Native PAGE Analysis Research Articles

Purification and Characterization of Cold-Adapted Metalloprotease from Deep Sea Water Lactic Acid Bacteria Enterococcus Faecalis TN-9

This paper investigated a 3-step purification and characterization of a protease from Enterococcus faecalis TN-9, abathypelagic lactic acid bacteria. The purification procedure includes precipitation with (NH4)2SO4, then ion-exchangechromatography with DEAE-Sephadex A-25 and DEAE Cellulofine A-500. Native PAGE analysis indicates a singleprotease band. The molecular weight is 30 kDa by SDS-PAGE analysis, and 69 kDa by gel chromatography analysis. Itproves that the optimal temperature for protease reaction is 30 oC, and the optimal pH is 7.5-8.0. The reaction is stablewhile pH is 6.0-9.5 and temperature is under 45 oC. The relative activity is 6.1% at 0 oC. The enzyme is totallydeactivated with heat treatment at 60 oC or over. The protease is partially inhibited by EDTA-2Na, Hg2+, Cu2+, Ni2+,Ag2+, Co2+ and Pepstatin A. Zn2+ shows obvious activation to the protease. Km and Vmax of purified protease acting onazocasein are 0.098 % and 72 mg/(h.mg) respectively. This protease is one of gelatinase with N-terminal sequence ofVGSEVTLKNS, and shows characteristics of a cold-adapted metalloprotease.

Analysis of Assembly and Trafficking of Native P2X4 and P2X7 Receptor Complexes in Rodent Immune Cells

P2X4 and P2X7 are the predominant P2X receptor subtypes expressed in immune cells. Having previously shown a structural and functional interaction between the two recombinant receptors, our aims here were to identify the preferred assembly pathway of the endogenous receptors in macrophage-like cells and to investigate the trafficking of these receptors between the plasma membrane and intracellular sites. We exploited the difference in size between the two subunits, and we used a combination of cross-linkers and blue native-PAGE analysis to investigate the subunit composition of complexes present in primary cultures of rat microglia and macrophages from wild type and P2X7(-/-) mice. Our results indicate that the preferred assembly pathway for both receptors is the formation of homotrimers. Homotrimers of P2X7 were able to co-immunoprecipitate with P2X4, suggesting that an interaction occurs between rather than within receptor complexes. In both macrophages and microglia, P2X7 receptors were predominantly at the cell surface, whereas P2X4 receptors were predominantly intracellular. There were clear cell type-dependent differences in the extent to which P2X4 receptors trafficked to and from the surface; trafficking was much more dynamic in microglia than in the macrophages, and further activation of cultured microglia with relatively short (3-h) incubations with lipopolysaccharide caused an approximately 4-fold increase in the fraction of receptors at the surface with only a 1.2-fold increase in total expression. The redistribution of intracellular receptors is thus an efficient means of enhancing the functional expression of P2X4 at the plasma membrane of microglia.

Membrane surface charge modulates lipoprotein complex forming capability of peptides derived from the C-terminal domain of apolipoprotein E

Apolipoprotein E (apoE) plays a major role in the transport and metabolism of lipid by acting as a ligand for low density lipoprotein-receptors. The amphipathic helical regions of its C-terminal domain are necessary for the lipoprotein binding and assembly of nascent lipoprotein particles. Lipoproteins in the plasma are known to possess a net negative charge, determined by both its protein and lipid components, which regulates the metabolism of lipoproteins. The role of membrane surface charge on the interaction of apoE has not been studied previously. Also the importance of individual amphipathic helical regions of its C-terminal domain in binding to negatively charged lipid membrane is not addressed. In this study we have compared the interaction of four peptide segments of apoE C-terminal domain (apoE(202–223), apoE(223–244), apoE(245–266), and apoE(268–289)) with zwitterionic and negatively charged model membranes by employing UV–visible and fluorescence spectroscopy, circular dichroism, and native PAGE analysis. Our results show that the peptide sequence 202–223, 245–266 and 268–289 of apoE has higher affinity towards negatively charged lipid membrane and are independently capable of forming lipoprotein particles of 17±2 nm Stokes diameter. The results suggest that surface charge of lipoprotein regulates its metabolism possibly by modulating the recruitment of apoE on its surface.

Reduction-sensitive and cysteine residue-mediated Streptococcus pneumoniae HrcA oligomerization in vitro.

In both gram-positive and several gram-negative bacteria, the transcription of dnaK and groE operons is negatively regulated by HrcA; however, the mechanism modulating HrcA protein activity upon thermal stress remains elusive. Here, we demonstrate that HrcA is modulated via reduction and oligomerization in vitro. Native-PAGE analysis was used to reveal the oligomeric structure of HrcA. The oligomeric HrcA structure became monomeric following treatment with the reducing agent dithothreitol, and this process was reversed by treatment with hydrogen peroxide. Moreover, the mutant HrcA C118S exhibited reduced binding to CIRCE elements and became less oligomerized, suggesting that cysteine residue 118 is important for CIRCE element binding as well as oligomerization. Conversely, HrcA mutant C280S exhibited increased oligomerization. An HrcA double mutant (C118S, C280S) was monomeric and exhibited a level of oligomerization and CIRCE binding similar to wild type HrcA, suggesting that cysteine residues 118 and 280 may function as checks to one another during oligomer formation. Biochemical fractionation of E. coli cells overexpressing HrcA revealed the presence of HrcA in the membrane fraction. Together, these results suggest that the two HrcA cysteine residues at positions 118 and 280 function as reduction sensors in the membrane and mediate oligomerization upon stress.

Essential Roles for Coiled-coil Domains in STIM1 Oligomerization and CRAC Channel Activation

The calcium release-activated calcium (CRAC) channel is activated by depletion of Ca2+ from the ER. Store depletion causes the ER Ca2+ sensor, STIM1, to translocate to sites of close ER-plasma membrane apposition, where it interacts with Orai1, the pore-forming subunit of the CRAC channel and activates Ca2+ entry. STIM1 self-associates in resting cells (Baba et al., PNAS 103:16704, 2006) and further oligomerizes after store depletion (Liou et al., PNAS 104:9301, 2007), an event that triggers the self-assembly and activation of STIM1-Orai1 clusters at ER-PM junctions (Luik et al., Nature 454:538, 2008). STIM1 has several protein interaction domains, including a lumenal sterile alpha motif (SAM) and two putative cytosolic coiled-coil regions. The isolated lumenal EF hand - SAM region is known to oligomerize upon Ca2+ removal in vitro (Stathopulos et al., JBC 281:35855, 2006), but the roles of the coiled-coil domains in the functions of STIM1 in situ are not as well understood. Using fluorescence recovery after photobleaching, co-immunoprecipitation, and blue native PAGE analysis on truncated mutants of STIM1 we show that the two coiled-coil domains of STIM1 affect STIM1 oligomerization in different ways. The ER-proximal coiled-coil is sufficient for the self-association of STIM1 in resting cells but does not by itself support oligomerization in response to store depletion. The distal coiled-coil is required for depletion-induced oligomerization. Mutation of specific residues within the predicted hydrophobic interface of the distal coiled-coil prevents the formation of STIM1 puncta and the activation of CRAC channels. These results reveal an essential role for the distal coiled-coil of STIM1 in the oligomerization step that controls store-operated Ca2+ entry.

The orientation of the ends of G-quadruplex structures investigated using end-extended oligonucleotides

Human telomere DNA is of intense interest because of its role in the biology of both cancer and aging. The single-stranded telomere terminus can adopt the structure of a G-quadruplex, which is of particular important for anticancer drug discovery many researchers have reported various G-quadruplex structures in the human telomere. Although the human telomere consists of a number of tandem repeats, higher-order G-quadruplex structures are less discussed due to the complexity of the structures. Here we examined the orientation of the ends of the G-quadruplex structures with consideration given to higher-order structures. We prepared end-extended and BrG-substituted oligonucleotides. Native PAGE analysis, CD measurements and NMR spectroscopy showed that the ends of stable G-quadruplex structures point in opposite directions. Our results indicate that the human telomere DNA is likely to form rod-like higher-order structures. This may provide important information for understanding telomere structure and the development of telomere G-quadruplex-binding molecules as telomerase inhibitors.

Superoxide Dismutase and Catalase Activities in Vibrio Cholerae Non-O1 Strains

ABSTRACTAntioxidant enzymes are essential for living cells, producing protection from reactive oxygen species such as superoxide, which cause oxidative damage to cell structures. Superoxide dismutase (SOD) and catalase (CAT) activities were determined for three Vibrio strains (Vibrio cholerae non O1/29, V. cholerae non O1/29-T and V. cholerae non O1/26) aerobically grown at 30°C (optimal level) and 10 °C (cold stress). All strains tested expressed both antioxidant enzymes under normophysiological and stress conditions, but the cell response is more strain-dependent than dependent on temperature. Levels of SOD in cultures of V. cholerae non O1/26 grown at 30°C were about 2.5 to 9.5-times higher than those in the cultures grown at 10°C. In contrast, SOD activity in V. cholerae non O1/29 increased by 7.5-fold under stress conditions in comparison to that at optimal temperature. The strain V. cholerae non O1/29-T did not show any significant difference in the cell response depending on the growth temperature. CAT activity in cells of V. cholerae non O1/26 and V. cholerae non O1/29-T exhibited a similar tendency suggesting that this enzyme is not included in antioxidant response against cold stress. Contrary to the above observations, V. cholerae non 01/29 demonstrated higher CAT activity in response to temperature downshift. Only one SOD isoenzyme was detected in each of the three Vibrio strains by native PAGE analysis.

Quaternary Structure Assessment of ICln by Fluorescence Resonance Energy Transfer (FRET) in vivo

ICln is a ubiquitously expressed multifunctional protein that plays a critical role in regulatory volume decrease after cell swelling. The majority of ICln is localized in the cytosol and a small fraction of ICln associates with the plasma membrane. In artificial lipid bilayers ICln forms ion channels, and a putative channel model predicts the association of at least two ICln molecules to form a functional ion-conducting pore. Oligomers of ICln have been demonstrated in cytosolic fractions of different cells by native PAGE and gel filtration analysis, but these data have not yet been verified in vivo, and the basis of ICln homooligomerization is unknown. In silico prediction of the quaternary structure of ICln from its primary structure predicts that ICln forms a dimer, and that the C-terminus of ICln may be essential for the intermolecular interaction. To explore the quaternary structure of ICln in living NIH3T3 fibroblasts, we performed fluorescence resonance energy transfer (FRET) experiments using eCFP (donor) and eYFP (acceptor) fused to the C- and/or N-termini of both full length wild type ICln and of C-terminal truncation mutants thereof (ICln₁₅₉ and ICln₁₃₄). FRET was assessed by the acceptor photobleaching technique. Here we show that ICln forms oligomers in vivo, and demonstrate intermolecular FRET between the C-, but not the N-termini of full length ICln. In the truncation mutant ICln₁₅₉ oligomerization occurs and intermolecular FRET between N-termini can be detected, which indicates that the C-terminus of ICln sterically interferes with interactions between N-termini in full length ICln oligomers. In cells expressing the truncation mutant ICln₁₃₄ no FRET between C- and/or N-termini could be measured, suggesting the absence of interaction and a role of amino acids P135-Q159 in the oligomerization of ICln.

Dissociation and Reconstitution Studies of a Broad Substrate Specific Multimeric Alcohol Oxidase Protein Produced by Aspergillus terreus

A multimeric alcohol oxidase from Aspergillus terreus was dissociated and simultaneously deflavinated into catalytically inactive FAD-free subunits when incubated with 0.74 M beta-mercaptoethanol (beta-ME) for 8 h at 4 degrees C. This dissociation process had traversed through two FAD-associated intermediate proteins, between these one of them showed the enzyme activity. On removal of beta-ME, the multimeric apoprotein was regenerated, which was, however, catalytically inactive. Reactivation of the FAD supplemented apoprotein was accomplished only after incubating with the substrate. This catalytic reactivation was a slow process as evident from the prolonged FAD emission quenching. The dissociation and re-association phenomena were demonstrated by using dynamic light scattering, size exclusion chromatographic, confocal laser scan microscopic and native PAGE analyses. The solvent effect caused by the high concentration of beta-ME is attributed to the observed dissociation and linked deflavination of these multimeric alcohol oxidase protein particles.

Self-association of Chaetopterus variopedatus sperm histone H1-like. Relevance of arginine content and possible physiological role.

Self-association of histones H1 from calf thymus and from sperm of the marine worm Chaetopterus variopedatus was studied on native and glutaraldehyde cross-linked molecules by PAGE and by salt-induced turbidity measurements. Multiple polymers were generated by native sperm histone H1-like after glutaraldehyde cross-linking while the same treatment on its lysine- or arginine-modified derivatives and on somatic histone H1 failed to induce polymerization. This result suggests the relevance of arginine content in the formation of histone H1-like polymers particularly because Chaetopterus variopedatus and calf thymus histones H1 have similar content of lysine but different K/R ratio (2 and 15, respectively). Salt-induced turbidity experiments confirmed the high tendency of sperm histone H1-like to form oligomers, particularly in the presence of phosphate ions. Native PAGE analysis in the presence of phosphate supported this hypothesis. The reported results suggest that phosphate ions connecting lysine and arginine side chain groups contribute to the interaction of sperm histone H1-like with DNA in chromatin and play a key role in organization and stabilization of the chromatin higher order structures.

Stable Lariat Formation Based on a G-Quadruplex Scaffold

In the telomere region, T-loop structure is thought to participate in protecting chromosome ends. Although the tetraplex structure, called G-quadruplex, is suggested to form in the same region, the relationship between these two structures are less discussed. Here we designed the long lariat formation using G-quadruplex structure. The structural property was characterized by means of CD spectroscopy, FRET experiment, and native PAGE analysis. Because this lariat formation contains both loop structure and tetraplex structure, it can be a key structure linking T-loop structure to G-quadrurplex structure.

A novel streptococcal leucine zipper protein (Lzp) binds to human immunoglobulins

Streptococcus pyogenes is an important pathogen that causes pharyngitis, scarlet fever, rheumatic fever, and streptococcal toxic shock syndrome. To survive within its host, S. pyogenes has developed several immune evasion mechanisms. Here, we identified a novel gene encoding a 66-kDa protein with many leucine zipper motifs, that we call streptococcal leucine zipper protein (Lzp). Lzp was expressed on the bacterial cell surface, and some was detected in the culture medium. Lzp was expressed by all the S. pyogenes strains we tested, but not by group B streptococcal strains. Western blotting and Biacore assay demonstrated that recombinant Lzp bound to human IgA, IgG, IgM, and Lzp. In addition, native-PAGE analysis suggested that the Lzp molecule formed dimer and trimer conformations. Thus, Lzp is a novel immunoglobulin-binding protein that may play a role in helping S. pyogenes escape detection by the host immune system.

Dissecting β-ring assembly pathway of the mammalian 20S proteasome

The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). Recent studies indicated that proteasome-specific chaperones and beta-subunit appendages assist in the formation of alpha-rings and dimerization of half-proteasomes, but the process involved in the assembly of beta-rings is poorly understood. Here, we clarify the mechanism of beta-ring formation on alpha-rings by characterizing assembly intermediates accumulated in cells depleted of each beta-subunit. Starting from beta2, incorporation of beta-subunits occurs in an orderly manner dependent on the propeptides of beta2 and beta5, and the C-terminal tail of beta2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as beta2 and is required for the structural integrity of early assembly intermediates. We propose a model in which beta-ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals.

DNase and RNase Responses of Petunia × hybrida During Transition to Flowering as Affected by Light Treatments

The responses of DNase and RNase isoforms and their specific activities following transition to flowering (1 to 6 weeks) were examined in Petunia × hybrida under different light conditions. Petunia × hybrida plants formed flower buds at the 4th week in the case of high light and at the 6th week in the far-red light treatment, while no flower bud formation was observed upon red light and control light treatments. The DNase and RNase activities decreased from the 1st to the 6th week during transition to flowering. Native-PAGE analysis revealed the appearance of one DNase (D1) and seven RNase (R1 - R7) isoforms in all light treatments. It is assumed that the progress of the flowering could be related to the disappearance or reduction of D1 DNase band intensity and disappearance of R1, R2 and R7 RNase isoforms. Consequently, these isoforms could be used as potent biochemical markers of flower bud formation under light intensity as well as light quality treatments.

Petunia × hybrida during transition to flowering as affected by light intensity and quality treatments

Petunia × hybrida was grown under high (H), medium (M) and low (L) light intensity [photoperiod; 16 h d−1, photosynthetic photon flux density (PPFD); 360, 120 and 40 μmol m−2 s−1, respectively] as well as under end-of-day (EOD) red (R) and far-red (FR) light quality treatments [photoperiod; 14.5 h d−1, PPFD; 30 μmol m−2 s−1 EOD; 15 min, Control (C) light; without EOD light treatment]. Shoot growth, leaf anatomical and photosynthetic responses as well as the responses of peroxidase (POD) isoforms and their specific activities following transition to flowering (1–6 weeks) were evaluated. Flower bud formation of Petunia × hybrida was achieved at the end of the 4th week for H light treatment and on the end of the 6th week for FR light treatment. No flower bud formation was noticed in the C and R light treatments. H and M light treatments induced lower chlorophyll (Chla, Chlb, Chla+b) concentrations in comparison to L light. On the other hand R and FR light chlorophyll content were similar to C light. Photosynthetic parameters [CO2 assimilation rate (A), transpiration rate (E) and stomatal conductance (gs) values] were higher in the H light treated plants in comparison to M and L light treated plants. A, E and gs values of R and FR light were similar to C light plants. Leaf anatomy revealed that total leaf thickness, thickness of the contained tissues (epidermis, palisade and spongy parenchyma) and relative volume percentages of the leaf histological components were differently affected within the light intensity and the light quality treatments. POD specific activities increased from the 1st to the 6th week during transition to flowering. Native-PAGE analysis revealed the appearance of four anionic POD (A1–A4) isoforms in all light treatments. On the basis of the leaf anatomical, photosynthetic and plant morphological responses, the production of high quality Petunia × hybrida plants with optimal flowering times could be achieved through the control of both light intensity and light quality. The appearance of A1 and A2 anionic POD isoforms could be also used for successful scheduling under light treatments.

Responses of MxPPO overexpressing transgenic tall fescue plants to two diphenyl-ether herbicides, oxyfluorfen and acifluorfen

We generated transgenic tall fescue (Festuca arundinacea Schreb. cv. Kentucky-31) plants harboring a synthetic Myxococcus xanthus protoporphyrinogen oxidase (MxPPO) gene through Agrobacterium-mediated gene transfer. Successful integration of the transgene into the genome of transgenic plants confirmed by polymerase chain reaction (PCR) and Southern blot analysis, and the functional expression of the MxPPO gene at the mRNA level in transgenic lines was validated by Northern blot analysis. Responses of transgenic and non-transgenic tall fescue plants to diphenyl-ether herbicides such as oxyfluorfen and acifluorfen have been evaluated in respect of various physiological and biochemical parameters. Differential responses were observed in chlorophyll content, in vivo H2O2 deposition and lipid peroxidation in both transgenic and non-transgenic plants exposed to oxyfluorfen or acifluorfen. Isozyme profiles of four antioxidant-enzymes, including peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), were also investigated in transgenic and non-transgenic plants using native PAGE analysis. Compared to the transgenic lines, higher staining activities of the examined antioxidant-enzymes observed in non-transgenic plants subjected to 100 μM of oxyfluorfen or acifluorfen suggests that non-transgenic plants are unable to prevent the photodynamic induced oxidative stress caused by herbicides. In addition, both transgenic and non-transgenic plants exposed to oxyfluorfen exhibited proportionally increased band-staining patterns in contrast to acifluorfen, which suggests that oxyfluorfen has relatively greater or more rapid effects on leaves than acifluorfen.

Response of a submersed macrophyte Hydrilla verticillata (L.f) Royle, to Sodium dodecylbenzene sulfonate stress

The acute toxicity of Sodium dodecylbenzene sulfonate (SDBS) to subcamersed macrophyte Hydrilla verticillata (L.f) Royle was studied. Chlorophyll contents and the activities of 3 antioxidant enzymes: superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) in the leaves were investigated under different concentrations of SDBS, The chlorophyll contents in leaves of experimental plants decreased gradually, while the electrical conductivity of culture solution increased gradually with the increased dose of SDBS. Under higher concentrations of SDBS (32 mg/L and 128 mg/L) treatment, SOD lost its activities completely after 24 h. The native PAGE analysis indicated the diversity of POD isoenzymes was obvious under different concentrations of SDBS stress.

Regulation of defense-related enzymes associated with bacterial spot resistance in Tomato

Twenty tomato (Solanum lycopersicon) cultivars were screened for resistance against bacterial spot disease incited byXanthomonas axonopodis pv.vesicatoria under field conditions with and without pathogen infection. Screening was done by artificially inoculating aX. axonopodis pv.vesicatoria suspension to 4-week-old tomato seedlings and observing them for typical symptoms of the disease. Seedlings were categorized into highly resistant, resistant, susceptible and highly susceptible cultivars on the basis of disease incidence. Tomato cultivars were screened for defense-related enzymes, total phenols and lignin contents. The temporal patterns of all these enzymes were estimated with a moderately susceptible tomato cultivar. Native PAGE analysis of both peroxidase (POX) and polyphenol oxidase (PPO) was carried out for the time course of enzyme activities and also by selecting three different tomato cultivars, following infection with the pathogen. Based on the inducible amounts of these enzymes upon pathogen infection, the tomato cultivars were correlated with the disease incidence under field conditions. A significant (P≤0.05) correlation was observed between the degree of host resistance and the enzyme levels. In highly resistant tomato cultivars the enzyme levels, total phenols and lignin contents were increased in comparison with highly susceptible tomato cultivars. Isoform analysis of POX and PPO enzymes indicated a clear difference between resistant and susceptible tomato cultivars in the number of isoforms and also in the intensity of each isoform in the presence of pathogen infection. The possible regulation of defense-related enzymes in imparting host resistance is discussed.

Paraquat-resistant lines in Pisum sativum cv. Alaska: biochemical and phenotypic characterization

In plants, the oxygen generated by photosynthesis can be excited to form reactive oxygen species (ROS) under excessive sunlight. Excess ROS including singlet oxygen (1O2) inhibit the growth, development and photosynthesis of plants. To isolate ROS-resistant crop plants, we used paraquat (PQ), a generator of O2 ·− as a source of screening and mutagen, and obtained two PQ-resistant lines in Pisum sativum, namely R3-1 and R3-2. Both lines showed greater resistance to PQ than their wild type (WT) siblings with respect to germination, root growth, and shoot growth. Biochemical analysis showed differences in these lines, in which ROS-scavenging enzymes undergo changes with a distinguishable increase in Mn-SOD. We further observed that the cytosolic catalases (CATs) in leaves in both lines were shifted in a native-PAGE analysis compared with that of the WT, indicating that the release of bound 1O2 was enhanced. Phenotypic analysis revealed distinguishable differences in leaf development, and in flowering time and position. In addition, R3-1 and R3-2 showed shorter individual internode lengths, dwarf plant height, and stronger branching compared with the WT. These results suggested that PQ-induced ROS-resistant Pisum have the potential pleiotropic effects on flowering time and stem branching, and that ROS including 1O2 plays not only important roles in plant growth and development as a signal transducer, but also appears as a strong inhibitor for crop yield.

High Mobility Group Box 1 Protein Binding to Lipopolysaccharide Facilitates Transfer of Lipopolysaccharide to CD14 and Enhances Lipopolysaccharide-Mediated TNF-α Production in Human Monocytes

LPS-binding protein (LBP) is a central mediator that transfers LPS to CD14 to initiate TLR4-mediated proinflammatory response. However, a possibility of another LPS transfer molecule has been suggested because LBP-deficient mice showed almost normal inflammatory response after LPS injection. In this study, we describe the novel finding that high mobility group box 1 protein (HMGB1) recently identified as a mediator of sepsis has a function of LPS transfer for a proinflammatory response. We used ELISA and surface plasmon resonance to show that HMGB1 binds LPS in a concentration-dependent manner and that the binding is stronger to lipid A moiety than to the polysaccharide moiety of LPS. This binding was inhibited by LBP and polymyxin B. Using native PAGE and fluorescence-based LPS transfer analyses, we show that HMGB1 can catalytically disaggregate and transfer LPS to both soluble CD14 protein and to human PBMCs in a dose-dependent manner. However, this effect was dramatically reduced to the baseline level when HMGB1 was heat inactivated. Furthermore, a mixture of HMGB1 and LPS treatment results in a higher increase in TNF-alpha production in human PBMCs and peripheral blood monocytes than LPS or HMGB1 treatment alone or their summation. Thus, we propose that HMGB1 plays an important role in Gram-negative sepsis by catalyzing movement of LPS monomers from LPS aggregates to CD14 to initiate a TLR4-mediated proinflammatory response.