Electron-transfer dissociation (ETD) delivers the unique attributes of electron capture dissociation to mass spectrometers that utilize radio frequency trapping-type devices (e.g., quadrupole ion traps). The method has generated significant interest because of its compatibility with chromatography and its ability to: (1) preserve traditionally labile post-translational modifications (PTMs) and (2) randomly cleave the backbone bonds of highly charged peptide and protein precursor ions. ETD, however, has shown limited applicability to doubly protonated peptide precursors, [M + 2H]2+, the charge and type of peptide most frequently encountered in "bottom-up" proteomics. Here we describe a supplemental collisional activation (CAD) method that targets the nondissociated (intact) electron-transfer (ET) product species ([M + 2H]+*) to improve ETD efficiency for doubly protonated peptides (ETcaD). A systematic study of supplementary activation conditions revealed that low-energy CAD of the ET product population leads to the near-exclusive generation of c- and z-type fragment ions with relatively high efficiency (77 +/- 8%). Compared to those formed directly via ETD, the fragment ions were found to comprise increased relative amounts of the odd-electron c-type ions (c+*) and the even-electron z-type ions (z+). A large-scale analysis of 755 doubly charged tryptic peptides was conducted to compare the method (ETcaD) to ion trap CAD and ETD. ETcaD produced a median sequence coverage of 89%-a significant improvement over ETD (63%) and ion trap CAD (77%).

Curcumin can reduce inflammation and neurodegeneration, but its chemical instability and metabolism raise concerns, including whether the more stable metabolite tetrahydrocurcumin (TC) may mediate efficacy. We examined the antioxidant, anti-inflammatory, or anti-amyloidogenic effects of dietary curcumin and TC, either administered chronically to aged Tg2576 APPsw mice or acutely to lipopolysaccharide (LPS)-injected wild-type mice. Despite dramatically higher drug plasma levels after TC compared with curcumin gavage, resulting brain levels of parent compounds were similar, correlating with reduction in LPS-stimulated inducible nitric-oxide synthase, nitrotyrosine, F2 isoprostanes, and carbonyls. In both the acute (LPS) and chronic inflammation (Tg2576), TC and curcumin similarly reduced interleukin-1beta. Despite these similarities, only curcumin was effective in reducing amyloid plaque burden, insoluble beta-amyloid peptide (Abeta), and carbonyls. TC had no impact on plaques or insoluble Abeta, but both reduced Tris-buffered saline-soluble Abeta and phospho-c-Jun NH(2)-terminal kinase (JNK). Curcumin but not TC prevented Abeta aggregation. The TC metabolite was detected in brain and plasma from mice chronically fed the parent compound. These data indicate that the dienone bridge present in curcumin, but not in TC, is necessary to reduce plaque deposition and protein oxidation in an Alzheimer's model. Nevertheless, TC did reduce neuroinflammation and soluble Abeta, effects that may be attributable to limiting JNK-mediated transcription. Because of its favorable safety profile and the involvement of misfolded proteins, oxidative damage, and inflammation in multiple chronic degenerative diseases, these data relating curcumin dosing to the blood and tissue levels required for efficacy should help translation efforts from multiple successful preclinical models.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones secreted in response to meal ingestion, thereby enhancing postprandial insulin secretion. Therefore, an attenuated incretin response could contribute to the impaired insulin responses in patients with diabetes mellitus. The aim of the present investigation was to investigate incretin secretion, in obesity and type 1 and type 2 diabetes mellitus, and its dependence on the magnitude of the meal stimulus. Plasma concentrations of incretin hormones (total, reflecting secretion and intact, reflecting potential action) were measured during two meal tests (260 kcal and 520 kcal) in eight type 1 diabetic patients, eight lean healthy subjects, eight obese type 2 diabetic patients, and eight obese healthy subjects. Both in diabetic patients and in healthy subjects, significant increases in GLP-1 and GIP concentrations were seen after ingestion of both meals. The incretin responses were significantly higher in all groups after the large meal, compared with the small meal, with correspondingly higher C-peptide responses. Both type 1 and type 2 diabetic patients had normal GIP responses, compared with healthy subjects, whereas decreased GLP-1 responses were seen in type 2 diabetic patients, compared with matched obese healthy subjects. Incremental GLP-1 responses were normal in type 1 diabetic patients. Increased fasting concentrations of GIP and an early enhanced postprandial GIP response were seen in obese, compared with lean healthy subjects, whereas GLP-1 responses were the same in the two groups. beta-cell sensitivity to glucose, evaluated as the slope of insulin secretion rates vs. plasma glucose concentration, tended to increase in both type 2 diabetic patients (29%, P = 0.19) and obese healthy subjects (22% P = 0.04) during the large meal, compared with the small meal, perhaps reflecting the increased incretin response. We conclude: 1) that a decreased GLP-1 secretion may contribute to impaired insulin secretion in type 2 diabetes mellitus, whereas GIP and GLP-1 secretion is normal in type 1 diabetic patients; and 2) that it is possible to modulate the beta-cell sensitivity to glucose in obese healthy subjects, and possibly also in type 2 diabetic patients, by giving them a large meal, compared with a small meal.

The neurodegeneration of Alzheimer's disease has been theorized to be mediated, at least in part, by insoluble aggregates of beta-amyloid protein that are widely distributed in the form of plaques throughout brain regions affected by the disease. Previous studies by our laboratory and others have demonstrated that the neurotoxicity of beta-amyloid in vitro is dependent upon its spontaneous adoption of an aggregated structure. In this study, we report extensive structure-activity analyses of a series of peptides derived from both the proposed active fragment of beta-amyloid, beta 25-35, and the full-length protein, beta 1-42. We examine the effects of amino acid residue deletions and substitutions on the ability of beta-amyloid peptides to both form sedimentable aggregates and induce toxicity in cultured hippocampal neurons. We observe that significant levels of peptide aggregation are always associated with significant beta-amyloid-induced neurotoxicity. Further, both N- and C-terminal regions of beta 25-35 appear to contribute to these processes. In particular, significant disruption of peptide aggregation and toxicity result from alterations in the beta 33-35 region. In beta 1-42 peptides, aggregation disruption is evidenced by changes in both electrophoresis profiles and fibril morphology visualized at the light and electron microscope levels. Using circular dichroism analysis in a subset of peptides, we observed classic features of beta-sheet secondary structure in aggregating, toxic beta-amyloid peptides but not in nonaggregating, nontoxic beta-amyloid peptides. Together, these data further define the primary and secondary structures of beta-amyloid that are involved in its in vitro assembly into neurotoxic peptide aggregates and may underlie both its pathological deposition and subsequent degenerative effects in Alzheimer's disease.

The term PEGylation describes the modification of biological molecules by covalent conjugation with polyethylene glycol (PEG), a non-toxic, non-immunogenic polymer, and is used as a strategy to overcome disadvantages associated with some biopharmaceuticals. PEGylation changes the physical and chemical properties of the biomedical molecule, such as its conformation, electrostatic binding, and hydrophobicity, and results in an improvement in the pharmacokinetic behavior of the drug. In general, PEGylation improves drug solubility and decreases immunogenicity. PEGylation also increases drug stability and the retention time of the conjugates in blood, and reduces proteolysis and renal excretion, thereby allowing a reduced dosing frequency. In order to benefit from these favorable pharmacokinetic consequences, a variety of therapeutic proteins, peptides, and antibody fragments, as well as small molecule drugs, have been PEGylated. This paper reviews the chemical procedures and the conditions that have been used thus far to achieve PEGylation of biomedical molecules. It also discusses the importance of structure and size of PEGs, as well as the behavior of linear and branched PEGs. A number of properties of the PEG polymer--e.g. mass, number of linking chains, the molecular site of PEG attachment--have been shown to affect the biological activity and bioavailability of the PEGylated product. Releasable PEGs have been designed to slowly release the native protein from the conjugates into the blood, aiming at avoiding any loss of efficacy that may occur with stable covalent PEGylation. Since the first PEGylated drug was developed in the 1970s, PEGylation of therapeutic proteins has significantly improved the treatment of several chronic diseases, including hepatitis C, leukemia, severe combined immunodeficiency disease, rheumatoid arthritis, and Crohn disease. The most important PEGylated drugs, including pegademase bovine, pegaspargase, pegfilgrastim, interferons, pegvisomant, pegaptanib, certolizumab pegol, and some of the PEGylated products presently in an advanced stage of development, such as PEG-uricase and PEGylated hemoglobin, are reviewed. The adaptations and applications of PEGylation will undoubtedly prove useful for the treatment of many previously difficult-to-treat conditions.

Type I diabetes may be an autoimmune disorder, although the evidence is largely circumstantial. The natural history of the disease after diagnosis includes partial remission in most patients, but only about 3 percent achieve transient insulin independence. beta Cell function, as indicated by the plasma concentration of C-peptide, is lost over 6 to 30 months and islet cell antibodies disappeared over 1 to 2 years. This article describes a pilot study in which 41 patients were treated with the immunosuppressive agent cyclosporine for 2 to 12 months. Of 30 patients treated within 6 weeks of diagnosis, 16 became insulin independent with concentrations of plasma C-peptide in the normal range and decreasing titers of islet cell antibodies. Of 11 patients who entered the study 8 to 44 weeks after diagnosis, two achieved this state. These results indicate that a controlled trial of the effects of cyclosporine in type I diabetes should be conducted.

Mature human aorta contains a 70-kDa versican fragment, which reacts with a neoepitope antiserum to the C-terminal peptide sequence DPEAAE. This protein therefore appears to represent the G1 domain of versican V1 (G1-DPEAAE(441)), which has been generated in vivo by proteolytic cleavage at the Glu(441)-Ala(442) bond, within the sequence DPEAAE(441)-A(442)RRGQ. Because the equivalent aggrecan product (G1-NITEGE(341)) and brevican product (G1-EAVESE(395)) are generated by ADAMTS-mediated cleavage of the respective proteoglycans, we tested the capacity of recombinant ADAMTS-1 and ADAMTS-4 to cleave versican at Glu(441)-Ala(442). Both enzymes cleaved a recombinant versican substrate and native human versican at the Glu(441)-Ala(442) bond and the mature form of ADAMTS-4 was detected by Western analysis of extracts of aortic intima. We conclude that versican V1 proteolysis in vivo can be catalyzed by one or more members of the ADAMTS family of metalloproteinases.

The detection and quantification of plasma (serum) proteins at or below the ng/ml concentration range are of critical importance for the discovery and evaluation of new protein biomarkers. This has been achieved either by the development of high sensitivity ELISA or other immunoassays for specific proteins or by the extensive fractionation of the plasma proteome followed by the mass spectrometric analysis of the resulting fractions. The first approach is limited by the high cost and time investment for assay development and the requirement of a validated target. The second, although reasonably comprehensive and unbiased, is limited by sample throughput. Here we describe a method for the detection of plasma proteins at concentrations in the ng/ml or sub-ng/ml range and their accurate quantification over 5 orders of magnitude. The method is based on the selective isolation of N-glycosites from the plasma proteome and the detection and quantification of targeted peptides in a quadrupole linear ion trap instrument operated in the multiple reaction monitoring (MRM) mode. The unprecedented sensitivity of the mass spectrometric analysis of minimally fractionated plasma samples is the result of the significantly reduced sample complexity of the isolated N-glycosites compared with whole plasma proteome digests and the selectivity of the MRM process. Precise quantification was achieved via stable isotope dilution by adding (13)C- and/or (15)N-labeled reference analytes. We also demonstrate the possibility of significantly expanding the number of MRM measurements during one single LC-MS run without compromising sensitivity by including elution time constraints for the targeted transitions, thus allowing quantification of large sets of peptides in a single analysis.

The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin alpha5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.

We have previously demonstrated that the Gag p9 protein of equine infectious anemia virus (EIAV) is functionally homologous with Rous sarcoma virus (RSV) p2b and human immunodeficiency virus type 1 (HIV-1) p6 in providing a critical late assembly function in RSV Gag-mediated budding from transfected COS-1 cells (L. J. Parent et al., J. Virol. 69:5455-5460, 1995). In light of the absence of amino acid sequence homology between EIAV p9 and the functional homologs of RSV and HIV-1, we have now designed an EIAV Gag-mediated budding assay to define the late assembly (L) domain peptide sequences contained in the EIAV p9 protein. The results of these particle budding assays revealed that expression of EIAV Gag polyprotein in COS-1 cells yielded extracellular Gag particles with a characteristic density of 1.18 g/ml, while expression of EIAV Gag polyprotein lacking p9 resulted in a severe reduction in the release of extracellular Gag particles. The defect in EIAV Gag polyprotein particle assembly could be corrected by substituting either the RSV p2b or HIV-1 p6 protein for EIAV p9. These observations demonstrated that the L domains of EIAV, HIV-1, and RSV were interchangeable in mediating assembly of EIAV Gag particles in the COS-1 cell budding assay. To localize the L domain of EIAV p9, we next assayed the effects of deletions and site-specific mutations in the p9 protein on its ability to mediate budding of EIAV Gag particles. Analyses of EIAV Gag constructs with progressive N-terminal or C-terminal deletions of the p9 protein identified a minimum sequence of 11 amino acids (Q20N21L22Y23P24D25L26S27E28I29K30) capable of providing the late assembly function. Alanine scanning studies of this L-domain sequence demonstrated that mutations of residues Y23, P24, and L26 abrogated the p9 late budding function; mutations of other residues in the p9 L domain did not substantially affect the level of EIAV Gag particle assembly. These data indicate that the L domain in EIAV p9 utilizes a YXXL motif which we hypothesize may interact with cellular proteins to facilitate virus particle budding from infected cells.

A surface protein antigen of Streptococcus mutans having two sets of antigenic determinants (antigens I and II) was purified by column chromatography from culture supernatants of S. mutans serotype c. The protease-resistant component, antigen II, was purified from pronase-digested antigen I/II. The antigens were analyzed chemically and immunologically, and their physicochemical properties were investigated. Antigen I/II consisted of more than 80% protein, and its peptide chain molecular weight was estimated to be 185,000. Antigen II consisted of approximately 60% protein, with a peptide chain molecular weight of 48,000. Antisera to antigens I/II and II were raised in rabbits and used to investigate the presence of the antigens in cells of other streptococci. This indicated that not only serotype c but also serotypes e and f possessed antigen I and II determinants, whereas serotypes a, d, and g possessed a determinant related to antigen I but not one related to antigen II.

We performed a large scale study of electron transfer dissociation (ETD) performance, as compared with ion trap collision-activated dissociation (CAD), for peptides ranging from approximately 1000 to 5000 Da (n approximately 4000). These data indicate relatively little overlap in peptide identifications between the two methods ( approximately 12%). ETD outperformed CAD for all charge states greater than 2; however, regardless of precursor charge a linear decrease in percent fragmentation, as a function of increasing precursor m/z, was observed with ETD fragmentation. We postulate that several precursor cation attributes, including peptide length, charge distribution, and total mass, could be relevant players. To examine these parameters unique ETD-identified peptides were sorted by length, and the ratio of amino acid residues per precursor charge (residues/charge) was calculated. We observed excellent correlation between the ratio of residues/charge and percent fragmentation. For peptides of a given residue/charge ratio, there is no correlation between peptide mass and percent fragmentation; instead we conclude that the ratio of residues/charge is the main factor in determining a successful ETD outcome. As charge density decreases so does the probability of non-covalent interactions that can bind a newly formed c/z-type ion pair. Recently we have described a supplemental activation approach (ETcaD) to convert these non-dissociative electron transfer product ions to useful c- and z-type ions. Automated implementation of such methods should remove this apparent precursor m/z ceiling. Finally, we evaluated the role of ion density (both anionic and cationic) and reaction duration for an ETD experiment. These data indicate that the best performance is achieved when the ion trap is filled to its space charge limit with anionic reagents. In this largest scale study of ETD to date, ETD continues to show great promise to propel the field of proteomics and, for small- to medium-sized peptides, is highly complementary to ion trap CAD.

Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in [Ca2+]i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.

Dendritic cells (DCs) are highly efficient antigen-presenting cells (APCs) that collect antigen in body tissues and transport them to draining lymph nodes. Antigenic peptides are loaded onto major histocompatibility complex (MHC) molecules for presentation to naive T cells, resulting in the induction of cellular and humoral immune responses. DCs take up antigen through phagocytosis, pinocytosis, and endocytosis via different groups of receptor families, such as Fc receptors for antigen-antibody complexes, C-type lectin receptors (CLRs) for glycoproteins, and pattern recognition receptors, such as Toll-like receptors (TLRs), for microbial antigens. Uptake of antigen by CLRs leads to presentation of antigens on MHC class I and II molecules. DCs are well equipped to distinguish between self- and nonself-antigens by the variable expression of cell-surface receptors such as CLRs and TLRs. In the steady state, DCs are not immunologically quiescent but use their antigen-handling capacities to maintain peripheral tolerance. DCs are continuously sampling and presenting self- and harmless environmental proteins to silence immune activation. Uptake of self-components in the intestine and airways are good examples of sites where continuous presentation of self- and foreign antigens occurs without immune activation. In contrast, efficient antigen-specific immune activation occurs upon encounter of DCs with nonself-pathogens. Recognition of pathogens by DCs triggers specific receptors such as TLRs that result in DC maturation and subsequently immune activation. Here we discuss the concept that cross talk between TLRs and CLRs, differentially expressed by subsets of DCs, accounts for the different pathways to peripheral tolerance, such as deletion and suppression, and immune activation.

Flap EndoNuclease-1 (FEN-1) and the processivity factor proliferating cell nuclear antigen (PCNA) are central to DNA replication and repair. To clarify the molecular basis of FEN-1 specificity and PCNA activation, we report here structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results. FEN-1 binds the unpaired 3' DNA end (3' flap), opens and kinks the DNA, and promotes conformational closing of a flexible helical clamp to facilitate 5' cleavage specificity. Ordering of unstructured C-terminal regions in FEN-1 and PCNA creates an intermolecular beta sheet interface that directly links adjacent PCNA and DNA binding regions of FEN-1 and suggests how PCNA stimulates FEN-1 activity. The DNA and protein conformational changes, composite complex structures, FRET, and mutational results support enzyme-PCNA alignments and a kinked DNA pivot point that appear suitable to coordinate rotary handoffs of kinked DNA intermediates among enzymes localized by the three PCNA binding sites.

Cytolysis of target cells by natural killer (NK) cells and by some cytotoxic T cells occurs unless prevented by inhibitory receptors that recognize MHC class I on target cells. Human NK cells express a p58 inhibitory receptor specific for HLA-C. We report association of the tyrosine phosphatase HCP with the p58 receptor in NK cells. HCP association was dependent on tyrosine phosphorylation of p58. Phosphotyrosyl peptides corresponding to the p58 tail bound and activated HCP in vitro. Furthermore, introduction of an inactive mutant HCP into an NK cell line prevented the p58-mediated inhibition of target cell lysis. These data imply that the inhibitory function of p58 is dependent on its tyrosine phosphorylation and on recruitment and activation of HCP.

Experimental allergic encephalomyelitis (EAE) serves as a model for autoimmune diseases mediated by T lymphocytes. Following sensitization to rat, mouse or guinea pig myelin basic protein (MBP) in complete Freund's adjuvant, inbred mouse strains PL/J (H-2u), SJL/J (H-2s) and (PL/J X SJL/J)F1((PLSJ)F1) develop EAE. Whereas sensitization to the N-terminal 37 amino-acid peptide of rat or guinea pig MBP [MBP(1-37)] induces EAE in PL/J mice, immunization to the C-terminal peptide (89-169) leads to EAE in SJL/J mice. The immune response to MBP in (PLSJ)F1 mice is not co-dominant; sensitization to the N-terminal peptide induces EAE, while sensitization to the C-terminal peptide does not. We have generated MBP-specific T-cell clones restricted to class II (Ia) antigens of the major histocompatibility complex (MHC) from PL/J and (PLSJ)F1 mice following sensitization to rat MBP. Two such I-Au-restricted T-cell clones that proliferate in response to the encephalitogenic N-terminal MBP peptide and recognize a shared determinant with mouse (self) MBP cause paralysis in 100% of (PLSJ)F1 mice tested. Paralysis is induced even when recipients are injected with as few as 1 X 10(5) cloned T cells. Relapsing paralysis followed in two-thirds of the recipients after recovery from acute paralysis, whereas one-third developed chronic persistent paralysis, a form of EAE not usually seen. Histopathology revealed intense perivascular inflammation, demyelination and remyelination within the central nervous system of paralysed mice. The experimental disease induced with these clones shares important features with human demyelinating diseases such as multiple sclerosis. This is the first demonstration that T-cell clones that respond to a defined self-antigen can induce clinical and histological autoimmune disease.

In a wide variety of cellular settings, from organelle transport to muscle contraction, Ca2+ binding to members of the EF hand family of proteins controls the interaction between actin and different myosins that are responsible for generating movement. In vertebrate skeletal and cardiac muscle the Ca(2+)-binding protein troponin C (TnC) is one subunit of the ternary troponin complex which, through its association with actin and tropomyosin on the thin filament, inhibits the actomyosin interaction at submicromolar Ca2+ concentrations and stimulates the interaction at micromolar Ca2+ concentrations. Because TnC does not interact directly with actin or tropomyosin, the Ca(2+)-binding signal must be transmitted to the thin filament via the other two troponin subunits: troponin I (TnI), the inhibitory subunit, and troponin T (TnT), the tropomyosin-binding subunit. Thus, the troponin complex is a Ca(2+)-sensitive molecular switch and the structures of and interactions between its components have been of great interest for many years. Although the crystal structure of TnC has been known for almost a decade, the molecular structures of TnI and TnT are not known and therefore convincing models of the organization of the troponin complex and the Ca(2+)-induced changes in its structure have not been forthcoming. Recent advances on a wide variety of fronts including 1) the bacterial expression and characterization of mutants of TnC, TnI, and TnT; 2) cross-linking and fluorescence studies; and 3) the determination of the crystal and nuclear magnetic resonance structures of synthetic and recombinant troponin fragments and complexes between EF hand proteins and their target peptides have provided new insights into the nature of the interactions between troponin subunits. This review discusses these recent advances with the aim of critically assessing molecular models of the nature of the Ca(2+)-induced structural transition in troponin.

The Legionella pneumophila Dot/Icm system is a type IV secretion apparatus that transfers bacterial proteins into eukaryotic host cells. The RalF protein is a substrate engaged and translocated into host cells by the Dot/Icm system. In this study, the mechanism of Dot/Icm-mediated translocation of RalF has been investigated. It was determined that RalF translocation into host cells occurs before bacterial internalization. Sequences essential for RalF translocation were located at the C terminus of the RalF protein. A fusion protein consisting of a 20-aa C-terminal RalF peptide appended to the calmodulin-dependent adenylate cyclase domain of the Bordetella pertussis adenylate cyclase protein was translocated into host cells by the Dot/Icm system. A leucine (L372) residue at the -3 position in relation to the RalF C terminus was critical for translocation. Consistent with RalF L372 playing an important role in substrate recognition by the Dot/Icm system, most other Dot/Icm substrates were found to have amino acid residues with similar physical properties at their -3 or -4 C-terminal positions. These data demonstrate that the Dot/Icm system can transfer bacterial proteins that modulate host cellular functions before uptake and indicate that substrate recognition involves a C-terminal translocation signal. Thus, Legionella has the ability to engage synthesized substrate proteins and transfer them into host cells on contact, enabling Legionella to rapidly alter transport of the vacuole in which it resides.

Hepatocyte growth factor (HGF) induces a three-phase response leading to the formation of branched tubular structures in epithelial cells. The HGF receptor tyrosine kinase works through a Src homology (SH2) docking site that can activate several signalling pathways. The first phase of the response (scattering), which results from cytoskeletal reorganization, loss of intercellular junctions and cell migration, is dependent on phosphatidylinositol-3-OH kinase and Rac activation. The second phase (growth) requires stimulation of the Ras-MAP kinase cascade. Here we show that the third phase (tubulogenesis) is dependent on the STAT pathway. HGF stimulates recruitment of Stat-3 to the receptor, tyrosine phosphorylation, nuclear translocation and binding to the specific promoter element SIE. Electroporation of a tyrosine-phosphorylated peptide, which interferes with both the association of STAT to the receptor and STAT dimerization, inhibits tubule formation in vitro without affecting either HGF-induced 'scattering' or growth. The same result is obtained using a specific 'decoy' oligonucleotide that prevents STAT from binding to DNA and affecting the expression of genes involved in cell-cycle regulation (c-fos and waf-1). Activation of signal transducers that directly control transcription is therefore required for morphogenesis.

The light-harvesting complex (LHC) of barley photosystem II (PS II) was fractionated by Deriphat-polyacrylamide gel electrophoresis into five different pigmented components: one subcomplex (LHC IIb) and four pigment-proteins (LHC IIa, -c, -d, and -e). No loss of chorophyll from the components occurred during fractionation, and violaxanthin is the only photosynthetic pigment that apparently occurs in thylakoids free of association with protein. Each LHC II component has a distinct stoichiometry of neoxanthin, violaxanthin, lutein, chlorophyll a, and chlorophyll b. LHC IIa, -d, and -e were obtained as monomeric pigment-proteins, each of which contained one apoprotein Mr 31,000, 21,000, and 13,000, respectively. LHC IIc was also isolated as a monomer but it contained two poly-peptides of Mr 29,000 and 26,500, whereas the trimeric LHC IIb subcomplex (Mr 72,000) contained three subunits of Mr 28,000, 27,000, and 25,000, but not in equal stoichiometry. How the LHC II subunits are organized in PS II was examined. We isolated PS II subcomplexes which contained four of the LHC II subunits and the core complex (CC II) in unit stoichiometry; the relative strengths of association of the LHC II subunits with CC II are: LHC IIa greater than LHC IIc greater than LHC IIb greater than LHC IId. The LHC II subunits were associated with the native dimeric and not with the derived monomeric form of CC II. In addition, a multimeric LHC II sub-complex composed of the LHC IIa, LHC IIb, and LHC IId pigment-proteins was isolated. We propose that this LHC II subcomplex, which contained the Mr 28,000 and 25,000 subunits but lacked the Mr 27,000 subunit of LHC IIb and CC II. An LHC IIb pigmented fraction of LHC IIb and CC II. An LHC IIb pigmented fraction of Mr 250,000 was isolated which contained only the Mr 28,000 and 27,000 subunits. The LHC IIb subunits in this complex were the most highly phosphorylated on a protein basis. These data together with analyses of the chlorophyll b-less barley chlorina f2 mutant were used to construct a model for the LHC II pigment-protein arrangement in higher plant PS II.

Serum levels of free and total insulin as well as total C-peptide immunoreactivity (C-peptide and proinsulin) and C- peptide were measured in insulin-treated diabetics with circulating insulin antibodies by the addition of polyethylene glycol (PEG) before and after acidification. PEG resulted in complete precipitation of insulin antibodies from serum and made it possible to measure free insulin in the supernatant. Incubation of serum at 37 degrees C. for two hours before addition of PEG resulted in values for free insulin that probably resembled the in-vivo levels most closely. The same method could also be used to remove proinsulin bound to circulating insulin antibodies and permitted the measurement of C-peptide in the supernatant. Clinical studies using this approach indicate that combined measurements of serum free and total insulin and C-peptide provide information that is helpful in understanding the contribution of endogenous and exogenous insulin to the course and metabolic control of insulin-requiring diabetic patients.

Molecular dynamics simulations of the protein chymotrypsin inhibitor 2 in 8 M urea at 60 degrees C were undertaken to investigate the molecular basis of chemical denaturation. The protein unfolded rapidly under these conditions, but it retained its native structure in a control simulation in water at the same temperature. The overall process of unfolding in urea was similar to that observed in thermal denaturation simulations above the protein's T(m) of 75 degrees C. The first step in unfolding was expansion of the hydrophobic core. Then, the core was solvated by water and later by urea. The denatured structures in both urea and at high temperature contained residual native helical structure, whereas the beta-structure was completely disrupted. The average residence time for urea around hydrophilic groups was six times greater than around hydrophobic residues and in all cases greater than the corresponding water residence times. Water self-diffusion was reduced 40% in 8 M urea. Urea altered water structure and dynamics, thereby diminishing the hydrophobic effect and encouraging solvation of hydrophobic groups. In addition, through urea's weakening of water structure, water became free to compete with intraprotein interactions. Urea also interacted directly with polar residues and the peptide backbone, thereby stabilizing nonnative conformations. These simulations suggest that urea denatures proteins via both direct and indirect mechanisms.

A growing body of evidence supports a role for mitochondria and mitochondria-derived factors in the cell death process. In particular, much attention has focused on cytochrome c, a key component of the electron transport chain, that has been reported to translocate from the mitochondria to the cytosol in cells undergoing apoptosis. The mechanism for this release is, as yet, unknown. Here we report that ectopic expression of Bax induces apoptosis with an early release of cytochrome c preceding many apoptosis-associated morphological alterations as well as caspase activation and subsequent substrate proteolysis. A loss of mitochondrial transmembrane potential was detected in vivo, although no mitochondrial swelling or loss of transmembrane potential was observed in isolated mitochondria treated with Bax in vitro. Caspase inhibitors, such as endogenous XIAP and synthetic peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), although capable of altering the kinetics and perhaps mode of cell death, had no influence on this release, suggesting that if cytochrome c plays a role in caspase activation it must precede this step in the apoptotic process. Mitochondrial permeability transition was also shown to be significantly prevented by caspase inhibition, indicating that the translocation of cytochrome c from mitochondria to cytosol is not a consequence of events requiring mitochondrial membrane depolarization. In contrast, Bcl-xL was capable of preventing cytochrome c release while also significantly inhibiting cell death. It would therefore appear that the mitochondrial release of factors such as cytochrome c represents a critical step in committing a cell to death, and this release is independent of permeability transition and caspase activation but is inhibited by Bcl-xL.