Exposure of cryptic domains in the alpha 1-chain of laminin-1 by elastase stimulates macrophages urokinase and matrix metalloproteinase-9 expression

Degradation of the extracellular matrix leads to the release of fragments, which elicit biological responses distinct from intact molecules. We have reported that alpha1:Ser(2091)-Arg(2108), a peptide derived from the alpha1-chain of laminin-1, triggers protein kinase C-dependent activation of MAPK(erk1/2), leading to the up-regulation of macrophage urokinase type plasminogen activator and matrix metalloproteinase (MMP)-9 expression. Since intact laminin-1 failed to trigger these events, we hypothesized that alpha1:Ser(2091)-Arg(2108) is cryptic or assumes a conformation not recognized by macrophages. Here we demonstrate that elastase cleavage of laminin-1 generates fragments, which stimulate proteinase expression by RAW264.7 macrophages and peritoneal macrophages. In contrast, fragments generated by MMP-2, MMP-7, or plasmin had no effect on macrophage proteinase expression. Elastase-generated laminin-1 fragments were fractionated by heparin-Sepharose chromatography. Heparin-binding fragments stimulated macrophages' proteinase expression severalfold greater than nonbinding fragments. The heparin binding fragments reacted with antibodies directed against regions of the alpha1-chain including alpha1:Ser(2091)-Arg(2108) and the globular domain. A peptide from the first loop of the globular domain (alpha1:Ser(2179)-Ser(2198)) triggered the phosphorylation of MAPK(erk1/2) and stimulated the expression of macrophage urokinase type plasminogen activator and MMP-9. Moreover, a heparin-binding fraction isolated from an aortic aneurysm contained fragments of alpha1-chain and stimulated macrophages' proteinase expression. Based on these data, we conclude that cryptic domains in the COOH-terminal portion of the alpha1-chain of laminin are exposed by proteolysis and stimulate macrophages' proteinase expression.     

Similarity of expression patterns of knotted1 and ZmLEC1 during somatic and zygotic embryogenesis in maize ( Zea mays L.)

Expression of knotted1 ( kn1) and ZmLEC1, a maize homologue of the Arabidopsis LEAFY COTYLEDON1 ( LEC1) was studied using in situ hybridization during in vitro somatic embryogenesis of maize ( Zea mays L.) genotype Hi-II. Expression of kn1 was initially detected in a small group of cells (5-10) in the somatic embryo proper at the globular stage, in a specific region where the shoot meristem is initiating at the scutellar stage, and specifically in the shoot meristem at the coleoptilar stage. Expression of ZmLEC1 was strongly detected in the entire somatic embryo proper at the globular stage, gradually less in the differentiating scutellum at the scutellar and coleoptilar stages. The results of analyses show that the expression pattern of kn1 during in vitro somatic embryogenesis of maize is similar to that of kn1 observed during zygotic embryo development in maize. The expression pattern of ZmLEC1 in maize during in vitro development is similar to that of LEC1 in Arabidopsis during zygotic embryo development. These observations indicate that in vitro somatic embryogenesis likely proceeds through similar developmental pathways as zygotic embryo development, after somatic cells acquire competence to form embryos. In addition, based on the ZmLEC1 expression pattern, we suggest that expression of ZmLEC1 can be used as a reliable molecular marker for detecting early-stage in vitro somatic embryogenesis in maize.     

Aged men display blunted biorhythmic variation of muscle performance and physiological responses.

Aging is known to disrupt the "biological clock" that governs physiological variables at rest. This study sought to determine whether aged men demonstrated biorhythmic variation in muscle performance during resistance exercise and physiological responses to that stimulus. Ten aged (75.6 +/- 1.6 yr; mean +/- SE) men completed an isokinetic testing protocol of knee extensors and flexors at 0800, 1200, 1600, and 2000 h. Although time of day variation in peak torque was detectable, significant (P < or = 0.05) oscillation was established only in the knee flexors at 3.14 rad/s. Heart rate, blood pressure, and rectal temperature displayed no significant variation, but trends (P < 0.10) in oscillation of postexercise blood pressure and rectal temperature were noted. Temporal patterns in biorhythmic variation of muscle performance, as well as thermal and cardiovascular measures, emulated those observed in a previous study involving young men where the magnitude of variation was sufficient to achieve statistical significance. Similar to our earlier findings in young men, however, pre- and postexercise testosterone and cortisol concentrations demonstrated significant variation among aged men. These data confirm the blunting of biorhythmic variation in muscle performance and physiological variables, except for circulating hormones, in aged men.     

Inhibition of p38 MAP kinase by utilizing a novel allosteric binding site

The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics, consistent with the requirement for conformational change. Improving interactions in this allosteric pocket, as well as establishing binding interactions in the ATP pocket, enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series, BIRB 796, has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture.     

Structure and function in the isolated reaction center complex of Photosystem II: energy and charge transfer dynamics and mechanism

The dynamics of energy and charge transfer in the Photosystem II reaction center complex is an area of great interest today. These processes occur on a time scale ranging from femtoseconds to tens of picoseconds or longer. Steady-state and ultrafast spectroscopy techniques have provided a great deal of quantitative and qualitative data that have led to varied interpretations and phenomenological models. More recently, microscopic models that identify specific charge separated states have been introduced, and offer more insight into the charge transfer mechanism. The structure and energetics of PS II reaction centers are reviewed, emphasizing the effects on the dynamics of the initial charge transfer. This revised version was published online in June 2006 with corrections to the Cover Date.     

Covalent attachment of the heme prosthetic group in the CYP4F cytochrome P450 family

We demonstrated earlier that the heme in cytochrome P450 enzymes of the CYP4A family is covalently attached to the protein through an I-helix glutamic acid residue [Hoch, U., and Ortiz de Montellano, P. R. (2001) J. Biol. Chem. 276, 11339-11346]. As the critical glutamic acid residue is conserved in many members of the CYP4F class of cytochrome P450 enzymes, we investigated covalent heme binding in this family of enzymes. Chromatographic analysis indicates that the heme is covalently bound in CYP4F1 and CYP4F4, which have the required glutamic acid residue, but not in CYP4F5 and CYP4F6, which do not. Catalytic turnover of CYP4F4 with NADPH-cytochrome P450 reductase shows that the heme is covalently bound through an autocatalytic process. Analysis of the prosthetic group in the CYP4F5 G330E mutant, into which the glutamic acid has been reintroduced, shows that the heme is partially covalently bound and partially converted to noncovalently bound 5-hydroxymethylheme. The modified heme presumably arises by trapping of a 5-methyl carbocation intermediate by a water molecule. CYP4F proteins thus autocatalytically bind their heme groups covalently in a process that requires a glutamic acid both to generate a reactive (cationic) form of the heme methyl and to trap it to give the ester bond.     

Introduction of the 305Arg-->305Ser mutation in the large extrinsic loop E of the CP43 protein of Synechocystis sp. PCC 6803 leads to the loss of cytochrome c(550) binding to Photosystem II

CP43, a component of Photosystem II (PSII) in higher plants, algae and cyanobacteria, is encoded by the psbC gene. Previous work demonstrated that alteration of an arginine residue occurring at position 305 to serine produced a strain (R305S) with altered PSII characteristics including lower oxygen-evolving activity, fewer assembled reaction centers, higher sensitivity to photoinactivation, etc. [Biochemistry 38 (1999) 1582]. Additionally, it was determined that the mutant exhibited an enhanced stability of its S2 state. Recently, we observed a significant chloride effect under chloride-limiting conditions. The mutant essentially lost the ability to grow photoautotrophically, assembled fewer fully functional PSII reaction centers and exhibited a very low rate of oxygen evolution. Thus, the observed phenotype of this mutation is very similar to that observed for the Delta(psb)V mutant, which lacks cytochrome c550 (Biochemistry 37 (1998) 1551). A His-tagged version of the R305S mutant was produced to facilitate the isolation of PSII particles. These particles were analyzed for the presence of cytochrome c550. Reduced minus oxidized difference spectroscopy and chemiluminescence examination of Western blots indicated that cytochrome c550 was absent in these PSII particles. Whole cell extracts from the R305S mutant, however, contained a similar amount of cytochrome c550 to that observed in the control strain. These results indicate that the mutation R305S in CP43 prevents the strong association of cytochrome c550 with the PSII core complex. We hypothesize that this residue is involved in the formation of the binding domain for the cytochrome.     

Thyrotropin-releasing hormone receptor processing: role of ubiquitination and proteasomal degradation

These studies were designed to characterize ubiquitination of the G protein-coupled TRH receptor (TRHR). TRHRs and ubiquitin coprecipitated with antibodies to either receptor or ubiquitin in Chinese hamster ovary or pituitary GHFT cells. Inhibition of the proteasome with MG-132 resulted in an accumulation of total TRHRs and the appearance of a small amount of cytosolic receptor. MG-132 caused an increase in newly synthesized receptors, detected by microscopy using a TRHR coupled to Timer, a DsRed that undergoes a spontaneous time-dependent color change. Misfolded TRHRs were particularly heavily ubiquitinated. These results show that the proteasome participates in TRHR quality control early after receptor synthesis. Under normal circumstances, most ubiquitinated TRHRs were absorbed to wheat germ agglutinin, indicating that they had undergone complex glycosylation in the Golgi apparatus. When cells were treated with tunicamycin to block glycosylation, a ladder of ubiquitinated species was detectable. Cell surface receptors, which were labeled selectively with either radioligand or antibody, showed no detectable ubiquitin modification. To determine if ubiqutination plays a role in TRH-induced receptor endocytosis, the receptor was expressed in Ts20 cells, which have a temperature-sensitive ubiquitin pathway. TRH induced a significant calcium response and rapid and extensive receptor internalization at both the permissive and nonpermissive temperatures, indicating that ligand-dependent ubiquitination of the receptor, or any other protein, is not necessary for TRHR signaling or internalization. These results show that ubiquitin modification targets misfolded receptors for degradation and suggest a possible role for ubiquitination in receptor trafficking.