Modeling and simulation of fed-batch protein refolding process

The simplified kinetic model that assumes competition between first-order folding and third-order aggregation was used to model the fed-batch refolding of denatured-reduced lysozyme. It was found that the model was able to describe the process at limited concentration ranges, i.e., 1-2 and 5-7 mg mL(-)(1), respectively, at extensive guanidinium chloride (GdmCl) concentrations and controlled concentrations of oxidizing and reducing agents. The folding or aggregation rate constant was different at the two protein concentration ranges and strongly dependent on the denaturant concentration. As a result, both rate constants at the two concentration ranges were expressed as functions of GdmCl concentration. The rate constants determined by fed-batch experiments could be employed for the prediction of the fed-batch process but were not able to be extended to a batch refolding by direct dilution. Computer simulations show that the denaturant concentration and fed-batch flow rate are important factors influencing the refolding yield. Prolonged fed-batch time is beneficial to keep the transient intermediate concentration at a low level and to increase the yield of correctly folded protein. This is of importance when the denaturant concentration in refolding buffer solution is low. Thus, at a low denaturant concentration, fed-batch time should be sufficiently long, whereas at an appropriately high GdmCl concentration, a short fed-batch time or a high feed rate of the denatured protein is effective to give a high refolding yield.     

Flow cytometric assessment of the signaling status of human B lymphocytes from normal and autoimmune individuals

Abnormalities in lymphocyte signaling cascades are thought to play an important role in the development of autoimmune disease. However, the large amount of cellular material needed for standard biochemical assessment of signaling status has made it difficult to evaluate putative abnormalities completely using primary lymphocytes. The development of technology to employ intracellular staining and flow cytometry to assess the signaling status of individual cells has now made it possible to delineate the perturbations that are present in lymphocytes from patients with autoimmune disease. As an example, human B cells from the Ramos B cell line and the periphery of systemic lupus erythematosus (SLE) patients or normal nonautoimmune controls were assessed for activation of the NF-kappaB and mitogen activated protein kinase (MAPK) signaling cascades by intracellular multiparameter flow cytometric analysis and biochemical Western blotting. In combination with fluorochrome conjugated antibodies specific for surface proteins that define B cell subsets, antibodies that recognize activated, or phosphorylated inhibitors of kappaB (IkappaB) as well as the extracellular regulated kinase (ERK), jun N-terminal kinase (JNK) or p38 MAPKs were used to stain fixed and permeabilized human B cells and analyze them flow cytometrically. Examination of the known signaling pathways following engagement of CD40 on human B cells confirmed that intracellular flow cytometry and Western blotting equivalently assay CD154-induced phosphorylation and degradation of IkappaB proteins as well as phosphorylation of the MAPKs ERK, JNK and p38. In addition, B cells from the periphery of SLE patients had a more activated status immediately ex vivo as assessed by intracellular flow cytometric analysis of phosphorylated ERK, JNK and p38 when compared with B cells from the periphery of normal, nonautoimmune individuals. Together, these results indicate that multiparameter intracellular flow cytometric analysis of signaling pathways, such as the NF-kappaB and MAPK cascades, can be used routinely to assess the activation status of a small number of cells and thus delineate abnormalities in signaling molecules expressed in primary lymphocytes from patients with autoimmune disease.     

Construction, molecular modeling, and simulation of Mycobacterium tuberculosis cell walls

The mycobacterial cell wall is extraordinarily thick and tight consisting mainly of (1). long chain fatty acids, the mycolic acids, and (2). a unique polysaccharide, arabinogalactan (AG). These two chemical constituents are covalently linked through ester bonds. Minnikin (The Biology of the Mycobacteria; Academic: London, 1982) proposed that the mycobacterial cell wall is composed of an asymmetric lipid bilayer. The inner leaflet of the cell wall contains mycolic acids covalently linked to AG. This inner leaflet is believed to have the lowest permeability to organic compounds of the overall cell wall. Conformational search and molecular dynamics simulation were used to explore the conformational profile of AG and the conformations and structural organization of the mycolic acid-AG complex, and overall, an inner leaflet molecular model of the cell wall was constructed. The terminal arabinose residues of AG that serve as linkers between AG and mycolic acids were found to exist in four major chemical configurations. The mycolate hydrocarbon chains were determined to be tightly packed and perpendicular to the "plane" formed by the oxygen atoms of the 5-hydroxyl groups of the terminal arabinose residues. For Mycobacterium tuberculosis, the average packing distance between mycolic acids is estimated to be approximately 7.3 A. Thus, Minnikin's model is supported by this computational study. Overall, this modeling and simulation approach provides a way to probe the mechanism of low permeability of the cell wall and the intrinsic drug resistance of M. tuberculosis. In addition, monolayer models were built for both dipalmitoylphosphatidylethanolamine and dimyristoylphosphatidylcholine, two common phospholipids in bacterial and animal membranes, respectively. Structural comparisons of these cell wall phospholipid membrane models were made to the M. tuberculosis cell wall model.     

Near-infrared optical imaging of integrin alphavbeta3 in human tumor xenografts

In vivo optical imaging is potentially useful for evaluating the presence of tumor markers that are targets of molecular medicine. Here we report the synthesis and characterization of integrin alphavbeta3-targeted peptide cyclo(Lys-Arg-Gly-Asp-Phe) [c(KRGDf )] labeled with fluorescence dyes with wavelength spanning from the visible/near infrared (Cy5.5) to the true near infrared (IRDye800) for optical imaging. In vitro, the peptide-dye conjugates bound specifically to tumor cells expressing alphavbeta3. When administered intravenously into mice at a dose of 6 nmol /mouse, the conjugates accumulated in tumors expressing alphavbeta3. The tumor-to-background ratios for human KS1767 Kaposi's sarcoma in mice injected with Cy5.5-c(KRGDf ) and Cy5.5 were 5.5 and 1.5, respectively. Preinjection of c(KRGDf ) blocked the uptake of Cy5.5-c(KRGDf ) in tumors by 89%. In alphavbeta3-positive M21 and alphavbeta3-negative M21-L human melanoma, fluorescence intensity in the tumor of mice injected with IRDye800 - c(KRGDf ) was 2.3 and 1.3 times that in normal tissue, respectively. Dynamic imaging revealed that Cy5.5- c(KRGDf ) was rapidly taken up by KS1767 tumor immediately after bolus injection. The rate of its uptake in the tumor was reduced by preinjection of c(KRGDf ) in an interval time-dependent manner. Our data suggest that near-infrared fluorescence imaging may be applied to the detection of tumors expressing integrin alphavbeta3 and to the assessment of the optimal biological dose and schedule of targeted therapies.     

Homoepiboxidines: further potent agonists for nicotinic receptors

Homoepiboxidine (3) and the corresponding N-methyl (4) and N-benzyl (5) derivatives were prepared from a 6beta-carbomethoxynortropane (8). Affinities and functional activities at neuromuscular, central neuronal and ganglionic-type nicotinic receptors were compared to those of epibatidine 1, and epiboxidine 2. Homoepiboxidine had equivalent affinity/activity to epiboxidine at neuromuscular, neuronal alpha4beta2, and most alpha3-containing ganglionic-type nicotinic receptors. The N-substituted derivatives showed reduced affinity/activity at most receptor subtypes. Replacement of the methylisoxazole moiety of 3 and 4 with a methyloxadiazole moiety provided analogues 6 and 7, which had greatly reduced affinity/activity in virtually all assays at nicotinic receptors. Marked analgetic activity in mice occurred at the following ip doses: epibatidine 10 microg/kg; epiboxidine 25 microg/kg; homoepiboxidine 100 microg/kg; N-methylhomoepiboxidine 100 microg/kg; the methyloxadiazole (6) 100 microg/kg. The time course at such ip doses was significantly longer for homoepiboxidine 3 with marked analgesia still manifest at 30 min post-injection. Epiboxidine and the homoepiboxidines were less toxic than epibatidine.     

Reaction trajectory of pyrophosphoryl transfer catalyzed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg(2+)-dependent pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). The reaction follows a bi-bi mechanism with ATP as the first substrate and AMP and HP pyrophosphate (HPPP) as the two products. HPPK is a key enzyme in the folate biosynthetic pathway and is essential for microorganisms but absent from mammals. For the HPPK-catalyzed pyrophosphoryl transfer, a reaction coordinate is constructed on the basis of the thermodynamic and transient kinetic data we reported previously, and the reaction trajectory is mapped out with five three-dimensional structures of the enzyme at various liganded states. The five structures are apo-HPPK (ligand-free enzyme), HPPK.MgATP(analog) (binary complex of HPPK with its first substrate) and HPPK.MgATP(analog).HP (ternary complex of HPPK with both substrates), which we reported previously, and HPPK.AMP.HPPP (ternary complex of HPPK with both product molecules) and HPPK.HPPP (binary complex of HPPK with one product), which we present in this study.     

Crystal structure of a FYVE-type zinc finger domain from the caspase regulator CARP2

The caspase-associated ring proteins (CARP1 and CARP2) are distinguished from other caspase regulators by the presence of a FYVE-type zinc finger domain. FYVE-type domains are divided into two known classes: FYVE domains that specifically bind to phosphatidylinositol 3-phosphate in lipid bilayers and FYVE-related domains of undetermined function. Here, we report the crystal structure of the N-terminal region of CARP2 (44-139) including the FYVE-type domain and its associated helical bundle at 1.7 A resolution. The structure reveals a cramped phosphoinositide binding pocket and a blunted membrane insertion loop. These structural features indicate that the domain is not optimized to bind to phosphoinositides or insert into lipid bilayers. The CARP2 FYVE-like domain thus defines a third subfamily of FYVE-type domains that are functionally and structurally distinct. Structural analyses provide insights into the possible function of this unique subfamily of FYVE-type domains.     

Regulation of alternative splicing of Bcl-x by IL-6, GM-CSF and TPA

The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinct requirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficiently increased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 of the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effect. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron. Thus, IL-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.     

Binding activity of H-Ras is necessary for in vivo inhibition of ASK1 activity

H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals. In this study, we found that H-Ras interacted with ASK1 to cause the inhibition of both ASK1 activity and ASKl-induced apoptosis in vivo, which was reversed only partially by addition of RafS621 A, an antagonist of Raf, whereas MEK inhibitor, PD98059, and PI3K inhibitor, LY294002, did not disturb the inhibitory effect of H-Ras on ASK-1-induced apoptosis. Furthermore, by means of immunoprecipitate and kinase assays, we demonstrated that the interaction between H-Ras and ASK1 as well as the inhibition of ASKI activity were dependent on the binding activity of H-Ras. These results suggest that a novel mechanism may be involved in H-Rasmediated cell survival in addition to the well established MEK/ERK and PI3K/Akt kinase-dependent enhancement of cell survival.