Correlation between Shiga toxin B-subunit stability and antigen crosspresentation: a mutational analysis

The homopentameric B-subunit of Shiga toxin (STxB) is used as a tool to deliver antigenic peptides and proteins to the cytosolic compartment of dendritic cells (DCs). In this study, a series of interface mutants of STxB has been constructed. All mutants retained their overall conformation, while a loss in thermal stability was observed. This effect was even more pronounced in trifluoroethanol solutions that mimic the membrane environment. Despite this, all mutants were equally efficient at delivering a model antigenic protein into the MHC class I-restricted antigen presentation pathway of mouse DCs, suggesting that the structural stability of STxB is not a key factor in the membrane translocation process.     

Inhibition of apoptosis, activation of NKT cell and upregulation of CD40 and CD40L mediated by M. leprae antigen(s) combined with Murabutide and Trat peptide in leprosy patients

Protective immunity against intracellular pathogen Mycobacterium leprae is dependent on the activation of T cells. Repeated stimulation of T cells by M. leprae antigens MLCwA (M. leprae total cell wall antigen) and ManLAM (mannose-capped lipoarabinomannan), may lead to apoptosis in leprosy patients. In the present study, inhibition of the Fas-induced apoptosis of peripheral blood mononuclear cells of leprosy patients was investigated using above M. leprae antigen(s), in combination with immunomodulators murabutide (MB) and a Trat peptide in particulate form (liposome). Incubation of the cells with antigen containing the two immunomodulators in particulate form (liposomes) led to decrease in percentage of propidium iodide positive cells and T cells expressing Fas–FasL as well as decreased caspase-8/-3 activities in lepromatous patients, thereby inhibiting apoptosis, while converse was true upon stimulation with soluble antigen. Concurrently, there was an upregulation of antiapoptotic protein Bcl-x_L in lepromatous patients, leading to the inhibition of apoptosis. It was also observed that same formulation upregulated the expression of CD40 on B cells and monocytes-macrophages and CD40L on T cells of lepromatous leprosy patients. The same liposomal formulation significantly increased the expression of CD1b and CD1d on monocytes-macrophages as well as percentage of NKT cells secreting IFN-γ in lepromatous leprosy patients. Thus, the liposomal formulation of antigen with the immunomodulators in vitro promoted the activation of CD40:CD40L pathways and NKT cell function involved in providing cell-mediated immunity to these patients. The same formulation also caused reversal of T cell anergy by inhibiting apoptosis through decreased expression of death receptors (Fas–FasL) and caspase activities (3 and 8) and increased expression of antiapoptotic protein Bcl-x_L in these patients.     

Structural stability and reversible unfolding of recombinant porcine S100A12

Porcine S100A12 is a member of the S100 proteins, family of small acidic calcium-binding proteins characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events such as the regulation of protein phosphorylation, enzymatic activity, protein-protein interaction, Ca2+ homeostasis, inflammatory processes and intermediate filament polymerization. In addition, members of this family bind Zn2+ or Ca2+ with cooperative effect on binding. In this study, the gene sequence encoding porcine S100A12 was obtained by the synthetic gene approach using E. coli codon bias. Additionally, we report a thermodynamic study of the recombinant S100A12 using circular dichroism, fluorescence and isothermal titration calorimetry. The results of urea and temperature induced unfolding and refolding processes indicated a reversible two-state process. Also, the ANS fluorescence studies showed that in presence of divalent ions the protein exposes hydrophobic sites which could facilitate the interaction with other proteins and trigger the physiological responses.     

The path toward identifying prostatic stem cells

Abstract The concept that stem cells reside in prostate epithelia is well accepted and has led to significant efforts aimed at isolating and characterizing adult prostate stem/progenitor cells from both human and rodent prostate tissues. Understanding how prostatic stem and/or progenitor cells give rise to prostate epithelia during development and maintain homeostasis of the prostate gland in adulthood is a major research focus. Furthermore, the ability to identify and manipulate prostatic stem cells (PSCs) holds enormous promise for the development of new approaches to manage and treat prostate cancer. This review maps the pathways to identifying, isolating, characterizing, and understanding the differentiation capacity of PSCs.     

CD14 major role during lipopolysaccharide-induced inflammation in chick embryo cardiomyocytes

CD14 is a surface differentiation antigen that functions as a receptor for bacterial lipopolysaccharide. The cellular signaling events that lead to lipopolysaccharide-induced production of inflammatory mediators are the primary cause of myocardial dysfunction observed in sepsis. Here, we evaluated the role of CD14 in chick embryo cardiomyocytes stimulated with lipopolysaccharide. CD14 expression was detected by confocal laser microscopy observation and by immunoblotting analysis. Moreover, we provided evidence for CD14-dependent functional responses of lipopolysaccharide-stimulated cardiomyocytes in terms of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) production. Attenuated TNF-alpha and NO secretion, following anti-CD14 treatment of cardiomyocytes, suggested a role for this receptor in lipopolysaccharide-mediated cell responses. We also evidenced that labeled lipopolysaccharide was internalized and localized next to the Golgi complex, at the level of lysosomes, and in the perinuclear zone. The intracytoplasmatic transport seems to depend on the contractile apparatus, because cell pretreatment with cytochalasin D prevented lipopolysaccharide internalization and reduced both TNF-alpha and NO release. Lipopolysaccharide internalization was dependent on CD14 receptor, since anti-CD14 pre-treatment prevented endotoxin uptake by cardiomyocytes. Results demonstrated: (1) CD14 is expressed on the surface membrane of cardiomyocytes; (2) CD14 is involved in cytoskeletal dependent lipopolysaccharide internalization at specific cytoplasmatic locations; (3) CD14 plays a role in lipopolysaccharide-mediated responses by cardiomyocytes after lipopolysaccharide internalization.     

Self-assembling peptides: sequence, secondary structure in solution and film formation

Peptides of alternating charge and hydrophobic amino acids have a tendency to adopt unusually stable beta-sheet structures that can form insoluble macroscopic aggregates under physiological conditions. In this study, analogues of a well-known self-assembling peptide, characterized by the same polar/nonpolar periodicity but with different residues, were designed to study the relationship between sequence, conformation in solution and film-forming capacity in saline solution. Peptide conformation, evaluated by circular dichroism, correlated with film forming capacity observed by inverted optical microscopy after addition of saline solution and subsequent drying. We found that polar/nonpolar periodicity of several analogues is not criterion enough to induce beta-sheet and thus film formation and that conformations different from beta-sheet also allow self-assemblage. Furthermore, addition of the short adhesive sequence RGD to a known self-assembling sequence was shown to not prevent the self-assembling process. This finding might prove useful for the design of biomimetic scaffolds. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 906-915, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Improved reactor performance and operability in the biotransformation of carveol to carvone using a solid-liquid two-phase partitioning bioreactor

In an effort to improve reactor performance and process operability, the microbial biotransformation of (-)-trans-carveol to (R)-(-)-carvone by hydrophobic Rhodococcus erythropolis DCL14 was carried out in a two phase partitioning bioreactor (TPPB) with solid polymer beads acting as the partitioning phase. Previous work had demonstrated that the substrate and product become inhibitory to the organism at elevated aqueous concentrations and the use of an immiscible second phase in the bioreactor was intended to provide a reservoir for substrates to be delivered to the aqueous phase based on the metabolic rate of the cells, while also acting as a sink to uptake the product as it is produced. The biotransformation was previously undertaken in a two liquid phase TPPB with 1-dodecene and with silicone oil as the immiscible second phase and, although improvement in the reactor performance was obtained relative to a single phase system, the hydrophobic nature of the organism caused the formation of severe emulsions leading to significant operational challenges. In the present work, eight types of polymer beads were screened for their suitability for use in a solid-liquid TPPB for this biotransformation. The use of selected solid polymer beads as the second phase completely prevented emulsion formation and therefore improved overall operability of the reactor. Three modes of solid-liquid TPPB operation were considered: the use of a single polymer bead type (styrene/butadiene copolymer) in the reactor, the use of a mixture of polymer beads in the reactor (styrene/butadiene copolymer plus Hytrel(R) 8206), and the use of one type of polymer beads in the reactor (styrene/butadiene copolymer), and another bead type (Hytrel(R) 8206) in an external column through which fermentation medium was recirculated. This last configuration achieved the best reactor performance with 7 times more substrate being added throughout the biotransformation relative to a single aqueous phase benchmark reactor and 2.7 times more substrate being added relative to the best two liquid TPPB case. Carvone was quantitatively recovered from the polymer beads via single stage extraction into methanol, allowing for bead re-use.     

Chitin binding by Thermobifida fusca cellulase catalytic domains

Cellulose is a linear homopolymer of beta 1-4 linked glucose residues. Chitin is similar to cellulose in structure, and can be described as cellulose with the hydroxyl group on the C2 carbon replaced by an acetylamine group. Both cellulose and chitin form tightly packed, extensively hydrogen-bonded micro-fibrils. Up to now, binding of cellulase catalytic domains (CDs) to chitin has not been reported. In this article, binding of the CDs of Thermobifida fusca Cel6A, Cel6B, Cel48A, Cel5A, and Cel9A to alpha-chitin was investigated. The CDs of endocellulases, Cel6A and Cel5A did not bind to alpha-chitin; one exocellulase, Cel48A CD bound alpha-chitin moderately well; and the exocellulase Cel6B CD and the processive endocellulase Cel9A CD bound extremely tightly to alpha-chitin. Only mutations of Cel6B W329C, W332A and G234S and Cel9A Y206F, Y206S and D261A/R378K caused weaker binding to alpha-chitin than wild-type, and all these mutations were of residues near the catalytic center. One mutant enzyme, Cel9A D261A/R378K had weak chitinase activity, but no soluble products were detected. Chitotriose and chitotetraose were docked successfully to the catalytic cleft of Cel9A. In general, the positioning of the sugar residues in the model structures matched the cellooligosaccharides in the X-ray structure. Our results show that the binding of chitin by a cellulase can provide additional information about its binding to cellulose.     

A convenient preparation of 6-oligo(lactic acid)cyclomaltoheptaose as kinetically degradable derivative for controlled release of amoxicillin

6-Oligo(lactic acid)cyclomaltoheptaose (6-OLA-βCD) with an average substitution of about 7.0 lactic acid units was prepared as a new water-soluble cyclomaltoheptaose (βCD) derivative (solubility of about 70.7-fold that of βCD), based on the ring-opening polymerization of 3,6-dimethyl-1,4-dioxane-2,5-dione (lactide). The product was characterized by ¹H NMR, ¹³C NMR, IR, and MS spectroscopy. The complexation of amoxicillin with 6-OLA-βCD was found to be much stronger than that with βCD at first, and then 6-OLA-βCD was shown to decompose moderately into βCD and lactic acid. 6-OLA-βCD might be greatly valuable in a controlled release system for Amoxicillin (AMX).     

The role of extracellular matrix metalloproteinase inducer protein in prostate cancer progression

Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) is a multifunctional membrane glycoprotein overexpressed in many solid tumors, and involved in tumor invasion and angiogenesis. We investigated EMMPRIN expression in human prostate cancer (CaP) tissues and cells, and evaluated whether EMMPRIN expression is related to tumor progression and matrix metalloproteinase (MMPs) expression in human CaP. An immunohistochemical study using tissue microarrays of 120 primary CaPs of different grades and 20 matched lymph node metastases from untreated patients was performed. The association of EMMPRIN expression with clinicopathological parameters was evaluated. Co-immunolocalization for EMMPRIN and MMP-1, MMP-2 or MMP-9 in primary tumors was examined using confocal microscopy. Flow cytometry and immunoblotting were used to examine EMMPRIN expression in 11 metastatic CaP cell lines. Heterogeneous expression of EMMPRIN was found in 78/120 (65%) CaPs, correlated significantly with progression parameters including pre-treatment PSA level (P < 0.05) and increased with progression of CaP (Gleason score, P < 0.05; pathological stage, P < 0.01; nodal involvement, P < 0.05 and surgical margin, P < 0.05). Heterogeneous cytoplasmic MMP-1, MMP-2 and MMP-9 associated with EMMPRIN immunolabeling was observed, particularly in tumors with Gleason scores >3 + 4. Metastatic CaP cell lines, except DuCaP, expressed abundant EMMPRIN protein, indicating highly ( approximately 45 to approximately 65 kDa) and less ( approximately 30 kDa) glycosylated forms, although with no relationship to cells being either androgen responsive or nonresponsive. Our results suggest that EMMPRIN may regulate MMPs and be involved in CaP progression, and as such, could provide a target for treating metastatic CaP disease.