Investigations into self-association of vancomycin covalent dimers using surface plasmon resonance technology.

Covalent dimers of vancomycin linked through the vancosamine sugar moieties of the glycopeptide antibiotic have been synthesized in one step in 67-69% yield. The propensity for self-association of these and related vancomycin covalent dimers is evaluated using surface plasmon resonance technology.     

Uptake kinetics of 99Tc in common duckweed

The uptake of the nuclear waste product technetium-99 was studied in common duckweed (Lemna minor). In addition to measurements, a model involving two compartments in duckweed with different chemical forms of technetium was derived. The model was tested by chemical speciation, i.e. differentiating between reduced Tc-compounds and Tc(VII)O(4)(-). The TcO(4)(-) concentrations measured were in good agreement with those predicted by the model. Two processes determine technetium uptake: (1) transport of Tc(VII)O(4)(-) across the cell membrane, and (2) reduction of Tc(VII). The TcO(4)(-) concentration in duckweed reaches a steady state within 2 h while reduced Tc-compounds are stored, as a result of absence of release or re-oxidation processes. Bioaccumulation kinetic properties were derived by varying 99Tc concentration, temperature, nutrient concentrations, and light intensity. The reduction of technetium in duckweed was highly correlated with light intensity and temperature. At 25 degrees C the maximum reduction rate was observed at light intensities above 200 μmol m(-2) s(-1) while half of the maximum transformation rate was reached at 41 μmol m(-2) s(-1). Transport of TcO(4)(-) over the cell membrane requires about 9.4 kJ mol(-1), indicating an active transport mechanism. However, this mechanism behaved as first-order kinetics instead of Michaelis-Menten kinetics between 1x10(-14) and 2.5x10(-5) mol l(-1) TcO(4)(-). Tc uptake could not be inhibited by 10(-3) mol l(-1) nitrate, phosphate, sulphate or chloride.     

Collagen cross-links. Synthesis of immunoreagents for development of assays for deoxypyridinoline, a marker for diagnosis of osteoporosis

(+)-Deoxypyridinoline (Dpd, 2) is a cross-link of bone collagen, which is released and excreted in urine during process of bone resorption. It has been shown that this bone collagen degradation product, Dpd (2) is a useful marker for diagnosis of osteoporosis and other metabolic bone diseases. In this paper, the design and synthesis of two immunogens (3, 4) via of conjugation of succinimidyl ester (14) to carrier protein, bovine serum albumin, or keyhole limphet hemocyanin, was presented. Additionally, fluorescent (5) and chemiluminescent (6) tracers were prepared from (-)-acid (13) via in situ activation and subsequent reaction with 6-Fln-CH(2)NH(2) (17) or Acr-NH(2) (18) and hydrolysis. The key hapten (-)-acid (13) was prepared by quaternization of 3-hydroxypyridine derivative (S,S)-(-)-11 with iodide (S)-(-)-10 followed by selective hydrolysis. These immunreagents (immunogens 3 and 4 and tracers 5 and 6) are useful for the development of sensitive and high throughput immunoassays, such as FPIA and CLIA for Dpd (2).     

Region-selective labeling of antibodies as determined by electrospray ionization-mass spectrometry (ESI-MS)

The electrospray ionization-mass spectrometry (ESI-MS) analysis of three sets of monoclonal antibody-acridinium-9-carboxamide conjugates is described. The conjugates (nine total) were enzymatically digested using papain and the resulting fragments [Fc heavy chain, Fab, or F(ab')(2)] were analyzed using liquid chromatography/ESI-MS. The average number of labels per fragment were calculated using Sigma nx%, where n is the number of acridinium molecules covalently bound to the fragment and x% is the percent relative area of the corresponding peaks in the mass spectrum. When these values were normalized against the molecular weight of their respective region, antibody-dependent labeling patterns were observed. For antibodies T (anti-L-T(4)) and F (anti-FITC), there was a preference for conjugation of the Fab region over the Fc region. For antibody B (anti-biotin), the trend was reversed.     

A chemoselective method for site-specific immobilization of peptides via aminooxy group

Site-specific modification of peptides and proteins is an important area of basic research for preparation of well-defined biosensors and probes. The unique properties of aminooxy group present an opportunity for chemoselective site-specific immobilization of peptides to prepare well-defined biosensors. We have prepared FLAG peptide derivatives containing L-epsilon-aminooxylysine (L-epsilon-AOLys, 1a) and L-lysine units in their sequence at the C- and N-terminals via solid-phase synthesis. Site-specific modification of peptides through aminooxy group was demonstrated in the preparation of biosensors and selective conjugation in the preparation of biotinylated probes. Effect of the incorporation of L-epsilon-AOLys (1a) into the peptide sequence and its subsequent labeling on the FLAG epitopic character was measured using a surface plasmon resonance detector. It was found that incorporation of L-epsilon-AOLys (1a) into the FLAG peptide and site-specific immobilization through aminooxy group preserved the integrity of FLAG epitope.     

Correlating differences in larval survival and development of bollworm (Lepidoptera: Noctuidae) and fall armyworm (Lepidoptera: Noctuidae) to differential expression of Cry1A(c) delta-endotoxin in various plant parts among commercial cultivars of transgenic Bacillus thuringiensis cotton

Differences in larval survival and development of bollworm, Helicoverpa zea (Boddie), and fall armyworm, Spodoptera frugiperda (J. E. Smith), respectively, were found to exist among commercially available Cry1A(c) transgenic Bacillus thuringiensis Berliner (Bt) varieties. Using a quantification assay (ELISA) to measure the levels of delta-endotoxin in two of these varieties ('DP 451B/RR' and 'NuCOTN 33B'), differences in the amount of delta-endotoxin present in various plant parts was correlated with larval survival of bollworms and larval development of fall armyworms throughout the growing season. Larvae that were fed on DP 451B/RR completed development faster and exhibited better survivorship than those larvae fed NuCOTN 33B, whereas lower levels of delta-endotoxin were generally detected in plant parts from DP 451B/RR compared with NuCOTN 33B. These differences may impact population dynamics of these pests which may be a critical factor in managing resistance to Bt. Furthermore, the utility of using this system for providing information to the grower concerning varietal choices may be more common in the future.     

Functional studies on sciatic nerve blood flow in respect to its vascular supply and tonic neural activity.

An introduction of laser flow meters for a continuous measurements of a tissue blood flow has opened new avenues for an accurate assessment blood flow in peripheral nerves. The aim of our study was: 1) to carry out a functional verification of anatomical sources of a sciatic nerve blood supply in the rat; 2) develop a measurement technique to facilitate standardisation of results; 3) to determine the role of nerve fibres tonic activity in the maintenance of a resting blood flow in the sciatic nerve. Based on results of the present study the following conclusions have been drawn out: 1) in order to obtain a real values of the blood flow through the sciatic nerve it is necessary to remove its muscular fascia; 2) an uninjured epineurium plays a crucial role in maintaining the resting blood flow; 3) major blood supply of sciatic nerve comes from inferior gluteal and popliteal arteries; 4) the tonic neural activity plays a role in the maintenance of the resting sciatic nerve blood flow in anaesthetised rats.