Induction of transplantation tolerance in non-human primate preclinical models

Short-term outcomes following organ transplantation have improved considerably since the availability of cyclosporine ushered in the modern era of immunosuppression. In spite of this, many of the current limitations to progress in the field are directly related to the existing practice of relatively non-specific immunosuppression. These include increased risks of opportunistic infection and cancer, and toxicity associated with long-term immunosuppressive drug exposure. In addition, long-term graft loss continues to result in part from a failure to adequately control the anti-donor immune response. The development of a safe and reliable means of inducing tolerance would ameliorate these issues and improve the lives of transplant recipients, yet given the improving clinical standard of care, the translation of new therapies has become appropriately more cautious and dependent on increasingly predictive preclinical models. While convenient and easy to use, rodent tolerance models have not to date been reliably capable of predicting a therapy's potential efficacy in humans. Non-human primates possess an immune system that more closely approximates that found in humans, and have served as a more rigorous preclinical testing ground for novel therapies. Prior to clinical adaptation therefore, tolerance regimens should be vetted in non-human primates to ensure that there is sufficient potential for efficacy to justify the risk of its application.     

Alpha-synuclein and parkin contribute to the assembly of ubiquitin lysine 63-linked multiubiquitin chains

Mutations in alpha-synuclein, Parkin, and UCH-L1 cause heritable forms of Parkinson disease. Unlike alpha-synuclein, for which no precise biochemical function has been elucidated, Parkin functions as a ubiquitin E3 ligase, and UCH-L1 is a deubiquitinating enzyme. The E3 ligase activity of Parkin in Parkinson disease is poorly understood and is further obscured by the fact that multiubiquitin chains can be formed through distinct types of linkages that regulate diverse cellular processes. For instance, ubiquitin lysine 48-linked multiubiquitin chains target substrates to the proteasome, whereas ubiquitin lysine 63-linked chains control ribosome function, protein sorting and trafficking, and endocytosis of membrane proteins. It is notable in this regard that ubiquitin lysine 63-linked chains promote the degradation of membrane proteins by the lysosome. Because both Parkin and alpha-synuclein can regulate the activity of the dopamine transporter, we investigated whether they influenced ubiquitin lysine 63-linked chain assembly. These studies revealed novel biochemical activities for both Parkin and alpha-synuclein. We determined that Parkin functions with UbcH13/Uev1a, a dimeric ubiquitin-conjugating enzyme, to assemble ubiquitin lysine 63-linked chains. Our results and the results of others indicate that Parkin can promote both lysine 48- and lysine 63-linked ubiquitin chains. alpha-Synuclein also stimulated the assembly of lysine 63-linked ubiquitin chains. Because UCH-L1, a ubiquitin hydrolase, was recently reported to form lysine 63-linked conjugates, it is evident that three proteins that are genetically linked to Parkinson disease can contribute to lysine 63 multiubiquitin chain formation.     

Disease-related modifications in tau affect the interaction between Fyn and Tau

Microtubule-associated protein tau is the major component of the neurofibrillary tangles of Alzheimer disease (AD) and is genetically linked to frontotemporal dementias (FTDP-17). We have recently shown that tau interacts with the SH3 domain of Fyn, an Src family non-receptor tyrosine kinase, and is tyrosine-phosphorylated by Fyn on Tyr-18. Also, tyrosine-phosphorylated tau is present in the neuropathology of AD. To determine whether alterations in the tau-Fyn interaction might correlate with disease-related factors in AD and FTDP-17, we have performed real-time surface plasmon resonance studies on a panel of 21 tau constructs with Fyn SH3. We report that the interaction between Fyn SH3 and 3R-tau was 20-fold higher than that with 4R-tau. In addition, the affinity between 4R-tau and Fyn SH3 was increased 25-45-fold by phosphorylation-mimicking mutations or by FTDP-17 mutations. In vitro kinase reactions show that tau, with lower affinity SH3 interactions, exhibited a lower level of Tyr-18 phosphorylation under our reaction conditions. Lastly, we have demonstrated that tau is phosphorylated on Tyr-18 in the tau P301L mouse model for tauopathy (JNPL3). In summary, our results suggest that disease-related phosphorylation and missense mutations of tau increase association of tau with Fyn. Because these effects are mediated through the 4R component of the tau population, these results also have implications for the FTDP-17 diseases caused by increased expression of 4R-tau. Our data support a role for the Fyn-tau interaction in neurodegeneration.     

Stereo-specificity for pro-(R) hydrogen of NAD(P)H during enzyme-catalyzed hydride transfer to CL-20

A dehydrogenase from Clostridium sp. EDB2 and a diaphorase from Clostridium kluyveri were reacted with CL-20 to gain insights into the enzyme-catalyzed hydride transfer to CL-20, and the enzyme's stereo-specificity for either pro-R or pro-S hydrogens of NAD(P)H. Both enzymes biotransformed CL-20 at rates of 18.5 and 24nmol/h/mg protein, using NADH and NADPH as hydride-source, respectively, to produce a N-denitrohydrogenated product with a molecular weight of 393Da. In enzyme kinetics studies using reduced deuterated pyridine nucleotides, we found a kinetic deuterium isotopic effect of 2-fold on CL-20 biotransformation rate using dehydrogenase enzyme against (R)NADD as a hydride-source compared to either (S)NADD or NADH. Whereas, in case of diaphorase, the kinetic deuterium isotopic effect of about 1.5-fold was observed on CL-20 biotransformation rate using (R)NADPD as hydride-source. In a comparative study with LC-MS, using deuterated and non-deuterated NAD(P)H, we found a positive mass-shift of 1Da in the N-denitrohydrogenated product suggesting the involvement of a deuteride (D(-)) transfer from NAD(P)D. The present study thus revealed that both dehydrogenase and diaphorase enzymes from the two Clostridium species catalyzed a hydride transfer to CL-20 and showed stereo-specificity for pro-R hydrogen of NAD(P)H.     

Antiinflammatory and antiulcer activities of phytic acid in rats

Maximum antiinflammatory activity of phytic acid (PA) was seen at an oral dose of 150 mg/kg in the carrageenan induced rat paw edema model. Although PA showed ability to prevent denaturation of proteins, it showed less antiinflammatory activity than ibuprofen. Ability of PA to bring down thermal denaturation of proteins might be a contributing factor in the mechanism of action against inflammation. PA, at all the doses tested, showed significant protection from ulcers induced by ibuprofen, ethanol and cold stress, with a maximum activity at 150 mg/kg. There was a significant increase in gastric tissue malondialdehyde levels in ethanol treated rats but these levels decreased following PA pretreatment. Moreover, pretreatment with PA significantly inhibited various effects of ethanol on gastric mucosa, such as, reduction in the concentration of nonprotein sulfhydryl groups, necrosis, erosions, congestion and hemorrhage. These results suggested that gastro-protective effect of PA could be mediated by its antioxidant activity and cytoprotection of gastric mucosa.     

Optimized protocols for the isolation of specific protein-binding peptides or peptoids from combinatorial libraries displayed on beads

Many methods have been published by which combinatorial libraries may be screened for compounds capable of manipulating the function(s) of a target protein. One of the simplest approaches is to identify compounds in a library that bind the protein of interest, since these binding events usually occur on functionally important surfaces of the protein. These protein-binding compounds could also be of utility as protein capture agents in the construction of protein-detecting microarrays or related analytical devices and as reagents for the affinity purification of proteins from complex mixtures. In this article, we provide optimized methods for screening libraries of molecules displayed on the beads on which they were synthesized. This is a particularly convenient format for library screening for laboratories with limited budgets and modest robotics capabilities.     

Honokiol potentiates apoptosis, suppresses osteoclastogenesis, and inhibits invasion through modulation of nuclear factor-kappaB activation pathway

Recent reports have indicated that honokiol can induce apoptosis, suppress tumor growth, and inhibit angiogenesis. In this report, we found that honokiol potentiated the apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents, suppressed TNF-induced tumor cell invasion, and inhibited RANKL-induced osteoclastogenesis, all of which are known to require nuclear factor-kappaB (NF-kappaB) activation. Honokiol suppressed NF-kappaB activation induced by a variety of inflammatory stimuli, and this suppression was not cell type specific. Further studies showed that honokiol blocked TNF-induced phosphorylation, ubiquitination, and degradation of IkappaBalpha through the inhibition of activation of IkappaBalpha kinase and of Akt. This led to suppression of the phosphorylation and nuclear translocation of p65 and NF-kappaB-dependent reporter gene expression. Magnolol, a honokiol isomer, was equally active. The expression of NF-kappaB-regulated gene products involved in antiapoptosis (IAP1, IAP2, Bcl-x(L), Bcl-2, cFLIP, TRAF1, and survivin), proliferation (cyclin D1, cyclooxygenase-2, and c-myc), invasion (matrix metalloproteinase-9 and intercellular adhesion molecule-1), and angiogenesis (vascular endothelial growth factor) were also down-regulated by honokiol. Honokiol also down-regulated NF-kappaB activation in in vivo mouse dorsal skin model. Thus, overall, our results indicate that NF-kappaB and NF-kappaB-regulated gene expression inhibited by honokiol enhances apoptosis and suppresses osteoclastogenesis and invasion.     

Substrate specificity of nonribosomal peptide synthetase modules responsible for the biosynthesis of the oligopeptide moiety of cephabacin in Lysobacter lactamgenus

Lysobacter lactamgenus produces cephabacins, a class of beta-lactam antibiotics which have an oligopeptide moiety attached to the cephem ring at the C-3 position. The nonribosomal peptide synthetase (NRPS) system, which comprises four distinct modules, is required for the biosynthesis of this short oligopeptide, when one takes the chemical structure of these antibiotics into consideration. The cpbI gene, which has been identified in a region upstream of the pcbAB gene, encodes the NRPS - polyketide synthase hybrid complex, where NRPS is composed of three modules, while the cpbK gene -- which has been reported as being upstream of cpbI-- comprises a single NRPS module. An in silico protein analysis was able to partially reveal the specificity of each module. The four recombinant adenylation (A) domains from each NRPS module were heterologously expressed in Escherichia coli and purified. Biochemical data from ATP-PPi exchange assays indicated that L-arginine was an effective substrate for the A1 domain, while the A2, A3 and A4 domains activated L-alanine. These findings are in an agreement with the known chemical structure of cephabacins, as well as with the anticipated substrate specificity of the NRPS modules in CpbI and CpbK, which are involved in the assembly of the tetrapeptide at the C-3 position.     

Acetyl-11-keto-beta-boswellic acid potentiates apoptosis, inhibits invasion, and abolishes osteoclastogenesis by suppressing NF-kappa B and NF-kappa B-regulated gene expression

Acetyl-11-keto-beta-boswellic acid (AKBA), a component of an Ayurvedic therapeutic plant Boswellia serrata, is a pentacyclic terpenoid active against a large number of inflammatory diseases, including cancer, arthritis, chronic colitis, ulcerative colitis, Crohn's disease, and bronchial asthma, but the mechanism is poorly understood. We found that AKBA potentiated the apoptosis induced by TNF and chemotherapeutic agents, suppressed TNF-induced invasion, and inhibited receptor activator of NF-kappaB ligand-induced osteoclastogenesis, all of which are known to require NF-kappaB activation. These observations corresponded with the down-regulation of the expression of NF-kappaB-regulated antiapoptotic, proliferative, and angiogenic gene products. As examined by DNA binding, AKBA suppressed both inducible and constitutive NF-kappaB activation in tumor cells. It also abrogated NF-kappaB activation induced by TNF, IL-1beta, okadaic acid, doxorubicin, LPS, H2O2, PMA, and cigarette smoke. AKBA did not directly affect the binding of NF-kappaB to the DNA but inhibited sequentially the TNF-induced activation of IkappaBalpha kinase (IKK), IkappaBalpha phosphorylation, IkappaBalpha ubiquitination, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation. AKBA also did not directly modulate IKK activity but suppressed the activation of IKK through inhibition of Akt. Furthermore, AKBA inhibited the NF-kappaB-dependent reporter gene expression activated by TNFR type 1, TNFR-associated death domain protein, TNFR-associated factor 2, NF-kappaB-inducing kinase, and IKK, but not that activated by the p65 subunit of NF-kappaB. Overall, our results indicated that AKBA enhances apoptosis induced by cytokines and chemotherapeutic agents, inhibits invasion, and suppresses osteoclastogenesis through inhibition of NF-kappaB-regulated gene expression.