Stiffness of γ subunit of F1-ATPase

F₁-ATPase is a molecular motor in which the γ subunit rotates inside the α₃β₃ ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F₁-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the γ subunit (Hirono-Hara et al. 2005; Iko et al. 2009). These findings provide important insight into how individual reaction steps release energy to power F₁ and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the γ subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F₁ from thermophilic Bacillus PS3: the internal part of the γ subunit embedded in the α₃β₃ ring, and the complex of the external part of the γ subunit and the α₃β₃ ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the γ subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F_o and F₁-ATPase.     

Effects of Mutating Leucine to Threonine in the M2 Segment of α1 and β1 Subunits of GABAAα1β1 Receptors

The conserved leucine residues at the 9′ positions in the M2 segments of α₁ (L264) and β₁ (L259) subunits of the human GABA_A receptor were replaced with threonine. Normal or mutant α₁ subunits were co-expressed with normal or mutant β₁ subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher ``resting' chloride conductance than cells expressing wild-type α₁β₁ receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABA_A channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β₁ with mutated α₁ subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type α₁β₁ receptors. In cells expressing mutated β₁ subunits with wild-type or mutated α₁ subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABA_A receptors in the plasmalemma because the cells bound [³H]-muscimol. It was concluded that in GABA_A channels containing the L9′T mutation in the β₁ subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an open-channel state of the GABA_A receptor in the absence of ligand. Received: 17 April 1996/Revised: 5 July 1996     

Functional assessment of genetic variants of α1-Antitrypsin

Summary The specific activity of thirteen genetic variants of the protease inhibitor ₁-antitrypsin (₁-AT) has been determined. Elastase inhibitor activity was assayed using protein substrates (elastin and gelatin) and the synthetic substrate N-tert-butoxycarbonyl-l-alanine p-nitrophenyl ester. The synthetic substrate -N-benzoyl-dl-arginine p-nitroanilide HCl was used to assay trypsin inhibitor activity. The specific activity of ₁-AT was expressed as serum inhibition/immunological concentration of ₁-AT. Sera of PI type FM had reduced specific activity with elastase, but not with trypsin. With the possible exception of MP, no other variants showed significant differences in specific activity when compared with normal PI type M.This research was supported by the Medical Research Council of Canada (No. MA 5426)     

Synthesis of 2′-Deoxyribonucleoside Derivatives of 1-Deazapurine

Abstract

5, 7-Dichloro-3H-imidazo[4, 5-b]pyridine (4) is a versatile base which can be coupled with a variety of sugar moieties and transformed in a series of 7-alkyl(aryl)amino-derivatives by reacting with the corresponding amines. In this paper synthesis, structure elucidation and ADA inhibitory activity of 2′-deoxyribonucleoside derivatives of N⁶-substituted 1-deazaapurines are described.     

Patterns of associations of clinical features in neurofibromatosis 1 (NF1)

Neurofibromatosis 1 (NF1) is a common, fully penetrant autosomal dominant disease. The clinical course is generally progressive but highly variable, and the pathogenesis is poorly understood. We studied statistical associations among 13 of the most common or important clinical features in data from four separate sets of NF1 patients: a "developmental sample" of 1,413 probands from the NNFF International Database, an independent "validation sample" of 1,384 probands from the same database, 511 affected relatives of these probands, and 441 patients from a population-based registry in northwest England. We developed logistic regressive models for each of the 13 features using the developmental sample and attempted to validate these models in the other three samples. Age and gender were included as covariates in all models. Models were successfully developed and validated for ten of the 13 features analysed. The results are consistent with grouping nine of the clinical features into three sets: (1) café-au-lait spots, intertriginous freckling and Lisch nodules; (2) cutaneous, subcutaneous and plexiform neurofibromas; (3) macrocephaly, optic glioma and other neoplasms. In addition, three-way interactions among café-au-lait spots, intertriginous freckling and subcutaneous neurofibromas indicate that the first two groups are not independent. Our studies show that some individuals with NF1 are more likely than others to develop certain clinical features of the disease. Some NF1 features appear to share pathogenic mechanisms that are not common to all features.     

Identification of LRP1B-interacting proteins and inhibition of protein kinase Cα-phosphorylation of LRP1B by association with PICK1

Recent studies show LDL receptor-related protein 1B, LRP1B as a transducer of extracellular signals. Here, we identify six interacting partners of the LRP1B cytoplasmic region by yeast two-hybrid screen and confirmed their in vivo binding by immunoprecipitation. One of the partners, PICK1 recognizes the C-terminus of LRP1B and LRP1. The cytoplasmic domains of LRP1B are phosphorylated by PKCα about 100 times more efficiently than LRP1. Binding of PICK1 inhibits phosphorylation of LRP1B, but does not affect LRP1 phosphorylation.This study presents the possibility that LRP1B participates in signal transduction which PICK1 may regulate by inhibiting PKCα phosphorylation of LRP1B.

Structured summary

MINT-6801075: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with SNTG2 (uniprotkb:Q925E0) by two hybrid (MI:0018)MINT-6801030, MINT-6801468: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Pick1 (uniprotkb:Q80VC8) by two hybrid (MI:0018)MINT-6801284: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with RanBPM (uniprotkb:P69566) by anti tag coimmunoprecipitation (MI:0007)MINT-6801108: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Grb7 (uniprotkb:Q03160) by two hybrid (MI:0018)MINT-6801090: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with RanBPM (uniprotkb:P69566) by two hybrid (MI:0018)MINT-6801008: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-1b (uniprotkb:Q9WVI9-1) by two hybrid (MI:0018)MINT-6801052: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-2 (uniprotkb:Q9ERE9) by two hybrid (MI:0018)MINT-6801258, MINT-6801271: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with Pick1 (uniprotkb:Q80VC8) by anti tag coimmunoprecipitation (MI:0007)MINT-6801244: RanBPM (uniprotkb:P69566) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)MINT-6801131, MINT-6801158: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-1b (uniprotkb:Q9WVI9-1) by anti tag coimmunoprecipitation (MI:0007)MINT-6801231: PICK1 (uniprotkb:Q80VC8) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)MINT-6801173: Jip-1b (uniprotkb:Q9WVI9-1) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)     

Glycosylation of Skp1 Promotes Formation of Skp1–Cullin-1–F-box Protein Complexes in Dictyostelium

O₂ sensing in diverse protozoa depends on the prolyl 4 hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of five sugars. In yeast, plants, and animals, Skp1 is associated with F-box proteins. The Skp1–F-box protein heterodimer can, for many F-box proteins, dock onto cullin-1 en route to assembly of the Skp1–cullin-1–F-box protein–Rbx1 subcomplex of E3^SCFUb ligases. E3^SCFUb ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S proteasome. In the social amoeba Dictyostelium, we found that O₂ availability was rate-limiting for the hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knockout mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells able to fully glycosylate Skp1 had a greater abundance of an SCF complex containing the cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, than the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA–Skp1CulA complex was also more abundant in glycosylation-competent cells. The CulE interactome also included higher levels of proteasomal regulatory particles when Skp1 was glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modified when it harbored an F-box mutation that compromised Skp1 binding, consistent with an effect on the abundance of potential substrate proteins. We propose that O₂-dependent posttranslational glycosylation of Skp1 promotes association with F-box proteins and their engagement in functional E3^SCFUb ligases that regulate O₂-dependent developmental progression.Timely protein degradation is a cornerstone of cell cycling and the regulation of numerous physiological and developmental processes. Eukaryotes have evolved an extensive array of polyubiquitination enzymes to tag proteins on a protein-by-protein basis as a recognition marker for degradation in the 26S proteasome. The cullin-RING ubiquitin ligases (CRLs)¹ are a prominent subgroup of these enzymes (1) and consist of an E3 architecture that includes a substrate receptor, an adaptor (in most cases), the cullin scaffold, the RING protein, and an exchangeable E2 ubiquitin donor that has been charged with ubiquitin (Ub) by an E1 enzyme. The first discovered and still prototypic example is the CRL1 class (2), also referred to as SCF on account of the names of its founding subunits, Skp1, cullin-1, and F-box proteins (FBPs). The CRL1 (or SCF) complexes utilize FBPs as substrate receptors, Skp1 as the adaptor linking the FBP to the N-terminal region of cullin-1 (Cul1), and Rbx1 as the RING protein that tethers the E2 Ub donor to the Cul1 C-terminal region (see Fig. 2B). CRL1s can be activated by neddylation of Cul1 by a Nedd8-specific E2, which mobilizes Rbx1 to afford rotational flexibility of the E2 and displaces the inhibitor Cand1, permitting docking of the Skp1–FBP heterodimer (3–5). Deneddylation mediated by the eight-subunit COP9 signalosome is required for in vivo activity, suggesting that Cand1 serves as a substrate exchange factor to allow for re-equilibration of SCF complexes from preexisting subunits. Each reaction cycle requires the exchange of a new E2-Ub and typically assembles a K48-linked polyUb chain that is recognized by the proteasome. Substrate specificity is conferred by FBPs, a gene family that numbers 69 in humans, 20 in budding yeast, 300 in Caenorhabditis elegans, and ∼800 in Arabidopsis. Some characterized FBPs can recognize perhaps a dozen or more substrates, and the coding of recognition and the meaning of their control by the same FBP is under intense investigation (6). Recognition is often activated by posttranslational modification of the substrate (often phosphorylation). Regulation of SCF Ub ligases has centered on the neddylation cycle, which potentially influences all seven known CRLs. Regulation of Skp1, investigated in this paper, would be specific to CRLs possessing Skp1, which include CRL1 and possibly the minor class CRL7 (7).Open in a separate windowFig. 2.Skp1 modification pathway and global analysis of Skp1 interactions. A, Skp1 is sequentially modified by the indicated enzymes (in blue), resulting in the formation of a pentasaccharide at Pro143. B, model of the SCF complex in the context of the overall E3 Ub ligase, from studies in yeast, plants, and animals. Catalysis involves transfer of Ub from an exchangeable Ub-E2 conjugate to the substrate. Removal of Nedd8 by the COP9 signalosome facilitates binding of Cand1 to Cul1, which inhibits binding of Skp1 to Cul1. C, D, vegetative (growth stage) cells were filter-lysed, and a cytosolic fraction prepared via ultracentrifugation was chromatographed on a Superose 12 gel filtration column. Fractions were analyzed via Western blotting (representative examples are shown in C) followed by densitometry (D). The elution position of free Skp1 from a separate trial is indicated.The basic SCF model is thought to be widespread among eukaryotes but has been extensively studied only in fungi/yeasts, plants, and animals. The broad phylogeny represented by protists includes many benign and pathogenic unicellular organisms of great economic, health, and environmental impact. Emerging evidence reveals that Skp1 in some of these groups is subject to a novel form of prolyl 4(trans)-hydroxylation and complex glycosylation (8). The roles of these Skp1 modifications have been most studied in the social amoeba Dictyostelium, which undergoes a starvation-induced developmental program during which individual amoebae chemotactically aggregate into an initial mound that then elongates into a migratory slug. Under appropriate conditions, the slug reorganizes to form a fruiting body consisting of a ball of spores supported by a vertical cellular stalk. The slug-to-fruit switch, referred to as culmination, and sporulation are regulated by checkpoints that are sensitive to multiple factors, including O₂ (9–11). Functional studies of Dictyostelium Skp1 hydroxylation and glycosylation reveal roles in regulating the O₂ dependence of culmination and sporulation (12–14). For example, wild-type (wt) cells require 7% to 10% O₂ and phyA^– requires 18% to 21% O₂ in order to achieve 50% spore formation (a quantitative measure of fruiting body formation), whereas glycosylation mutants exhibit a complex pattern of intermediate requirements (13). In addition, at 21% O₂, phyA^– cells require an additional 3 to 4 h to complete development relative to their wt counterparts (14). In the apicomplexan Toxoplasma gondii, PhyA is also required for Skp1 glycosylation, and phyA^– parasites are deficient in proliferation, especially at low O₂ (15).The idea that O₂ availability is rate limiting for Skp1 modification was originally based on the observation that the Dictyostelium phyA^– phenotype mimics that of wt cells in low O₂ (9). However, the majority of Skp1 is hydroxylated and glycosylated in wt cells even at low O₂ levels where culmination is blocked or delayed. Further analysis of a submerged development model, in which terminal development depended on an atmosphere of 70% to 100% O₂ in order to overcome the diffusion barrier posed by the water layer, showed that at atmospheric O₂ levels of 5% to 21% where sporulation was blocked, unmodified Skp1 accumulated to a higher level than at permissive O₂ levels (10). As Skp1 modifications are thought to be irreversible, this likely resulted from slow hydroxylation of newly synthesized Skp1. To address this in a more physiological setting, we investigated nascent Skp1 directly using metabolic labeling with [³⁵S]Met/Cys and verified that the rate of hydroxylation of newly synthesized Skp1 polypeptide was indeed inversely proportional to O₂ levels, which makes PhyA-mediated hydroxylation of Skp1 an excellent candidate for the primary O₂ sensor for culmination.These modifications of Skp1 are of interest as a novel mechanism regulating the SCF ligase. Previously, we showed that hydroxylation and glycosylation of Dictyostelium Skp1 affect its conformation and promote binding to a soluble FBP, guinea pig Fbs1, in studies of purified proteins (16). Here we show that Dictyostelium Skp1 is indeed a subunit of a canonical SCF complex, as expected. The significance of undermodified Skp1 was examined via interactome analysis of Skp1 isoforms that accumulate in modification pathway mutants. Our findings revealed a lower abundance of SCF complexes than in wt cells, suggesting that Skp1 modification may promote SCF assembly and E3^SCFUb ligase activities that control timely turnover of select proteins involved in developmental progression.     

Identification of a Peptide Inhibitor of the RPM-1·FSN-1 Ubiquitin Ligase Complex

The Pam/Highwire/RPM-1 (PHR) proteins include: Caenorhabditis elegans RPM-1 (Regulator of Presynaptic Morphology 1), Drosophila Highwire, and murine Phr1. These important regulators of neuronal development function in synapse formation, axon guidance, and axon termination. In mature neurons the PHR proteins also regulate axon degeneration and regeneration. PHR proteins function, in part, through an ubiquitin ligase complex that includes the F-box protein FSN-1 in C. elegans and Fbxo45 in mammals. At present, the structure-function relationships that govern formation of this complex are poorly understood. We cloned 9 individual domains that compose the entire RPM-1 protein sequence and found a single domain centrally located in RPM-1 that is sufficient for binding to FSN-1. Deletion analysis further refined FSN-1 binding to a conserved 97-amino acid region of RPM-1. Mutagenesis identified several conserved motifs and individual amino acids that mediate this interaction. Transgenic overexpression of this recombinant peptide, which we refer to as the RPM-1·FSN-1 complex inhibitory peptide (RIP), yields similar phenotypes and enhancer effects to loss of function in fsn-1. Defects caused by transgenic RIP were suppressed by loss of function in the dlk-1 MAP3K and were alleviated by point mutations that reduce binding to FSN-1. These findings suggest that RIP specifically inhibits the interaction between RPM-1 and FSN-1 in vivo, thereby blocking formation of a functional ubiquitin ligase complex. Our results are consistent with the FSN-1 binding domain of RPM-1 recruiting FSN-1 and a target protein, such as DLK-1, whereas the RING-H2 domain of RPM-1 ubiquitinates the target.     

Mode of action of Chrysosporium lucknowense C1 α-l-arabinohydrolases

The mode of action of four Chrysosporium lucknowense C1 α-L-arabinohydrolases was determined to enable controlled and effective degradation of arabinan. The active site of endoarabinanase Abn1 has at least six subsites, of which the subsites -1 to +2 have to be occupied for hydrolysis. Abn1 was able to hydrolyze a branched arabinohexaose with a double substituted arabinose at subsite -2. The exo acting enzymes Abn2, Abn4 and Abf3 release arabinobiose (Abn2) and arabinose (Abn4 and Abf3) from the non-reducing end of reduced arabinose oligomers. Abn2 binds the two arabinose units only at the subsites -1 and -2. Abf3 prefers small oligomers over large oligomers. It is able to hydrolyze all linkages present in beet arabinan, including the linkages of double substituted residues. Abn4 is more active towards polymeric substrate and releases arabinose monomers from single substituted arabinose residues. Depending on the combination of the enzymes, the C1 arabinohydrolases can be used to effectively release branched arabinose oligomers and/or arabinose monomers.     

Origin of the multiple components of human α1-antitrypsin

Multiple components of human ₁-antitrypsin were separated by preparative starch gel electrophoresis, and the sialic acid contents of these components were determined. The acidic components contained more sialic acid per molecule than the basic components. The molecular sizes of these components were identical, excluding the possibility of polymerization of the inhibitor in the formation of the multiple components. Consequently, the multiple components of the inhibitor are primarily due to differences in the sialic acid content of each component. Three major components contain eight, seven, and six sialic acid residues per molecule, respectively.This work was supported by Grant HL-17535 from the National Institutes of Health.     

Topographical analysis of As-induced folding of α-MT1a

Metallothionein binds multiple metals into two clustered domains. While the structure of the fully metalated protein is well known for the Cd- and Zn-containing protein, there is little known about the structures of the metal-free protein (apo-metallothionein) and even less about the partially metalated forms. However, the partially-metalated species are vitally important intermediates in the passage of the protein from translational synthesis to its homeostatic buffer or metal chaperone roles. Because multiple metals bind to metallothioneins, the partially-metalated species span a wide range depending on the metal bound. Up to 3 As³⁺ bind stepwise to the α-domain fragment in a manner that allows measurement of each of the 4 species simultaneously with the number of free cysteines diminishing by 3 for every As³⁺ bound: apo- (11 Cys), As₁- (8 Cys), As₂- (5 Cys) and As₃-α-MT (2 Cys). The cysteine modifier benzoquinone (Bq), was used to determine the relative accessibility of the free cysteines in the α-MT fragment as a function of the number of As³⁺ bound. The effect of each As³⁺ was to induce folding in the protein. The ESI-MS results show that the whole protein folds significantly even when just one of the three As³⁺ has bound. The profile of the Bq reacting with the unbound cysteines shows effects of steric hindrance in slowing down the reaction. By freezing the reaction midway to the endpoint, the mass spectral data show the ‘mid-flight’ concentrations of all the key species, 27 in all. Analysis of this mid-flight reaction profile gives insight into the topology of the partially metalated MT from the differential access to the unbound cysteinyl thiols by the Bq. Significantly, the metal-free, apo-α-MT also adopts a folded structure in the presence of the As³⁺ even though there is no As³⁺ bound. This can only happen if the apo-protein wraps around other metalated proteins in solution via protein–protein interactions.     

Persistence of TGF-β1 induction of increased fibroblast contractility

Fibroblast contraction of collagen gels is regarded as a model of wound contraction. Transforming growth factor (TGF)-beta added to such gels can augment contraction consistent with its suggested role as a mediator of fibrotic repair. Since fibroblasts isolated from fibrotic tissues have been suggested to express a "fibrotic phenotype," we hypothesized that TGF-beta exposure may lead to a persistent increase in fibroblasts' contractility. To evaluate this question, confluent human fetal lung fibroblasts were treated with serum-free Dulbecco modified Eagle medium (DMEM), with or without 100 pM [corrected] TGF-beta1, TGF-beta2, or TGF-beta3 for 48 h. Fibroblasts were then trypsinized and cast into gels composed of native type I collagen isolated from rat tail tendons. After 20 min for gelation, the gels were released and maintained in serum-free DMEM. TGF-beta-pretreated fibroblasts caused significantly more rapid gel contraction (52.5+/-0.6, 50.9+/-0.2, and 50.3+/-0.5% by TGF-beta1, -beta2, and -beta3 pretreated fibroblasts, respectively) than control fibroblasts (74.0+/-0.3%, P < 0.01). This effect is concentration dependent (50-200 nM), and all three isoforms had equal activity. The effect of TGF-beta1, however, persisted for only a short period of time following the removal of TGF-beta, and was lost with sequential passage. These observations suggest that the persistent increase in collagen-gel contractility, mediated by fibroblasts from fibrotic tissues, would not appear to be solely due to previous exposure of these cells to TGF-beta.     

Microbial 1β-Hydroxylation of Digitoxigenin

l-Deoxy-l-l-proIino-d-fructose was isolated from flue-cured tobacco leaves in crystalline form. The structure was confirmed by comparison with synthetic compound.     

The development of the antigenic structures of human α1-antichymotrypsin and C 1 q

Summary The cross-reactions of human ₁-antichymotrypsin and C 1 q with their homologues in the plasmas of the chimpanzee, several Old World monkeys and nine non-primate eutheria were investigated by standard procedures. The results show that cross-reactions are limited to the first species mentioned. Comparative Ouchterlony tests and absorption controls revealed the presence of two (human) determinants on both human and chimpanzee molecules, while the cercopithecoids analyzed carried only one of them on their homologue. The results are discussed briefly with reference to earlier findings from this laboratory.

---

Zusammenfassung Die Kreuzreaktionen des menschlichen ₁-Antichymotrypsin und des C 1 q mit seinen Homologen im Plasma des Schimpansen, einiger Altweltaffen und demjenigen von 9 Nichtprimaten (Eutheria) wurden mit Standardmethoden untersucht. Die Ergebnisse zeigen, daß Kreuzreaktionen auf die zuerst genannten Species beschränkt sind. Vergleichende Ouchterlony-Tests und Absorptionskontrollen ließen die Anwesenheit zweier (menschlicher) Determinaten auf den Molekülen des Menschen und des Schimpansen erkennbar werden, während die untersuchten Cercopithecoidea nur eine dieser Determinanten besitzen. Die Ergebnisse werden kurz im Zusammenhang mit früheren Befunden aus unserem Laboratorium diskutiert.

     

Release of Interleukin-1α or Interleukin-1β Depends on Mechanism of Cell Death

The cytokine interleukin-1 (IL-1) has two main pro-inflammatory forms, IL-1α and IL-1β, which are central to host responses to infection and to damaging sterile inflammation. Processing of IL-1 precursor proteins to active cytokines commonly occurs through activation of proteases, notably caspases and calpains. These proteases are instrumental in cell death, and inflammation and cell death are closely associated, hence we sought to determine the impact of cell death pathways on IL-1 processing and release. We discovered that apoptotic regulation of caspase-8 specifically induced the processing and release of IL-1β. Conversely, necroptosis caused the processing and release of IL-1α, and this was independent of IL-1β processing and release. These data suggest that the mechanism through which an IL-1-expressing cell dies dictates the nature of the inflammatory mechanism that follows. These insights may allow modification of inflammation through the selective targeting of cell death mechanisms during disease.     

Cloning of DNA encoding a catalytic subunit of SNF1-related protein kinase-1 (SnRK1-α1), and immunological analysis of multiple forms of the kinase,in spinach leaf

Plant Molecular Biology - Using a PCR approach, we have cloned DNA encoding a catalytic subunit isoform (SnRK1-α1) of SNF1-related protein kinase-1 from spinach leaf. The predicted amino acid...