In addition, the bioactivity of the isolated laminin-111 was demonstrated by (a) using the material as a substrate for outgrowth of NG 108-15 neuronal cell lines and (b) promoting the formation of interconnected vascular networks by GFP-expressing human umbilical vein endothelial cells.In summary, the isolation procedure of laminin-111 as described here from human placenta tissue, fulfills many demands for various tissue engineering and regenerative medicine approaches and therefore may represent a human alternative to various classically used xenogenic standard materials.

Apatite-forming ability of vinylphosphonic acid-based copolymer in simulated body fluid: effects of phosphate group content.

Phosphate groups on materials surfaces are known to contribute to apatite formation upon exposure of the materials in simulated body fluid and improved affinity of the materials for osteoblast-like cells. Typically, polymers containing phosphate groups are organic matrices consisting of apatite-polymer composites prepared by biomimetic process using simulated body fluid. Ca(2+) incorporation into the polymer accelerates apatite formation in simulated body fluid owing because of increase in the supersaturation degree, with respect to apatite in simulated body fluid, owing to Ca(2+) release from the polymer. However, the effects of phosphate content on the Ca(2+) release and apatite-forming abilities of copolymers in simulated body fluid are rather elusive. In this study, a phosphate-containing copolymer prepared from vinylphosphonic acid, 2-hydroxyethyl methacrylate, and triethylene glycol dimethacrylate was examined. The release of Ca(2+) in Tris-NaCl buffer and simulated body fluid increased as the additive amount of vinylphosphonic acid increased. However, apatite formation was suppressed as the phosphate groups content increased despite the enhanced release of Ca(2+) from the polymer. This phenomenon was reflected by changes in the surface zeta potential. Thus, it was concluded that the apatite-forming ability of vinylphosphonic acid-2-hydroxyethyl methacrylate-triethylene glycol dimethacrylate copolymer treated with CaCl2 solution was governed by surface state rather than Ca(2+) release in simulated body fluid.

Large-Scale Purification, Characterization, and Spore Outgrowth Inhibitory Effect of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361.

Large-scale purification of the highly hydrophobic bacteriocin thurincin H was accomplished via a novel and simple two-step method: ammonia sulfate precipitation and C18 solid-phase extraction. The inhibition spectrum and stability of thurincin H as well as its antagonistic activity against Bacillus cereus F4552 spores were further characterized. In the purification method, secreted proteins contained in the supernatant of a 40 h incubated culture of B. thuringiensis SF361 were precipitated by 68 % ammonia sulfate and purified by reverse-phase chromatography, with a yield of 18.53 mg/l of pure thurincin H. Silver-stained SDS-PAGE, high-performance liquid chromatography, and liquid chromatography-mass spectrometry confirmed the high purity of the prepared sample. Thurincin H exhibited a broad antimicrobial activity against 22 tested bacterial strains among six different genera including Bacillus, Carnobacterium, Geobacillus, Enterococcus, Listeria, and Staphylococcus. There was no detectable activity against any of the selected yeast or fungi. The bacteriocin activity was stable for 30 min at 50 °C and decreased to undetectable levels within 10 min at temperatures above 80 °C. Thurincin H is also stable from pH 2-7 for at least 24 h at room temperature. Thurincin H is germicidal against B. cereus spores in brain heart infusion broth, but not in Tris-NaCl buffer. The efficient purification method enables the large-scale production of pure thurincin H. The broad inhibitory spectrum of this bacteriocin may be of interest as a potential natural biopreservative in the food industry, particularly in post-processed and ready-to-eat food.

Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins

Unlike thioredoxins, glutaredoxins are involved in iron-sulfur cluster assembly and in reduction of specific disulfides (i.e. protein-glutathione adducts), and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, was shown to be localized in chloroplasts. A comparison of the biochemical, structural, and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicated that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the x-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys(32)) is required for cluster formation. In addition, thiol titrations, fluorescence measurements, and mass spectrometry analyses showed that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter- or intramolecular disulfide bond and that its activity exclusively relies on a monothiol mechanism.

Agarose slide elisa for diagnosis of HCV-AB in rural areas (65.5)

Abstract Hepatitis C (HCV) is a viral infection of the liver affecting 170 million people around the world. Commercially available tests for HCV today are based on enzyme immunosorbent assay (EIA) for detection of HCV-Ab. Regarding the sophisticated equipments required for diagnosis of HCV Thus this work targets offering a dependable field serological test can be used in urban areas as preliminary screening for HCV patients. 150 serum samples were collected from Jazan clinics in Saudi Arabia for HCV patients and 50 serum samples were collected from healthy volunteers. All samples were tested for HCV-Ab using slides coated with 1.5% agarose saturated with HCV Capside synthetic peptide (BIORAD). 50 µl of each sample were applied in spot of Ag saturate agarose and incubated in inverted position in humidified incubator for 90 min at 37\#959;C. After which the slides were washed gently by Tris NaCl buffer PH 7.4 for 20 min. mouse anti human IgG labeled conjugate was added to the slides and incubated in inverted position in humidified incubator for 30 min at 37C. After washing the slides same as before a substrate (BIORAD) was added for 30 min at room temperature then washed out as before. the slides were examined by necked eye and by light microscope against controls. The obtained results were compared with slandered MONOLISA HCV Ag-Ab kite and both results were match. These results proving that agarose slide EIA can be used for preliminary diagnosis of HCV in field clinics of rural areas.

Transmembrane Segment 11 Appears to Line the Purine Permeation Pathway of the Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1)

Purine transport is essential for malaria parasites to grow because they lack the enzymes necessary for de novo purine biosynthesis. The Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is a member of the equilibrative nucleoside transporter (ENT) gene family. PfENT1 is a primary purine transport pathway across the P. falciparum plasma membrane because PfENT1 knock-out parasites are not viable at physiologic extracellular purine concentrations. Topology predictions and experimental data indicate that ENT family members have eleven transmembrane (TM) segments although their tertiary structure is unknown. In the current work, we showed that a naturally occurring polymorphism, F394L, in TM11 affects transport substrate K(m). We investigated the structure and function of the TM11 segment using the substituted cysteine accessibility method. We showed that mutation to Cys of two highly conserved glycine residues in a GXXXG motif significantly reduces PfENT1 protein expression levels. We speculate that the conserved TM11 GXXXG glycines may be critical for folding and/or assembly. Small, cysteine-specific methanethiosulfonate (MTS) reagents reacted with four TM11 Cys substitution mutants, L393C, I397C, T400C, and Y403C. Larger MTS reagents do not react with the more cytoplasmic positions. Hypoxanthine, a transported substrate, protected L393C, I397C, and T400C from covalent modification by the MTS reagents. Plotted on an alpha-helical wheel, Leu-393, Ile-397, and Thr-400 lie on one face of the helix in a 60 degrees arc suggesting that TM11 is largely alpha helical. We infer that they line a water-accessible surface, possibly the purine permeation pathway. These results advance our understanding of the ENT structure.

Reduced erythrocyte deformability in active and untreated Behçet’s disease patients

Although the etiology of Behcet's disease is not clarified yet, the vascular endothelial damage and thrombosis are prominent features of the disease. The underlying mechanisms of increased risk of thrombosis in Behcet's disease are not completely understood. It is stated that the changes in blood rheology such as erythrocyte deformability take part in the thrombosis. There are limited numbers of studies with conflicting results about the erythrocyte deformability in Behcet's disease. The previous studies were not limited to the untreated patients with active disease. Blood samples were filtered immediately by "Imugard- leukocyte removal filter". After plasma and erythrocytes were separated by centrifugation, 10% erythrocyte suspensions were prepared with isotonic Tris NaCl buffer solution. These erythrocyte suspensions were filtered under gravitational force by "Nucleopore" to determine the filtration time. The filtration times of the erythrocyte and buffer solutions were referred as t1 and t2 respectively. Finally, deformability indices were obtained by dividing the t1 by t2. A decrease in the deformability of the erythrocytes would result in an increase in the filtration time of the erythrocyte suspension and deformability indices. The median deformability indices were 3.090 and 2.170 for patients and control subjects respectively and the difference was statistically significant (P = 0.000). We suggest that the decrease in erythrocyte deformability in Behcet's disease may be related to oxidative damage of erythrocytes and subsequently takes part in the development of thrombosis in Behcet's disease.

27 SODIUM CHLORIDE TREATMENT OF CELL MEMBRANE-PERMEABILIZED NUCLEAR DONOR CELLS FACILITATES THE DISPLACEMENT OF THE SOMATIC HISTONE H1 AND HMG-17 PROTEINS IN RECONSTRUCTED PORCINE EMBRYOS

Developmental efficiency of somatic cell-reconstructed embryos depends on extensive remodeling of chromatin structural components. Due to their importance for maintaining the high-order chromatin structure and controlling DNA functions, including replication, transcription, repair, and recombination, histones and other chromatin-binding proteins represent leading choice markers to investigate nuclear remodeling in reconstructed embryos. The main objective of this study was to investigate whether or not the exposure of cell membrane permeabilized nuclear donor cells to sodium chloride (salt-extraction) would facilitate the displacement of chromatin-binding proteins in reconstructed porcine embryos. Both linker histone H1 (H1) and high-mobility group (HMG) proteins are known to affect gene expression through the modulation of the high-order chromatin structure. Standard methods of oocyte enucleation and electrofusion were applied for embryo reconstruction using in vitro-matured oocytes and control or salt-extracted fetal fibroblast cells. For salt-extraction, confluent cell cultures were washed with Ca2+/Mg2+-free Hank's balanced salt solution (HBSS); cells were permeabilized by incubation with 1 µg/mL of streptolysin O at 37°C for 30 min in HBSS, and then maintained in Tris-NaCl buffer (10 mM Tris-HCl, 0.5 mM MgCl2, 0.7 M NaCl, 1 M sucrose) for 5 min. After salt-extraction, cells were rinsed and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 2 mM CaCl2 for 1 h at 37.5°C for membrane resealing prior to nuclear transfer. Reconstructed embryos were activated using ionomycin (15 µM/5 min) and strontium chloride (Sr2+; 10 mM/4 h), and then cultured in PZM-3 medium. Immunostaining for H1 and HMG-17 was performed in nuclear donor cells and embryos at different stages after reconstruction. The time required for H1 displacement in transplanted nuclei was reduced by the salt-extraction treatment (Table 1). Salt-extracted cells showed a stronger HMG-17 cytoplasmic signal compared to control cells. The proportion of HMG-17-positive reconstructed embryos at 1, 3, and 6 h was 54 vs. 19, 57 vs. 44, and 75 vs. 62, for control and salt-extracted cells, respectively. These data suggest that salt-extraction prior to nuclear transplantation enhances the remodeling of chromatin structure in embryos reconstructed with somatic cell nuclei. Table 1. Proportion (n) of H1-positive stained embryos after different times from parthenogenetic activation (PA) and nuclear transfer using control (NT-control) or salt-extracted (NT-extracted) cells This work was supported by a NSERC Discovery Grant to VB.

Mono- and di- N-acetamidomethylated derivatives of bis( l-alaninato)copper(II): Characterization and nucleolytic property : Crystal structure of bis[ N, N-di-( N′-methylacetamido)- l-alaninato]copper(II)

The Mannich aminomethylation of bis( l-alaninato)copper(II), Cu( l-ala) 2, with formaldehyde and acetamide yields the bis[ N-( N′-methylacetamido)- l-alaninato]copper(II) ( 1) and bis[ N, N-di-( N′-methylacetamido)- l-alaninato]copper(II) ( 2) complexes. The crystal structure of 2 shows the amide N–H of the uncoordinated N-methylacetamido substituents and that of the coordinated N-methylacetamido substituents forming intra- and inter-molecular hydrogen bonding. Both complexes have been characterized by elemental analysis, UV–Vis spectroscopy, magnetic susceptibility measurements and EI mass spectroscopy. A gel electrophoresis experiment on the soluble complex 1 in Tris–NaCl buffer at pH 8.3, from which 2 is formed, shows oxidative cleavage of DNA by 1 in the presence of hydrogen peroxide. The cleavage efficiency of 1 is greater than that of its precursor, Cu( l-ala) 2.

1-[3-(2-[18F]Fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione: Design, Synthesis, and Radiosynthesis of a New [18F]Fluoropyridine-Based Maleimide Reagent for the Labeling of Peptides and Proteins

FPyME (1-[3-(2-fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione) was designed as a [(18)F]fluoropyridine-based maleimide reagent for the prosthetic labeling of peptides and proteins via selective conjugation with a thiol (sulfhydryl) function. Its pyridinyl moiety carries the radioactive halogen (fluorine-18) which can be efficiently incorporated via a nucleophilic heteroaromatic substitution, and its maleimido function ensures the efficient alkylation of a free thiol function as borne by cysteine residues. [(18)F]FPyME (HPLC-purified) was prepared in 17-20% non-decay-corrected yield, based on starting [(18)F]fluoride, in 110 min using a three-step radiochemical pathway. The developed procedure involves (1) a high-yield nucleophilic heteroaromatic ortho-radiofluorination on [3-(3-tert-butoxycarbonylaminopropoxy)pyridin-2-yl]trimethylammonium trifluoromethanesulfonate as the fluorine-18 incorporation step, followed by (2) rapid and quantitative TFA-induced removal of the N-Boc-protective group and (3) optimized maleimide formation using N-methoxycarbonylmaleimide. Typically, 4.8-6.7 GBq (130-180 mCi) of radiochemically pure [(18)F]FPyME ([(18)F]-1) could be obtained after semipreparative HPLC in 110 min starting from a cyclotron production batch of 33.3 GBq (900 mCi) of [(18)F]fluoride (overall radiochemical yields, based on starting [(18)F]fluoride: 28-37% decay-corrected). [(18)F]FPyME ([(18)F]-1) was first conjugated with a small model hexapeptide ((N-Ac)KAAAAC), confirming the excellent chemoselectivity of the coupling reaction (CH(2)SH versus CH(2)NH(2)) and then conjugated with two 8-kDa proteins of interest, currently being developed as tumor imaging agents (c-AFIM-0 and c-STxB). Conjugation was achieved in high yields (60-70%, isolated and non-decay-corrected) and used optimized, short-time reaction conditions (a 1/9 (v/v) mixture of DMSO and 0.05 M aq Tris NaCl buffer (pH 7.4) or 0.1 M aq PBS (pH 8), at room temperature for 10 min) and purification conditions (a gel filtration using a Sephadex NAP-10 cartridge or a SuperDex Peptide HR 10/30 column), both compatible with the chemical stability of the proteins and the relatively short half-life of the radioisotope concerned. The whole radiosynthetic procedure, including the preparation of the fluorine-18-labeled reagent, the conjugation with the protein and the final purification took 130-140 min. [(18)F]FPyME ([(18)F]-1) represents a new, valuable, thiol-selective, fluorine-18-labeled reagent for the prosthetic labeling with fluorine-18 of peptides and proteins. Because of its excellent chemoselectivity, [(18)F]FPyME offers an interesting alternative to the use of the nonselective carboxylate and amine-reactive [(18)F]reagents and can therefore advantageously be used for the design and development of new peptide- and protein-based radiopharmaceuticals for PET.

17 FLOW CYTOMETRY ANALYSIS OF THE TURKEY SPERM GLYCOCALYX: EFFECTS OF NEURAMINIDASE TREATMENT

The surface of all eucaryotic cells consists of a carbohydrate-rich zone known as the glycocalyx which mediates a variety of specific cell-recognition processes, including sperm-egg interactions. Several carbohydrate residues are known to be necessary for poultry sperm to traverse the female reproductive tract and for sperm-egg recognition. Alteration of the sperm glycocalyx during semen storage could adversely impact fertility. Accordingly, the aim of this work was to elucidate the sugar residues existing in the surface of turkey spermatozoa as a first step in understanding the deleterious effects of semen storage on sperm physiology and function. Nine turkey toms were randomly assigned to 1 of 3 groups (3 males/group) and semen was pooled for each group. Seminal plasma was removed and isolated sperm were resuspended in Tris-NaCl buffer at either (1) pH 7.4 for immediate staining with 1 of 16 FITC-conjugated lectins (100 �g/mL; 30 min; 25�C; 100 � 106 cells/mL), or (2) pH 6.0 for neuraminidase treatment (30 min; 37�C; 1 IU/109 cells), followed by washing (700 g, 5 min; 4 times) and lectin staining. The control consisted of sperm cells incubated only in Tris-NaCl buffer. Stained and control samples were washed twice (700 g, 5 min) and counterstained with 12 �M propidium iodide (PI); the green fluorescence intensity of PI-negative cells was assessed using a Coulter EPICS XL-MCL" Flow Cytometer (Beckman Coulter, Inc., Fullerton, CA, USA) and single-parameter histogram analysis (6 replicates). Median fluorescence intensity (MdFI) was used to assess differences in the presence of sugar residues among the control, non-neuraminidase-treated, and neuraminidase-treated samples (Kruskal-Wallis test; Tukey's HSD test in post-hoc comparisons). Lectin staining occurred in the PI-positive subpopulation, or membrane-disrupted cells, of all samples. By contrast, only intact cells were labeled by LPA, ConA, RCA-I, and s-WGA lectins in the non-neuraminidase-treated samples, indicating that only residues of sialic acid, �-mannose/�-glucose, �-galactose, and N-acetylglucosamine oligomers were on the surface. As expected, removal of sialic acid significantly (P < 0.05) decreased the fluorescence intensity of LPA-labeled cells. This treatment significantly (P < 0.05) increased the affinity of ConA, RCA-I, and s-WGA lectins, and exposed sugar residues previously undetected using ECA (N-acetyllactosamine), GNA (�-mannose), GS-I (�-galactose), GS-II (N-acetylglucosamine monomers), LOTUS (�-l-fucose), PNA (�-galactose), SBA (�-N-acetylgalactosamine), and WFA (�-N-acetylgalactosamine) lectins. Low levels of MdFI were observed for cells labeled by JAC (�-galactose), PSA (�-mannose/�-glucose), STA (N-acetylglucosamine polymers) and UEA-I (�-l-fucose) irrespective of neuraminidase treatment (P > 0.05). These observations suggest that (1) the carbohydrate content of turkey sperm glycocalyx is extensively masked by sialic acid molecules, (2) it contains a diversity of sugar residues, and (3) discrimination between intact and damaged cells needs to be done in order to properly identify surface binding.

Distribution of copper in the diatom Haslea ostrearia simonsen

Haslea ostrearia is a pennate diatom important for oysters culture. This species was selected to study experimentally the distribution of copper in algal cells since the phytoplankton exhibits a high capacity for metal accumulation and thus influences metal transfer to consumers. This alga was grown in batch culture and exposed to copper (30 μg/l) added in the medium on the first day of experiment. In order to separate the soluble and insoluble fractions binding copper algal cells were centrifuged after homogenization with Tris NaCl buffer (pH 8.6). A part of the cytosol was heat-denatured and then centrifuged again to separate heat-stable and heat denaturable compounds. Gel filtration Sephadex (G-75SF:10 3−7 × 10 4 Da) was used to determine the molecular weight of soluble compounds binding copper. In controls, insoluble compounds were preponderant (63%) versus soluble compounds whereas in contamined cells, soluble compounds became preponderant (66%). In the cytosol of both controls and contamined cells, copper was mainly associated with thermoresistant compounds (about 80%). In contamined cells, gel filtration have shown that copper binding compounds had low molecular weights less than 3 KDa associated with sulfhydrl groups represented 65% of total cytosolic copper and few minority (8%) compounds binding copper between 3 and 9 KDa. In conclusion, contamination of Haslea ostrearia by copper induces an increase of copper-binding to cytosolic, heat stable, thiol-rich compounds of low molecular weight with characteristics of phytochelatins.

Rapid lysis of gram-positive cocci for pulsed-field gel electrophoresis using achromopeptidase.

To adapt pulsed-field gel electrophoresis (PFGE) to a busy clinical laboratory, we evaluated the use of achromopeptidase (ACP) as the sole agent for lysis of a variety of gram-positive cocci. Growth in an appropriate broth media was adjusted to a 3.0 McFarland density before aliquotting for washing and plug preparation. ACP incorporated into the agarose plugs, or added to the buffer solution, lysed cells in < 1 h when suspended in a Tris-NaCl buffer. Two 30-min washes in a Tris-EDTA buffer were sufficient to remove cellular debris without additional deproteination. Final PFGE results were obtained in approximately 48 h, including the initial broth subculturing.

Cobinding of bilirubin and laurate to human serum albumin: Spectroscopic characterization of stoichiometric complexes

Light absorption and CD spectra of bound bilirubin and albumin fluorescence spectra have been recorded from mixtures containing albumin, A, bilirubin, B, and laurate, L, in Tris-NaCl buffer at pH 8.2, 25 °C. Concentrations of the corresponding stoichiometric complexes, AB i L i , for i = 0 3 and j = 0 3 , have been calculated from previously determined stoichiometric cobinding constants (H. Sato et al. (1988) Arch. Biochem. Biophys. 260, 811–821). Spectral data of the complexes have finally been found by iterative computer fitting using the principle of several acceptable solutions (R. Brodersen et al. (1987) Eur. J. Biochem. 169, 487–495). The results were utilized at the microscopic level to investigate ligand-induced conformational changes. When laurate was bound to AB, a decrease of the distance between Trp-214 and the bound bilirubin occurred, as measured according to Förster's principle. The distances were 21.9 ± 0.3 Å in AB, 19.7 ± 0.3 Å in ABL, and 17.9 ± 0.2 Å in ABL 2.