Structural characterisation of a highly branched exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB2074

Lactobacillus delbrueckii subsp. bulgaricus NCFB2074 when grown in skimmed milk secretes a highly branched exopolysaccharide. The exopolysaccharide has a heptasaccharide repeat unit and is composed of glucose and galactose in the molar ratio 3:4. Using chemical techniques and 1D and 2D NMR spectroscopy the polysaccharide has been shown to possess the following repeat unit structure: [carbohydrate structure: see text].     

Purification of normal cellular prion protein from human platelets and the formation of a high molecular weight prion protein complex following platelet activation

A method for the extraction and purification of PrP(C), in its native monomeric form, from outdated human platelet concentrates is described. Both calcium ionophore platelet activation and lysis in Triton X-100 were evaluated as methods for the extraction of soluble platelet PrP(C) in its monomeric form. Following platelet activation, the majority of released PrP(C) was detected as a disulphide linked high molecular weight complex, which under reducing conditions could be separated into what appear to be stable non-disulphide linked PrP dimers or PrP covalently linked to another as yet unidentified protein. This phenomenon appears to be unique to activation since only monomeric PrP(C) was detected following lysis of resting platelets. Subsequently, PrP(C) was purified from the Triton X-100 lysate by sequential cation ion exchange and Cu2+ affinity chromatography. From 10 L of outdated platelet concentrate, we were able to recover 1.29 mg PrP(C) at a purity of 92%.     

Aberrant IgM signaling promotes survival of transitional T1 B cells and prevents tolerance induction in lupus-prone New Zealand black mice

New Zealand Black (NZB) mice develop a lupus-like syndrome. Although the precise immune defects leading to autoantibody production in these mice have not been characterized, they possess a number of immunologic abnormalities suggesting that B cell tolerance may be defective. In the bone marrow, immature self-reactive B cells that have failed to edit their receptors undergo apoptosis as a consequence of Ig receptor engagement. Splenic transitional T1 B cells are recent bone marrow emigrants that retain these signaling properties, ensuring that B cells recognizing self-Ags expressed only in the periphery are deleted from the naive B cell repertoire. In this study we report that this mechanism of tolerance is defective in NZB mice. We show that NZB T1 B cells are resistant to apoptosis after IgM cross-linking in vitro. Although extensive IgM cross-linking usually leads to deletion of T1 B cells, in NZB T1 B cells we found that it prevents mitochondrial membrane damage, inhibits activation of caspase-3, and promotes cell survival. Increased survival of NZB T1 B cells was associated with aberrant up-regulation of Bcl-2 after Ig receptor engagement. We also show that there is a markedly increased proportion of NZB T1 B cells that express elevated levels of Bcl-2 in vivo and provide evidence that up-regulation of Bcl-2 follows encounter with self-Ag in vivo. Thus, we propose that aberrant cell signaling in NZB T1 B cells leads to the survival of autoreactive B cells, which predisposes NZB mice to the development of autoimmunity.     

Immunodominance of poxviral-specific CTL in a human trial of recombinant-modified vaccinia Ankara

Many recombinant poxviral vaccines are currently in clinical trials for cancer and infectious diseases. However, these agents have failed to generate T cell responses specific for recombinant gene products at levels comparable with T cell responses associated with natural viral infections. The recent identification of vaccinia-encoded CTL epitopes, including a new epitope described in this study, allows the simultaneous comparison of CTL responses specific for poxviral and recombinant epitopes. We performed detailed kinetic analyses of CTL responses in HLA-A*0201 patients receiving repeated injections of recombinant modified vaccinia Ankara encoding a string of melanoma tumor Ag epitopes. The vaccine-driven CTL hierarchy was dominated by modified vaccinia Ankara epitope-specific responses, even in patients who had not received previous smallpox vaccination. The only recombinant epitope that was able to impact on the CTL hierarchy was the melan-A26-35 analog epitope, whereas responses specific for the weaker affinity epitope NY-ESO-1(157-165) failed to be expanded above the level detected in prevaccination samples. Our results demonstrate that immunodominant vaccinia-specific CTL responses limit the effectiveness of poxviruses in recombinant vaccination strategies and that more powerful priming strategies are required to overcome immunodominance of poxvirus-specific T cell responses.     

Preventing misfolding of the prion protein by trimethylamine N-oxide

Transmissible spongiform encephalopathies are a class of fatal neurodegenerative diseases linked to the prion protein. The prion protein normally exists in a soluble, globular state (PrP(C)) that appears to participate in copper metabolism in the central nervous system and/or signal transduction. Infection or disease occurs when an alternatively folded form of the prion protein (PrP(Sc)) converts soluble and predominantly alpha-helical PrP(C) into aggregates rich in beta-structure. The structurally disordered N-terminus adopts beta-structure upon conversion to PrP(Sc) at low pH. Chemical chaperones, such as trimethylamine N-oxide (TMAO), can prevent formation of PrP(Sc) in scrapie-infected mouse neuroblastoma cells [Tatzelt, J., et al. (1996) EMBO J. 15, 6363-6373]. To explore the mechanism of TMAO protection of PrP(C) at the atomic level, molecular dynamics simulations were performed under conditions normally leading to conversion (low pH) with and without 1 M TMAO. In PrP(C) simulations at low pH, the helix content drops and the N-terminus is brought into the small native beta-sheet, yielding a PrP(Sc)-like state. Addition of 1 M TMAO leads to a decreased radius of gyration, a greater number of protein-protein hydrogen bonds, and a greater number of tertiary contacts due to the N-terminus forming an Omega-loop and packing against the structured core of the protein, not due to an increase in the level of extended structure as with the PrP(C) to PrP(Sc) simulation. In simulations beginning with the "PrP(Sc)-like" structure (derived from PrP(C) simulated at low pH in pure water) in 1 M TMAO, similar structural reorganization at the N-terminus occurred, disrupting the extended sheet. The mechanism of protection by TMAO appears to be exclusionary in nature, consistent with previous theoretical and experimental studies. The TMAO-induced N-terminal conformational change prevents residues that are important in the conversion of PrP(C) to PrP(Sc) from assuming extended sheet structure at low pH.     

The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle

5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.     

Kinetic analysis-based quantitation of free radical generation in EPR spin trapping

Because short-lived reactive oxygen radicals such as superoxide have been implicated in a variety of disease processes, methods to measure their production quantitatively in biological systems are critical for understanding disease pathophysiology. Electron paramagnetic resonance (EPR) spin trapping is a direct and sensitive technique that has been used to study radical formation in biological systems. Short-lived oxygen free radicals react with the spin trap and produce paramagnetic adducts with much higher stability than that of the free radicals. In many cases, the quantity of the measured adduct is considered to be an adequate measure of the amount of the free radical generated. Although the intensity of the EPR signal reflects the magnitude of free radical generation, the actual quantity of radicals produced may be different due to modulation of the spin adduct kinetics caused by a variety of factors. Because the kinetics of spin trapping in biochemical and cellular systems is a complex process that is altered by the biochemical and cellular environment, it is not always possible to define all of the reactions that occur and the related kinetic parameters of the spin-trapping process. We present a method based on a combination of measured kinetic data for the formation and decay of the spin adduct alone with the parameters that control the kinetics of spin trapping and radical generation. The method is applied to quantitate superoxide trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). In principle, this method is broadly applicable to enable spin trapping-based quantitative determination of free radical generation in complex biological systems.     

Regulation of HAX-1 anti-apoptotic protein by Omi/HtrA2 protease during cell death

Omi/HtrA2 is a nuclear-encoded mitochondrial serine protease that has a pro-apoptotic function in mammalian cells. Upon induction of apoptosis, Omi translocates to the cytoplasm and participates in caspase-dependent apoptosis by binding and degrading inhibitor of apoptosis proteins. Omi can also initiate caspase-independent apoptosis in a process that relies entirely on its ability to function as an active protease. To investigate the mechanism of Omi-induced apoptosis, we set out to isolate novel substrates that are cleaved by this protease. We identified HS1-associated protein X-1 (HAX-1), a mitochondrial anti-apoptotic protein, as a specific Omi interactor that is cleaved by Omi both in vitro and in vivo. HAX-1 degradation follows Omi activation in cells treated with various apoptotic stimuli. Using a specific inhibitor of Omi, HAX-1 degradation is prevented and cell death is reduced. Cleavage of HAX-1 was not observed in a cell line derived from motor neuron degeneration 2 mice that carry a mutated form of Omi that affects its proteolytic activity. Degradation of HAX-1 is an early event in the apoptotic process and occurs while Omi is still confined in the mitochondria. Our results suggest that Omi has a unique pro-apoptotic function in mitochondria that involves removal of the HAX-1 anti-apoptotic protein. This function is distinct from its ability to activate caspase-dependent apoptosis in the cytoplasm by degrading inhibitor of apoptosis proteins.