The immunogenicity of p24 protein from HIV-1 virus is strongly supported and modulated by coupling with liposomes and mannan.

Anti-viral and anti-tumor vaccines aim to induce cytotoxic CD8+ T cells (CTL) and antibodies. Conserved protein antigens, such as p24 from human immunodeficiency virus, represent promising component for elicitation CTLs, nevertheless with suboptimal immunogenicity, if formulated as recombinant protein. To enhance immunogenicity and CTL response, recombinant proteins may be targeted to dendritic cells (DC) for cross presentation on MHCI, where mannose receptor and/or other lectin receptors could play an important role. Here, we constructed liposomal carrier-based vaccine composed of recombinant p24 antigen bound by metallochelating linkage onto surface of nanoliposomes with surface mannans coupled by aminooxy ligation. Generated mannosylated proteonanoliposomes were analyzed by dynamic light scattering, isothermal titration, and electron microscopy. Using murine DC line MutuDC and murine bone marrow derived DC (BMDC) we evaluated their immunogenicity and immunomodulatory activity. We show that p24 mannosylated proteonanoliposomes activate DC for enhanced MHCI, MHCII and CD40, CD80, and CD86 surface expression both on MutuDC and BMDC. p24 mannosylated liposomes were internalized by MutuDC with p24 intracellular localization within 1 to 3 h. The combination of metallochelating and aminooxy ligation could be used simultaneously to generate nanoliposomal adjuvanted recombinant protein-based vaccines versatile for combination of recombinant antigens relevant for antibody and CTL elicitation.

Imaging of transfection and intracellular release of intact, non-labeled DNA using fluorescent nanodiamonds.

Efficient delivery of stabilized nucleic acids (NAs) into cells and release of the NA payload are crucial points in the transfection process. Here we report on the fabrication of a nanoscopic cellular delivery carrier that is additionally combined with a label-free intracellular sensor device, based on biocompatible fluorescent nanodiamond particles. The sensing function is engineered into nanodiamonds by using nitrogen-vacancy color centers, providing stable non-blinking luminescence. The device is used for monitoring NA transfection and the payload release in cells. The unpacking of NAs from a poly(ethyleneimine)-terminated nanodiamond surface is monitored using the color shift of nitrogen-vacancy centers in the diamond, which serve as a nanoscopic electric charge sensor. The proposed device innovates the strategies for NA imaging and delivery, by providing detection of the intracellular release of non-labeled NAs without affecting cellular processing of the NAs. Our system highlights the potential of nanodiamonds to act not merely as labels but also as non-toxic and non-photobleachable fluorescent biosensors reporting complex molecular events.

Charge-sensitive fluorescent nanosensors created from nanodiamonds.

We show that fluorescent nanodiamonds (FNDs) are among the few types of nanosensors that enable direct optical reading of noncovalent molecular events. The unique sensing mechanism is based on switching between the negatively charged and neutral states of NV centers which is induced by the interaction of the FND surface with charged molecules.

The thin electrolyte layer approach to corrosion testing of dental materials--characterization of the technique.

An innovative technique for corrosion testing of metallic dental materials is introduced. The thin electrolyte layer technique (TET) simulates the physical characteristics of the oral environment by employing a still, thin layer of an electrolyte, in contrast to bulk electrolyte techniques (BET) which utilize relatively large quantities of fluid. Limiting current density tests on a platinum electrode revealed a lower surface oxygen content for TET. Borate buffer (pH 6.8) was employed as an electrolyte. The effect of lower oxygen content in TET on passivation and polarization characteristics of 316L SS in 0.9% saline was investigated. The results revealed differences in the polarization resistance and open circuit potential development with time, as well as in anodic and cathodic polarization behavior. Lower O2 concentration in TET was attributed to different electrolyte convection characteristics under both testing conditions. Additionally, use of the TET resulted in better data reproducibility. Overall, this investigation led to a deeper understanding of the electrochemical processes inherent in thin electrolytes such as those found in the oral environment.

Sarcomere length and capillary curvature of rat hindlimb muscles in vivo.

Mammalian skeletal muscle fibers have been reported to develop maximum force at a sarcomere length (Ls) of approximately 2.5 microns. However, the functional range of muscle length (Lm) and Ls encountered by skeletal muscle in vivo is not well defined. Changes in Ls markedly influence capillary geometry, but this effect has been shown only in fixed preparations. The purpose of this study was to evaluate the influence of limb position on Lm, Ls, and capillary geometry in living undisturbed hindlimb muscles. We tested the hypothesis that maximal excursion of the foot would have similar effects on Ls and capillary geometry of antagonistic soleus (Sol) and extensor digitorum longus (EDL) muscles in vivo. Female Sprague-Dawley rats (n = 9; 243 +/- 3 g) were anesthetized (pentobarbital sodium; 35 mg/kg). The right Sol and EDL muscles were exposed and irrigated with physiological saline solution (34 degrees C; pH 7.4). Sarcomeres and capillaries were observed with video microscopy (total magnification x 1,900; spatial resolution < 1 micron); sarcomeres were labeled with a fluorescent dye [4-(4-diethylaminostyryl)-N-methylpyridinium iodide]. As foot angle increased from 30 degrees (maximal dorsiflexion) to 170 degrees (maximal plantarflexion), Lm and Ls increased for EDL muscles (27.51 +/- 0.42 to 30.97 +/- 0.25 mm and 2.33 +/- 0.01 to 3.09 +/- 0.05 microns, respectively; P < 0.05) and decreased for Sol muscles (26.09 +/- 0.38 to 20.27 +/- 0.34 mm and 3.17 +/- 0.03 to 2.22 +/- 0.04 microns, respectively; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of O-[2-acetamido-2-deoxy-6-O-stearoyl- and -6-O-(2-tetradecylhexadecanoyl)-beta-D-glucopyranosyl]-(1-->4)-N- acetylnormuramoyl-L-alpha-aminobutanoyl-D-isoglutamine, lipophilic disaccharide analogues of MDP.

Silver triflate-promoted condensation of 3,4,6-tri-O-acetyl-2-deoxy-2- phthalimido-beta-D-glucopyranosyl bromide (1) with benzyl 2-acetamido-6-O-benzyl-2-deoxy-3-O- (methoxycarbonyl)methyl-alpha-D-glucopyranoside (4) afforded the key compound, benzyl 2-acetamido-6-O-benzyl-2-deoxy-3-O- (methoxy-carbonyl)methyl-4-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b eta-D- glucopyranosyl)-alpha-D-glucopyranoside (5), which after deprotection was transformed into acid 10. Condensation of 10 with the benzyl ester of L-alpha-aminobutanoyl-D-isoglutamine and deisopropylidenation of the product 11 afforded the benzyl ester of N-(2-O-[benzyl 2-acetamido-4-O-(2-acetamido-3-O-benzyloxymethyl-2- deoxy-beta-D-glucopyranosyl)-6-O-benzyl-2,3-dideoxy-alpha-D-glucopyra nosid-3- yl]glycoloyl)-L-alpha-aminobutanoyl-D-isoglutamine (12). Partial O-acylation of 12 and hydrogenolysis of protecting groups gave the 6-O-stearoyl- and 6-O-(2-tetradecylhexadecanoyl)-disaccharide-dipeptides 17 and 18, respectively. Pyrogenicity and adjuvant activity in cell-mediated immunity are reported.

Affinity chromatography of glycosidases.

The authors studied the separation of glucoamylase and beta-galactosidases from the interstinal mucosa of young rats by affinity chromatography. They tested the following chromatographic materials: p-aminophenyl-beta-D-thioglucoside bound to Sepharose 4B via hexamethylenediamine, gluconate and galactonate bound in different ways to Sepharose 4B and phlorizin bound by an azo-coupling reaction to a spacer attached to Sepharose 4B. The conditions of the adsorption of glycosidases to these materials and their subsequent elution were studied. Using chromatography on Sepharose 4B with a beta-thioglucoside affinant, we succeeded in purifying lactase preparation so that, in electrophoresis on polyacrylamide gel, it formed a single zone identical with 1-naphthyl-beta-glucosidase activity.

Stimulation of innate immunity in newborn kids against Cryptosporidium parvum infection-challenge by intranasal/per-oral administration of liposomal formulation of N-L18-norAbu-GMDP adjuvant.

The effects of a liposomal preparation of lipophilic immunomodulator beta-D-GlcNstearoyl-(1-4)-norMurNAc-L-Abu-D-isoGln (N-L18-norAbu-GMDP) were investigated on resistance to Cryptosporidium parvum infection in neonatal kids. The liposomal preparation was administered subcutaneously or intranasally/orally (i.n./p.o.) twice at doses of 100 microg, 200 microg, or 1000 microg per kid pre-infection challenge. The treatment schemes were (i) 72 and 24 h pre-infection challenge, (ii) 24 h pre-infection challenge and 24 h post-infection challenge (oral inoculation with 1 x 10(7) oocysts of C. parvum in 5 ml of PBS). Administration of liposomal N-L18-norAbu-GMDP by i.n./p.o. route at the cumulative dose of 2000 microg per kid 72 and 24 h pre-infection challenge, lead to substantially increased clearance of coccidian parasites from various parts of the intestine. On the basis of histological examination, the distribution of cryptosporidia in the intestine and the severity of the infection, treated kids were classified on day 5 as having a strong reduction in infection in comparison to the control group (P < 0.05). No cryptosporidia were found on the mucosal surface of treated kids by day 10, while the intestines of the control kids were still infected. All doses and routes of administration were judged effective with respect to suppression of cryptosporidia infections.

Preparation and isolation of dityrosine.

A new procedure for the isolation of dityrosine has been developed. Tyrosine was oxidized by means of incubation both with hydrogen peroxide and horse-radish peroxidase. The reaction mixture was separated by permeation chromatography on Sephadex G-10 being monitored at 280 and 310 nm spectrophotometrically. The dityrosine fraction was freeze-dried and purified on a cation-exchange column (in acidic citrate buffer). The purified fraction was desalted and freeze-dried. The yield was 96 mg of homogenous dityrosine per 1 g of D,L-tyrosine. Some physico-chemical constants of the preparation were measured (optical characteristics with U.V. and I.R. spectra, fluorescence spectra, chromatography on an amino acid analyzer).

Differences in elastase hydrolysates of elastin from ligamentum nuchae.

By means of molecular exclusive chromatography, a marked difference in the distribution of desmosine and isodesmosine in the products of proteolysis has been found between the partial enzymatic hydrolysates of elastin from the bovine nuchal ligament prepared by two different methods. In the preparation which was treated with hot NaOH the prevailing portion was localized in a macromolecular fraction corresponding to the void volume. An increased precipitation of this fraction with trichloracetic acid has been noticed. The autoclaved material forms substantial amounts of the coacervate during the digestion.

The content of dityrosine in chick and rabbit aorta proteins.

The possible presence of dityrosine in elastin derived by two different methods and in structural glycoproteins from aortas of 1 day old chicks, adult rabbits and fetal rabbits was determined by a sensitive spectrofluorimetric procedure. Only chick tissues were found to contain dityrosine, 0.3 residues/100,000 total amino acid residues in aortic elastin and 12-15 residues/100,000 residues in the structural glycoproteins. No dityrosine could be detected in any of the fetal or mature rabbit tissues. However, related fluorescent compounds with different excitation-emission maxima and different elution times were obtained by ion exchange chromatography of structural glycoproteins partially hydrolyzed under alkaline conditions.