Specific Conjugation Research Articles

Mechanism of RNA Cleavage Catalyzed by Sequence Specific Polyamide Nucleic Acid-Neamine Conjugate

In earlier studies, we found that a conjugate of neamine-polyamide nucleic acid targeting transactivation response element of HIV-1 RNA genome (HIV-1 TAR) displayed anti-HIV-1 activity and sequence-specific cleavage of the target RNA in vitro. Here we show that both the position of conjugation of polyamide nucleic acid (PNA) on neamine and the length of the spacer are critical parameters for conferring cleavage activity to the conjugate. The conjugation of PNA via a spacer incorporating 11 atoms to the 5-position of ring I of the neamine core conferred sequence-specific RNA cleavage activity on the conjugate, while conjugation to the 4'-position of ring II abolished this activity. Similarly, 5-neamine PNA complementary to TAR sequence of HIV-1 genome (PNA(TAR)) conjugates having either a 23-atom spacer or a bulky dansyl group between PNA and the neamine core also resulted in complete loss of cleavage activity. Based on these observations, we propose a mechanism for the observed RNA cleavage catalyzed by the conjugate involving unprotonated and protonated amino groups at the 3-position of ring I and the 6'-position of ring II of the neamine core, respectively.

A mixed collocation–finite difference method for 3D microscopic heat transport problems

Three-dimensional time-dependent initial-boundary value problems of a novel microscopic heat equation are solved by the mixed collocation–finite difference method in and on the boundaries of a particle when the thickness is much smaller than both the length and width. The collocation method on fixed grid size is used to approximate the space operator, whereas the finite difference scheme is used for time discretization. This new mixed method is applied to a novel heat problem in a particle, in order to compute the temperature distribution in and on the particle's surface. The second derivatives of the basis functions for the spectral approximation are derived. Direct substitution of derivatives in the model transforms the differential equation into a linear system of equations that is solved by the specific preconditioned conjugate gradient method. The high-order accuracy and resolution achieved by the proposed method allows one to obtain engineering-accuracy solution on coarse meshes. The consistency, stability and convergence analysis are provided and numerical results are presented.

Correction for Chowers et al., Specific polysaccharide conjugate vaccine-induced IgG antibodies prevent invasion of Shigella into Caco-2 cells and may be curative

Correction for Chowers et al., Specific polysaccharide conjugate vaccine-induced IgG antibodies prevent invasion of <i>Shigella</i> into Caco-2 cells and may be curative

Cochrane review: Conjugate vaccines for preventing meningococcal C meningitis and septicaemia

AbstractBackgroundMeningococcal polysaccharide (MPLS) vaccines protect against Serogroup C disease, but do not produce an immune response in infants less than two years of age. This limitation can be overcome by linking C polysaccharide to carrier proteins (‘conjugating’), to create meningococcal serogroup C conjugate (MCC) vaccines. In the absence of trial data, the immune response to vaccination has been considered to be a reasonable surrogate for vaccine protection.ObjectivesTo assess the immunogenicity, safety and efficacy of MCC vaccines for preventing meningitis and septicaemia.Search strategyWe searched the Cochrane Central Register Controlled Trials (CENTRAL) (The Cochrane Library Issue 3, 2005); MEDLINE (1966 to September, Week 1 2005); and EMBASE (1990 to June 2005) and references of studies.Selection criteriaRandomised controlled trials (RCTs) and controlled clinical trials (CCTs) in humans comparing MCC vaccines against a control vaccine or none. In the absence of any trials on vaccine efficacy, population‐based observational studies about effectiveness were included.Data collection and analysisTwo authors independently screened the results of the literature searches, selected eligible studies, extracted the data and evaluated the quality of them.Main resultsThe studies showed that MCC vaccine was highly immunogenic in infants after two and three doses, in toddlers after one and two doses and in older age groups after one dose. In general higher titres were generated after MCC than after MPLS vaccines. Immunological hypo‐responsiveness seen after repeated doses of MPLS vaccine may be overcome with MCC. Observational studies have documented a significant decline in meningococcal C disease in countries where MCC vaccines have been widely used. The timing of the vaccinations schedules, the specific conjugate used, and the vaccines given concomitantly or combined, may be important.Authors' conclusionsThe MCC vaccine appears to be safe, immunogenic and able to induce immunological memory in all age groups. Observational studies strongly suggest that MCC is clinically effective.Plain language summaryTraditional meningococcal polysaccharide (MPLS) vaccines protect against Serogroup C disease (caused by Neisseria meningitidis), but do not produce an immune response in infants less than two years of ageThis limitation can be overcome by linking C polysaccharide to carrier proteins (‘conjugating’), to create meningococcal serogroup C conjugate (MCC) vaccines. This review looks at MCC vaccines to protect young children against Serogroup C disease. Trials found that MCC induces an immune response against serogroup C in all age groups, but especially younger children for whom the immune response is greater than that of the traditional MPLS. It appears to be safe. Clinical efficacy of MCC could not be assessed from randomised controlled clinical trials (there were none). However, weaker evidence from observational studies showed a decrease in meningococcal C disease and meningococcal C carriage in countries where vaccines have been widely used.

Herd prevalence and risk factors of Leptospira infection in beef cow/calf operations in the United States

The herd prevalence of Leptospira borgpetersenii serovar hardjo infection and associated risk factors were determined for beef cow/calf operations in California, Florida, Mississippi, Missouri, South Dakota and Texas, USA. Three veterinary practitioners randomly selected in each state completed a questionnaire on herd management practices, and collected blood and urine from 10 to 15 cows in four randomly selected herds. Serum samples were tested for antibodies against Leptospira borgpetersenii serovars grippotyphosa, hardjo, icterohaemorrhagiae and pomona using the microscopic agglutination test. Urine sediments were stained with a non-serovar specific fluorescein-labeled anti-Leptospira antibody conjugate. Herd infection with L. borgpetersenii serovar hardjo was inferred if one or more cows had leptospires in their urine accompanied by either high antibody titres to the organism, or low titres to it and low titres against L. borgpetersenii serovars grippotyphosa, icterohaemorrhagiae and pomona. 28 (42%) of the 67 herds had results compatible with infection with L. borgpetersenii serovar hardjo. Herds in South Dakota had a significantly lower prevalence than herds in other states (P=0.01). A greater likelihood of infection with L. borgpetersenii serovar hardjo was found in herds with higher mean annual temperatures (P=0.02) and longer breeding seasons (P=0.02). It is recommended that altering risk factors that predispose herds to infection, such as shortening length of breeding season will increase the effectiveness of programmes to control this disease.

Active Antiviral T-Lymphocyte Response Can Be Redirected against Tumor Cells by Antitumor Antibody × MHC/Viral Peptide Conjugates

To redirect an ongoing antiviral T-cell response against tumor cells in vivo, we evaluated conjugates consisting of antitumor antibody fragments coupled to class I MHC molecules loaded with immunodominant viral peptides. First, lymphochoriomeningitis virus (LCMV)-infected C57BL/6 mice were s.c. grafted on the right flank with carcinoembryonic antigen (CEA)-transfected MC38 colon carcinoma cells precoated with anti-CEA x H-2D(b)/GP33 LCMV peptide conjugate and on the left flank with the same cells precoated with control anti-CEA F(ab')(2) fragments. Second, influenza virus-infected mice were injected i.v., to induce lung metastases, with HER2-transfected B16F10 cells, coated with either anti-HER2 x H-2D(b)/NP366 influenza peptide conjugates, or anti-HER2 F(ab')(2) fragments alone, or intact anti-HER2 monoclonal antibody. Third, systemic injections of anti-CEA x H-2D(b) conjugates with covalently cross-linked GP33 peptides were tested for the growth inhibition of MC38-CEA(+) cells, s.c. grafted in LCMV-infected mice. In the LCMV-infected mice, five of the six grafts with conjugate-precoated MC38-CEA(+) cells did not develop into tumors, whereas all grafts with F(ab')(2)-precoated MC38-CEA(+) cells did so (P = 0.0022). In influenza virus-infected mice, the group injected with cells precoated with specific conjugate had seven times less lung metastases than control groups (P = 0.0022 and P = 0.013). Most importantly, systemic injection in LCMV-infected mice of anti-CEA x H-2D(b)/cross-linked GP33 conjugates completely abolished tumor growth in four of five mice, whereas the same tumor grew in all five control mice (P = 0.016). The results show that a physiologic T-cell antiviral response in immunocompetent mice can be redirected against tumor cells by the use of antitumor antibody x MHC/viral peptide conjugates.

Site-directed photoproteolysis of 8-oxoguanine DNA glycosylase 1 (OGG1) by specific porphyrin-protein probe conjugates: A strategy to improve the effectiveness of photodynamic therapy for cancer

The specific light-induced, non-enzymatic photolysis of mOGG1 by porphyrin-conjugated or rose bengal-conjugated streptavidin and porphyrin-conjugated or rose bengal-conjugated first specific or secondary anti-IgG antibodies is reported. The porphyrin chlorin e6 and rose bengal were conjugated to either streptavidin, rabbit anti-mOGG1 primary specific antibody fractions or goat anti-rabbit IgG secondary antibody fractions. Under our experimental conditions, visible light of wavelengths greater than 600 nm induced the non-enzymatic degradation of mOGG1 when this DNA repair enzyme either directly formed a complex with chlorin e6-conjugated anti-mOGG1 primary specific antibodies or indirectly formed complexes with either streptavidin-chlorin e6 conjugates and biotinylated first specific anti-mOGG1 antibodies or first specific anti-mOGG1 antibodies and chlorin e6-conjugated anti-rabbit IgG secondary antibodies. Similar results were obtained when rose bengal was used as photosensitizer instead of chlorin e6. The rate of the photochemical reaction of mOGG1 site-directed by all three chlorin e6 antibody complexes was not affected by the presence of the singlet oxygen scavenger sodium azide. Site-directed photoactivatable probes having the capacity to generate reactive oxygen species (ROS) while destroying the DNA repair system in malignant cells and tumors may represent a powerful strategy to boost selectivity, penetration and efficacy of current photodynamic (PDT) therapy methodologies.

Electrogenerated indium tin oxide-coated glass surface with photosensitive interfaces: Surface analysis

We present herein a photo-immobilization technique for the localized and specific conjugation of biochip platforms with different proteinaceous bioreceptors, such as antigen or antibodies. This methodology based on a photoactivable electrogenerated polymer film, pyrrole-benzophenone, allows the covalent immobilization of biomolecules through light mediation. The surface-conductive glass platform electropolymerized with poly(pyrrole-benzophenone) thin film may then be used to affinity-coat the chip with molecular recognition probes. This glass chip electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide (ITO). Thereafter, pyrrole-benzophenone monomers are electropolymerized onto the conductive metal oxide surface and then exposed to an antigen Staphylococcal Enterotoxin B (SEB)) solution and illuminated with UV light (wavelength ∼345 nm) through a mask. As a result of the photochemical reaction, a pattern thin layer of the antigen was covalently bound to the benzophenone-modified surface. Then the sample to be analyzed, along with its specific target antibody (anti-SEB antibodies), is introduced onto the glass surface and left to react with the previously photo-immobilized antigen. When the immuno-reaction is completed, the specifically attached immunoglobulin analytes are detected by using secondary antibodies conjugated with Fluorescein isothiocyanate (FITC). The fluorescence signal emanating from the biochip surface is then quantified by two methods, using a filtered intensified charge-coupled device (CCD) camera and a grating spectrometer.

Well-defined protein–polymer conjugates—synthesis and potential applications

During the last decades, numerous studies have focused on combining the unique catalytic/functional properties and structural characteristics of proteins and enzymes with those of synthetic molecules and macromolecules. The aim of such multidisciplinary studies is to improve the properties of the natural component, combine them with those of the synthetic, and create novel biomaterials in the nanometer scale. The specific coupling of polymers onto the protein structures has proved to be one of the most straightforward and applicable approaches in that sense. In this article, we focus on the synthetic pathways that have or can be utilized to specifically couple proteins to polymers. The different categories of well-defined protein-polymer conjugates and the effect of the polymer on the protein function are discussed. Studies have shown that the specific conjugation of a synthetic polymer to a protein conveys its physico-chemical properties and, therefore, modifies the biodistribution and solubility of the protein, making it in certain cases soluble and active in organic solvents. An overview of the applications derived from such bioconjugates in the pharmaceutical industry, biocatalysis, and supramolecular nanobiotechnology is presented at the final part of the article.

The potential of a new stable ultrasound contrast agent for site-specific targeting. An in vitro experiment

Microbubble-based ultrasound contrast agents can be used for specific site targeting, but demonstrate time-limited opacification. We have previously demonstrated the potential of gold-bound microtubules to provide a stable ultrasound contrast effect. Aim of the present study was to test the feasibility of gold-bound microtubules specifically to bind to human thrombi and to inflammatory activated human umbilical vein endothelial cells (HUVEC) in vitro. HUVEC were incubated with tumor necrosis factor, to induce expression of adhesion molecules. Human clots and HUVEC were incubated with biotinylated monoclonal antifibrin and anti-E-selectin antibodies, respectively. Probes were incubated with excess avidin followed by biotinylated gold-bound microtubules and by secondary Cy3-anti-β-tubulin antibody and processed for immune fluorescence microscopy. Clots were transferred in copolymer foils filled with buffer and were ultrasonographically imaged before and after their treatment with the antifibrin antibody and with biotinylated microtubules, using a broadband harmonic transducer, transmitting and receiving at a mean frequency of 1.7 MHz and 3.2 MHz. The feasibility of specific gold-bound microtubules conjugation to antibody treated clots and HUVEC was confirmed using immune fluorescence analysis. Contrast intensities of the clots significantly increased after their treatment with antifibrin antibody and incubation with gold-bound microtubules (39 ± 2 dB versus 26 ± 2 dB, p < 0.001) and remained high after 20 min of ultrasound exposure (37 ± 2 dB versus 39 ± 2 dB, p = NS). Thus, gold-bound microtubules can specifically bind to human thrombi and to endothelial cells, providing a significant contrast effect which remains stable in the ultrasound field. This may be a promising approach to target thrombi and inflammatory active atherosclerotic plaques. (E-mail: gkorosoglou@hotmail.com)

HaloTag Protein‐Mediated Site‐Specific Conjugation of Bioluminescent Proteins to Quantum Dots

On the dot: A genetically engineered haloalkane dehalogenase was used to conjugate Renilla luciferase to quantum dots (see picture). The quantum dots can emit light through bioluminescence resonance energy transfer (BRET). This specific conjugation occurs upon simple mixing under mild conditions, and may be applied for specific in vivo labeling of proteins with quantum dots for imaging.

A novel R2R3 MYB transcription factor NtMYBJS1 is a methyl jasmonate‐dependent regulator of phenylpropanoid‐conjugate biosynthesis in tobacco

Target metabolic and large-scale transcriptomic analyses of tobacco (Nicotiana tabacum L.) Bright Yellow-2 (BY-2) cells were employed to identify novel gene(s) involved in methyl jasmonate (MJ)-dependent function in plants. At the metabolic level, we describe the specific accumulation of several phenylpropanoid-polyamine conjugates in MJ-treated BY-2 cells. Furthermore, global gene expression analysis of MJ-treated cells using a 16K cDNA microarray containing expressed sequence tags (ESTs) from BY-2 cells revealed 828 genes that were upregulated by MJ treatment within 48 h. Using time-course expression data we identified a novel MJ-inducible R2R3 MYB-type transcription factor (NtMYBJS1) that was co-expressed in a close temporal pattern with the core phenylpropanoid genes phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL). Overexpression of NtMYBJS1 in tobacco BY-2 cells caused accumulation of specific phenylpropanoid conjugates in the cells. Subsequent microarray analysis of NtMYBJS1 transgenic lines revealed that a limited number of genes, including PAL and 4CL, were specifically induced in the presence of the NtMYBJS1 transgene. These results, together with results of both antisense expression analysis and of gel mobility shift assays, strongly indicate that the NtMYBJS1 protein functions in tobacco MJ signal transduction, inducing phenylpropanoid biosynthetic genes and the accumulation of phenylpropanoid-polyamine conjugates during stress.

Sumoylation Inhibits Cleavage of Sp1 N-terminal Negative Regulatory Domain and Inhibits Sp1-dependent Transcription

Sp1 is a ubiquitously expressed transcription factor that binds GC-rich cis elements. Many posttranslational modifications have been implicated in the regulation of Sp1 activity. We now provide evidence for a novel mechanism of Sp1 regulation involving the small ubiquitin-like modifier (SUMO-1). Western blot analysis revealed a high molecular mass Sp1 of 125 kDa that is stabilized by a selective SUMO hydrolase inhibitor and destabilized by a specific SUMO-1 hydrolase. The covalent modification of Sp1 by endogenous SUMO-1 and SUMO-1 that has been fused to green fluorescent protein was demonstrated using transient transfection assays. A high probability sumoylation consensus motif, VK(16)IE(18), is located within the N-terminal negative regulatory domain of Sp1. Either arginine substitution for lysine 16 (Sp1(K16R)) or alanine substitution for glutamic acid 18 (Sp1(E18A)), abrogated Sp1 sumoylation. In vitro SUMO-1 covalently bound affinity-purified GST-Sp1, but not GST-Sp1(K16R). In vivo Sp1 was determined to be N-terminally cleaved, while Sp1(K16R) could not be cleaved indicating that sumoylation and cleavage are coupled through the key regulatory lysine 16. This coupling was evident by the demonstration of an inverse relationship between cellular SUMO-modified Sp1 and N-terminally cleaved Sp1. Compared with Sp1, sumoylation-deficient Sp1(E18A) exhibited enhanced cleavage and was a better transcriptional activator, while constitutively SUMO-1-modified Sp1 was deficient in proteolytic processing and repressed Sp1 transcriptional activity. The repressive effect of sumoylation on Sp1 activity is emphasized through the use of a GAL4 based transactivation assay. A model is proposed defining a mechanism by which sumoylation preserves the integrity of a negative regulatory domain thereby allowing for the inhibition of Sp-dependent transcription.

Selective disruption of wheat secondary metabolism by herbicide safeners

In wheat ( Triticum aestivum L.), treatment with herbicide safeners enhances the expression of enzymes involved in pesticide detoxification and reduces crop sensitivity to herbicides. Since these same enzymes are involved in plant secondary metabolism, it was of interest to determine whether or not the safener cloquintocet mexyl perturbed phenolic metabolism in wheat seedlings. LC/ESI/MS analysis identified 14 phenolic substrates in the shoots of young wheat plants. Fragmentation imposed by collision induced dissociation identified specific C-glycosidic conjugates of 4′,5,7-trihydroxflavone (apigenin), 3′,4′,5,7-tetrahydroxyflavone (luteolin) and 3′- O-methylluteolin. Treatment of 7-day-old wheat shoots with cloquintocet mexyl resulted in an accelerated depletion of the conjugates of all three flavones, most notably with the glycosides of luteolin. In contrast, safener treatment caused the selective accumulation of 4′,5,7-trihydroxy-3′,5′-dimethoxyflavone (tricin) and the phenylpropanoid ferulic acid. Changes in phenolic content were associated with an increase in O-methyltransferase and C-glucosyltransferase activity toward flavonoid substrates as well as the classic enhancement of detoxifying glutathione transferases. Our results suggest that in addition to altering the capacity of wheat to metabolise herbicides and other xenobiotics, safeners can also cause a selective shift in the metabolism of endogenous phenolics.

Headliners: Immune Response: Lead Disrupts T Cell Function

Farrer DG, Hueber SM, McCabe MJ Jr. 2005. Lead enhances CD4+ T cell proliferation indirectly by targeting antigen presenting cells and modulating antigen-specific interactions. Toxicol Appl Pharmacol 207:125–137. Although lead has been banned from use in products like house paint, gasoline, and water pipe solder in the United States, it is still present in older housing, and is used in products in other countries. Besides its widely studied neurotoxicity, lead is also a well-known immunotoxicant, though little is known about its mechanism of action. Now NIEHS grantee Michael McCabe and colleagues at the University of Rochester have discovered how lead may work to disturb T cell function in the body. Previous studies have suggested that lead’s immunotoxic effects may occur at exposures even lower than those required for neurotoxicity to occur; thus, suboptimal immune function may affect people who do not even realize they have been exposed to lead. Older adults and lactating, pregnant, and postmenopausal women are at greater risk for lead exposure as lead stored in the bones is released back into the blood and soft tissues. Children are also at heightened risk for lead exposure because they engage in more hand-to-mouth activity and absorb a larger proportion of ingested lead across the intestinal epithelium than do adults. The Rochester researchers used flow cytometry to analyze T cell division in cell cultures derived from lead-treated mice. T cells help regulate the body’s immune system by attacking bacteria, viruses, foreign tissue, and tumor cells. At day 4 of treatment, the frequency of proliferating T cells was much greater in treated than in nontreated cultures. Lead appeared to target a type of cell known as antigen presenting cells, and its effect was based on specific peptide-major histocompatibility complex conjugate. The results suggest that lead may pose even more long-term health threats than originally thought.

Highly Sensitive Surface Plasmon Resonance Sensor on Nanoscale Bioactive Surfaces for Specific Detection of Tri-Nitro Toluene

ABSTRACTA nano-scale biosensor chip surface was fabricated using dinitro-phenylated key hole limpet (DNP-KLH) protein conjugate as ligand supported by underlying 11-amino 1-undecanethiol hydrochloride(AUT) self assembled monolayer (SAM) and bis sulfosuccinimidyl suberate(BS3) as crosslinker. Bioactive thin films were fabricated over gold chip via layer-by-layer self assembly methods. Biomolecular interaction between substrate specific anti-TNT antibody and DNP-KLH conjugate surface was monitored through surface plasmon resonance based optical sensor. The quantitation of tri-nitro toluene(TNT) on this bioactive surface was done using the solution based competitive inhibition assay. The DNP-KLH surface biosensor has shown a detection limit of 0.14 ng/ml(140 ppt) for TNT molecule. The detection limit of surface plasmon resonance(SPR) biosensor was further enhanced by using goat anti mouse antibody to the 0.002 ng/ml for TNT analyte. This TNT specific biosensor holds the promise to be one of most sensitive sensor surface under indirect competitive assay format. A short injection (12 sec) of 10 mM Glycine-HCl solution was found adequate for regeneration of DNP-KLH surface for repeated use.

Glomérulopathies à dépôts organisés d'immunoglobulines

Glomerulonephritis with organized immunoglobulin deposits are heterogeneous and may be encountered in a variety of renal disorders. It is particularly important to determine the exact types of immunoglobulin deposited and their monotypy/monoclonality, using specific anti-light chain conjugates and in some instances, anti-heavy chain subclass conjugates. The histologic pattern and identification of monotypic Ig deposits in renal tissue are in some cases sufficient for the diagnosis of AL amyloidosis, type I cryoglobulinic glomerulonephritis or “Randall-type” monoclonal immunoglobulin deposition disease (MIDD). Electron microscopy is particularly valuable for the precise localization of Ig deposits and for determing the pattern of deposition: organized, homogeneous (non-organized or granular) or mixed. New entities in the broad spectrum of Ig deposition diseases are emerging from electron microscopy studies of renal diseases. We propose here a comprehensive classification for diseases featuring the organized deposition in tissues of Ig.

Fluorescence resonance energy transfer induced by conjugation of metalloproteins to nanoparticles

We show the possibility of realizing a hybrid system composed of a semiconductor nanoparticle (NP) and a metalloprotein, in which the photophysical properties of the two species can be exploited to elicit fluorescence resonance energy transfer mechanisms from the biomolecule to the NP. A specific conjugation process between CdSe/ZnS core/shell water soluble NPs, functionalized with surface exposed thiol groups (–SH), and the apo form of the metalloprotein azurin (Az) has been achieved, resulting in a fixed distance in the donor-acceptor pairs. The increase in the NP fluorescence intensity was found to be dependent on the Az to NP molar ratio.

Ubiquitin targeting of rat muscle proteins during short periods of unloading

The ubiquitin-proteasome system is known to be involved in many situations leading to skeletal muscle atrophy. However, the cellular mechanisms triggering the atrophic process initiation are still poorly understood. For short periods of rat hindlimb unloading, we assessed the specific ubiquitin targeting of sarcoplasmic or myofibrillar proteins in slow and fast rat muscle types. Adult Sprague Dawley rats were randomly assigned to three groups: control, hindlimb-unloaded for 4 days (HU4) and hindlimb-unloaded for 8 days (HU8). In fractionated extracts from soleus (SOL) and Extensor Digitorum Longus (EDL) muscles, the relative contents of free and conjugated ubiquitin were quantified by immunoblotting. Hindlimb unloading of short durations resulted in a preferential atrophy of slow-twitch fibres and bound ubiquitin levels were increased by 37 and 68% in the soleus myofibrillar fraction after respectively 4 and 8 days. The ubiquitin conjugation was shown to principally affect the high molecular weight proteins. Free and conjugated ubiquitin levels remained unchanged in sarcoplasmic fraction from SOL muscle after 8 days HU. For the fast muscle (EDL), ubiquitin contents were approximately twofold lower in control conditions, and did not significantly change during the hindlimb unloading periods considered. The postural SOL muscle was shown to contain higher constitutive sarcoplasmic ubiquitin levels than the phasic EDL. The high response to unloading of the slow twitch fibres rich SOL muscle was accompanied by a specific conjugation of its myofibrillar proteins that may participate in the initiation of skeletal muscle remodelling consequent to disuse.

A Peptide Carrier with a Built-In Vaccine Adjuvant: Construction of Immunogenic Conjugates

A multifunctional carrier combining B/T cell epitopes (i), a built-in vaccine adjuvant (ii), and a universal T cell epitope (iii) for the construction of potent and specific immunogenic conjugates is presented. The IL-1beta(163-171) fragment known to reproduce the immunostimulatory and adjuvant effects of the whole IL-1beta without possessing any of the pro-inflammatory properties of IL-1beta was covalently anchored to the N-terminus of the Sequential Oligopeptide Carrier, SOC(n), formed by the repeating tripeptide unit Lys-Aib-Gly. A promiscuous T cell epitope derived from the tetanus toxin, TT(593-599), was also positioned in the carboxy terminus of SOC(n) as a universal immunogen to provide broad immunogenicity. Selected B/T cell epitopes from the Sm and La/SSB autoantigens, against which is directed the humoral autoimmunity in patients with systemic lupus erythematosus and Sjögren's Syndrome, respectively, were coupled to the Lys-N(epsilon)H2 groups of the carrier, and the formulated constructs were administered in animals following the conventional immunization protocol of complete/incomplete Freund's adjuvant. The induced immune responses were compared with that produced when the Sm- and La/SSB-reconstituted immunogenic conjugates were injected alone. High titers of specific antibodies recognizing the priming construct, as well as the cognate autoantigen, were obtained when administered alone without the assistance of Freund's adjuvant. It is concluded that our approach provides the conceptual and experimental framework for the development of multifunctional immunogenic conjugates eliciting enhanced, specific, and prolonged humoral response for usage as human vaccine candidates.