A synthetic beta-casein phosphopeptide and analogues as model substrates for casein kinase-1, a ubiquitous, phosphate directed protein kinase

The phosphopeptide Ser (P)-Ser(P)-Ser-(P)-Glu-Glu-Ser22-Ile-Thr, reproducing the 17-24 segment of beta-casein A2 including the seryl residue (Ser-22) which is targeted by casein kinase-1 was synthesized and used as model substrate for this enzyme. Its phosphorylation efficiency is actually higher than that of intact beta-casein (similar Vmax and 14 microM Km). Conversely the fully dephosphorylated peptide SSSEESIT is not affected by CK-1 to any detectable extent and its glutamyl derivative EEEEESIT displays a more than 50-fold higher Km and a 5-fold lower Vmax as compared to the parent phosphopeptide. The relevance of the individual phosphoseryl residues has been assessed by comparing the phosphorylation efficiencies of the phosphopeptides EESpEESIT, ESpEEESIT and SpEEEESIT: while the first is a substrate almost as good as the tris Ser (P)-peptide (Km = 62 microM), and the third one is almost as poor as EEEEESIT (Km = 1.55 mM), ESpEEESIT displays a intermediate efficiency (Km = 277 microM). These data in conjunction with the finding that the phosphopentapeptide Ser(P)-Ser(P)-Ser(P)-Ser-Ser(P), but neither Ser(P)-Ser(P)-Ser-Ser(P) nor Ser-Ser(P)-Ser(P)-Glu-Glu and Ser-Ala-Ala-Ser(P)-Ser(P), is readily phosphorylated by CK-1, support the concept that CK-1 is a phosphate directed protein kinase recognizing the Ser(P)-X-X-Ser-X and, less efficiently, the Ser(P)-X-X-X-Ser-X motifs.     

Extracellular HIV-1 Nef increases migration of monocytes

Infiltration of human immunodeficiency virus type 1 (HIV-1)-infected and uninfected monocytes/macrophages in organs and tissues is a general phenomenon observed in progression of acquired immunodeficiency syndrome (AIDS). HIV-1 protein Nef is considered as a progression factor in AIDS, and is released from HIV-1-infected cells. Here, we show that extracellular Nef increases migration of monocytes. This effect is (i) concentration-dependent, (ii) reaches the order of magnitude of that induced by formyl-methyonyl-leucyl-proline (fMLP) or CC chemokine ligand 2 (CCL2)/monocyte chemotactic protein (MCP)-1, (iii) inhibited by anti-Nef monoclonal antibodies as well as by heating, and (iv) depends on a concentration gradient of Nef. Further, Nef does not elicit monocytic THP-1 cells to express chemokines such as CCL2, macrophage inhibitory protein-1alpha (CCL3) and macrophage inhibitory protein-1beta (CCL4). These data suggest that extracellular Nef may contribute to disease progression as well as HIV-1 spreading through affecting migration of monocytes.     

Glycine metabolism in intact leaves by in vivo 13C and 15N labeling

Solid-state (13)C NMR measurements of intact soybean leaves labeled by (13)CO(2) (at subambient concentrations) show that excess glycine from the photorespiratory C(2) cycle (i.e. glycine not part of the production of glycerate in support of photosynthesis) is either fully decarboxylated or inserted as (13)C-labeled glycyl residues in proteins. This (13)C incorporation in leaf protein, which is uniformly (15)N labeled by (15)NH(4)(15)NO(3), occurs as soon as 2 min after the start of (13)CO(2) labeling. In those leaves with lower levels of available nitrogen (as measured by leaf nitrate and glutamine-glutamate concentrations), the excess glycine is used primarily as glycyl residues in protein.     

Enzyme I of the phosphotransferase system: induced-fit protonation of the reaction transition state by Cys-502

Enzyme I (EI), the first component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of an N-terminal domain with the phosphorylation site (His-189) and a C-terminal domain with the PEP binding site. Here we use C3-substituted PEP analogues as substrates and inhibitors and the EI(C502A) mutant to characterize structure-activity relationships of the PEP binding site. EI(C502A) is 10 000 times less active than wild-type EI [EI(wt)] with PEP as the substrate, whereas the two forms are equally active with ZClPEP. Cys-502 acts as an acid-base catalyst which stereospecifically protonates the pyruvoyl enolate at C3. The electron-withdrawing chlorine of ZClPEP can compensate for the lack of Cys-502, and in this case, the released 3-Cl-enolate is protonated nonstereospecifically. Several PEP analogues were assayed as inhibitors and as substrates. The respective K(I)/K(m) ratios vary between 3 and 40 for EI(wt), but they are constant and around unity for EI(C502A). EI(wt) with PEP as the substrate is inhibited by oxalate, whereas EI(C502A) with ZClPEP is not. The different behavior of EI(wt) and EI(C502A) toward the PEP analogues and oxalate suggests that the PEP binding site of EI(wt) exists in a "closed" and an "open" form. The open to closed transition is triggered by the interaction of the substrate with Cys-502. The closed conformation is sterically disfavored by C3-modified substrate analogues such as ZClPEP and ZMePEP. If site closure does not occur as with EI(C502A) and bulky substrates, the transition state is stabilized by electron dispersion to the electron-withdrawing substituent at C3.     

The difluoromethylenesulfonic acid group as a monoanionic phosphate surrogate for obtaining PTP1B inhibitors

Three peptides, 7-9, bearing sulfono(difluoromethyl)phenylalanine (F(2)Smp, 2), a nonhydrolyzable, monoanionic phosphotyrosine mimetic, were prepared and evaluated as PTP1B inhibitors. The most effective inhibitor was the nonapeptide, ELEF(F(2)Smp)MDYE-NH(2), (9) which exhibited a K(i) of 360 nM. A comparison of F(2)Smp-bearing peptides 7 [DADE(F(2)Smp)LNH(2), K(i)=3.4 microM] and 8 [EEDE(F(2)Smp)LNH(2), K(i)=0.74 microM] with their phosphono(difluoromethyl)phenylalanine (F(2)Pmp)-bearing analogues indicated that F(2)Smp is not as effective a pTyr mimetic as F(2)Pmp by 100- to 130-fold. Although F(2)Smp is not as effective as F(2)Pmp, a comparison of peptide 7 with analagous peptides bearing other monoanionic pTyr mimetics recently reported in the literature indicates that F(2)Smp is about 65-fold more effective than any other non-hydrolyzable, monanionic pTyr mimetic reported to date. To further assess the difluoromethylenesulfonic acid (DFMS) group as a monoanionic phosphate mimetic, a series of 24 nonpeptidyl biaryl compounds bearing the DFMS group were prepared using polymer-supported methodologies and screened for PTP1B inhibition. Several of these compounds were selected for further study and their IC(50)'s compared to their difluoromethylenephosphonic (DFMP) analogues. The differences in IC(50)'s between the DFMS and DFMP non-peptidyl compounds was not as great as with the F(2)Smp- and F(2)Pmp-bearing peptides. Possible reasons for this and its implication to the design of small molecule PTP1B inhibitors is discussed.     

Expression of Integrin beta3 is correlated to the properties of quiescent hemopoietic stem cells possessing the side population phenotype

With significant attention paid to the field of tissue-specific stem cells, the identification of stem cell-specific markers is of considerable importance. Previously, the side population (SP) phenotype, with the capacity to efflux the DNA-binding dye Hoechst 33342, has been recognized as a common feature of adult tissue-specific stem cells. In this study, we show that high expression of integrin beta(3) (CD61) is an attribute of SP cells isolated from mouse bone marrow. Additionally, we confirmed that the expression of integrin beta(3) is correlated with properties of quiescent hemopoietic stem cells (HSCs) including the strength of the SP phenotype, cell cycle arrest, expression of HSC markers, and long-term hemopoiesis. Importantly, Lineage(-) (Lin(-))/integrin beta(3)(high) (beta(3)(high)) SP cells have as strong a capacity for long-term hemopoiesis as c-Kit(+)/Sca-1(+)/Lin(-) SP cells, which are regarded as one of the most highly enriched HSC populations. Finally, the integrin beta(3) subunit that is present in SP cells having the properties of HSCs, is associated with integrin alpha(v) (CD51). Therefore, our results demonstrate that high expression of integrin beta(3) is correlated to the properties of quiescent HSCs and suggest that the integrin beta(3) subunit is available as a common surface marker of tissue-specific stem cells.     

Activation of IKK by thymosin alpha1 requires the TRAF6 signalling pathway

Thymosin alpha1 (T(alpha)1) is noted for its immunomodulatory activities and therapeutic potential in treatment of infectious diseases and cancer. However, the molecular mechanism of its effectiveness is not completely understood. Here, we report that T(alpha)1 induces interleukin (IL)-6 expression through the I(kappa)B kinase (IKK) and nuclear factor-(kappa)B (NF-(kappa)B) pathway. Using IKK(beta)-deficient bone-marrow-derived macrophages and mouse embryo fibroblasts (MEFs), we show that IKK(beta) is essential for IKK and NF-(kappa)B activation as well as efficient IL-6 induction. Further analysis using tumour necrosis factor receptor-associated factor 6 (TRAF6)-deficient MEFs shows that TRAF6 is crucial for activation of IKK and induction of IL-6 by Talpha1. Intriguingly, T(alpha)1 triggers protein kinase C (PKC)iota/zeta activation, which is TRAF6 dependent and involves IKK. In addition, T(alpha)1 induces the formation of a signalsome composed of TRAF6, p62 and PKC(iota)/zeta as well as IKK. Thus, our study identifies T(alpha)1 as a unique activator of the TRAF6 signal pathway and provides a cohesive interpretation of the molecular basis of the therapeutic utility of T(alpha)1.     

Autotaxin的结构与功能

Autotaxin(ATX)是一个分泌型糖蛋白,具有磷酸二酯酶(PDE)活性,是胞外焦磷酸酶/磷酸二酯酶(ENPP)家族的一员.ATX还具有溶血磷脂酶D(lysoPLD)活性,能够以溶血磷脂酰胆碱(lysophosphatidylcholjne,LPC)为底物催化生成溶血磷脂酸(lysophosphatidic acid,LPA).ATX在很多肿瘤细胞中都有高表达,在肿瘤的发生、发展过程中有着重要作用,被认为是肿瘤治疗中一个可能的靶位.此外,ATX在神经系统、免疫系统中也发挥重要作用.目前已经建立了一系列快速检测ATX活性的方法,并在此基础上研发了相关疾病的诊断技术.基于ATX的多功能性,对其表达调控机理的研究和抑制剂的开发成为当前的研究热点.     

Synthesis of dermorphin-(1-4) derivatives catalyzed by proteases in organic solvents.

In order to extend the use of proteases to organic synthesis and seek the rules of enzymatic reactions in organic media, we focused on unnatural substrates for proteases to form amide bonds. In this paper, the study of unnatural substrates containing D-amino acid residue, which act as acyl acceptors as well as acyl donors for proteases in organic media, is reported. Dermorphin is a heptapeptide (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) with potent analgesic activity. The N-terminal tetrapeptide is the minimum sequence that retains dermorphin activity, and is selected as the model compound in our study. Two dermorphin-(1-4) derivatives, Boc-Tyr-D-Ala-Phe-Gly-N(2)H(2)Ph and Boc-Tyr-D-Ala-Phe-Gly-NH(2), which contained a d-amino acid residue, were synthesized by proteases in organic media for the first time. The synthesis of these two dermorphin-(1-4) derivatives could be catalyzed by subtilisin with Boc-Tyr-D-Ala-OCH(2)CF(3) as an acyl donor substrate in AcOEt. The synthesis of dermorphin-(1-2) derivative Boc-Tyr-D-Ala-N(2)H(2)Ph was catalyzed by alpha-chymotrypsin in different organic solvents and D-Ala-N(2)H(2)Ph was used as an acyl acceptor substrate. Factors influencing the above enzymatic reactions were systematically studied.     

Fluorescent virus precipitin test.

A fluorescent virus precipitin test (FVPT) for the serologic identification of small particulate antigens such as viruses has been described. The test has several advantageous characteristics: (a) It is probably as sensitive as any serologic test (i.e., aggregates with dimensions of 0.2 mum are detectable; therefore, complexes containing as few as three large viruses would give a positive test). (b) Cultivation of the virus is not required. (c) Since an indirect test can be used, only a single fluorescent conjugate is needed to permit the detection of a number of viruses. (d) The indirect test can be used to detect antiviral antibody. (e) The FVPT is rapid and reliable. (f) Its simplicity should enhance its general acceptance and application.     

Stem-loop IV in the 5' untranslated region is a cis-acting element in bovine coronavirus defective interfering RNA replication

The 210-nucleotide (nt) 5' untranslated region (UTR) in the positive-strand bovine coronavirus (BCoV) genome is predicted to contain four higher-order structures identified as stem-loops I to IV, which may function as cis-acting elements in genomic RNA replication. Here, we describe evidence that stem-loop IV, a bulged stem-loop mapping at nt 186 through 215, (i) is phylogenetically conserved among group 2 coronaviruses and may have a homolog in groups 1 and 3, (ii) exists as a higher-order structure on the basis of enzyme probing, (iii) is required as a higher-order element for replication of a BCoV defective interfering (DI) RNA in the positive but not the negative strand, and (iv) as a higher-order structure in wild-type (wt) and mutant molecules that replicate, specifically binds six cellular proteins in the molecular mass range of 25 to 58 kDa as determined by electrophoretic mobility shift and UV cross-linking assays; binding to viral proteins was not detected. Interestingly, the predicted stem-loop IV homolog in the severe acute respiratory syndrome (SARS) coronavirus appears to be group 1-like in that it is in part duplicated with a group 1-like conserved loop sequence and is not group 2-like, as would be expected by the SARS coronavirus group 2-like 3' UTR structure. These results together indicate that stem-loop IV in the BCoV 5' UTR is a cis-acting element for DI RNA replication and that it might function through interactions with cellular proteins. It is postulated that stem-loop IV functions similarly in the virus genome.     

Nitrogen-15 NMR studies of nitrogen metabolism in Picea glauca buds.

In vivo (15)N nuclear magnetic resonance (NMR) as well as (15)N solid-state magic angle spinning (MAS) NMR spectroscopy were used to investigate nitrogen metabolism in cultured white spruce (Picea glauca) buds. Long-term as well as short-term experiments were carried out involving the use of inhibitors of the nitrogen pathways such as methionine sulfoximine (MSO), azaserine (AZA) and aminooxyacetate (AOA). Both in vivo and solid-state NMR showed that when MSO blocked glutamine synthetase (GS) no NH(4)(+) is incorporated. When glutamate synthase (GOGAT) is blocked by AZA there is some incorporation into glutamine (Gln), but very little into alpha-amino groups (glutamate, Glu). The transamination inhibitor AOA does not affect the metabolism of (15)NH(4)(+) into Gln and Glu, but blocks the production of arginine (Arg), as would be expected. Proline (Pro) and gamma-aminobutyric acid (GABA), which are produced directly from Glu without a transamination step, were not affected. The solid-state NMR experiments showed that protein synthesis occurred. Collectively, our results show that NH(4)(+) can only be assimilated through the GS/GOGAT pathway in P. glauca buds.     

The effect of protein conformation change from alpha(II) to alpha(I) on the bacteriorhodopsin photocycle

The bacteriorhodopsin (bR) photocycle was followed by use of time-resolved Fourier-transform infrared (FTIR) spectroscopy as a function of temperature (15-85 degrees C) as the alpha(II) --> alpha(I) conformational transition occurs. The photocycle rate increases with increasing temperature, but its efficiency is found to be drastically reduced as the transition takes place. A large shift is observed in the all-trans left arrow over right arrow 13-cis equilibrium due to the increased stability of the 13-cis isomer in alpha(I) form. This, together with the increase in the rate of dark adaptation as the temperature increases, leads to a large increase in the 13-cis isomer concentration in bR in the alpha(I) form. The fact that 13-cis retinal has a much-reduced absorption cross-section and its inability to pump protons leads to an observed large reduction in the concentration of the observed photocycle intermediates, as well as the proton gradient at a given light intensity. These results suggest that nature might have selected the alpha(II) rather than the alpha(I) form as the helical conformation in bR to stabilize the all-trans retinal isomer that is a better light absorber and is capable of pumping protons.     

Modeling and measuring urban sustainability in multi-criteria based systems — A challenging issue

This paper addresses how urban sustainability is modeled and the ways criteria-based systems deal with its measurability for an effective and reliable assessment. Twelve sustainability models are reviewed and a subset is briefly presented. More importantly, this research work investigates five national rating systems of sustainable urban development compared with the newly developed CAMSUD system. The comparison focuses on the systems' structure, categorization, technical content and measurability. The main findings about the selected national rating systems thoroughly discussed in the paper are: (i) They all have a tree-like structure, (ii) their conceptualization and categorization follow three or four sustainability pillars models, sustainability topics or spatial scale; (iii) they use either planning-oriented or performance-oriented weighting approaches; (iv) the criteria are defined as sustainability goals, action measures or assignments to be fulfilled; (v) the sustainability items can hardly be juxtaposed since they are differently handled, (vi) overlapping criteria might occur, (vii) similar criteria can be categorized under different categories and this affects the emphasis put on these categories, (viii) all criteria are independently rated with no consideration of mutual interrelationships. In an attempt to solve some of these weaknesses, the newly developed CAMSUD system is introduced as alternative and relies on the following: (i) the system structure is considered as a network, (ii) the conceptualization and categorization is based on spatial scaling as well as on sustainability topics and pillars, (iii) many criteria are directly planning-relevant (23 of 40), (iv) the criteria are defined as sustainability goals rather than action measures and (v) the quantification of criteria is planned as to account for mutual interactions.     

Cybernetic formulation of the definition of life

A definition of life (a living individual) in cybernetic terms is proposed. In this formulation, life (a living individual) is defined as a network of inferior negative feedbacks (regulatory mechanisms) subordinated to (being at service of) a superior positive feedback (potential of expansion). It is suggested that this definition is the minimal definition, necessary and sufficient, for life to be distinguished from inanimate phenomena and, as such, it describes the essence of life. Subsequently, a quantitative expression for the amount of the biologically relevant ("purposeful") information (as opposed to the amount of information in the thermodynamic sense) is proposed. This is followed by the application of the formulated approach to different phenomena of a dubious status existing presently on the Earth as well as to the process of origination of life on our planet.     

Morphological and chiral symmetry breaking in reaction-diffusion systems

Morphological and chiral symmetry breaking in reaction-diffusion systems is considered on the basis of the theory of imperfect codimension-two bifurcations. A new type of pattern selection with two triggers is elucidated: (1) morphologically asymmetric structures displaying optical activity can probably be originated from initially racemic and homogeneous conditions when chiral interaction, having the characteristic strength delta (such as electroweak interaction and circularly polarized light) as well as external field, having the characteristic strength eta (such as gravitational field and electrostatic field) are considered; (2) the selective sensitivity of molecular chirality and morphological asymmetry is omicron(delta 1/3) and omicron(eta 1/3), respectively; the sensitivity of mode-mode interaction between chiral polarization and concentration vector is omicron(delta 2/3) or omicron(eta 2/3), respectively. The relation of these conclusions to the life problem is discussed briefly.     

Two distinct poly(A) polymerases isolated from the cytoplasm of Ehrlich ascites tumour cells

The poly(A) polymerases from the cytosol and ribosomal fractions of Ehrlich ascites tumour cells are isolated and partially purified by DEAE-cellulose and phosphocellulose column chromatography. Two distinct enzymes are identified: (a) a cytosol Mn2+-dependent poly(A) polymerase (ATP:RNA adenylyltransferase) and (b) a ribosome-associated enzyme defined tentatively as ATP(UTP): RNA nucleotidyltransferase. The cytosol poly(A) polymerase is strictly Mn2+-dependent (optimum at 1 mM Mn2+) and uses only ATP as substrate, poly(A) is a better primer than ribosomal RNA. The purified enzyme is free of poly(A) hydrolase activity, but degradation of [3H]poly(A) takes place in the presence of inorganic pyrophosphate. Most likely this enzyme is of nuclear origin. The ribosomal enzyme is associated with the ribosomes but it is found also in free state in the cytosol. The purified enzyme uses both ATP and UTP as substrates. The substrate specificity varies depending on ionic conditions: the optimal enzyme activity with ATP as substrate is at 1 mM Mn2+, while that with UTP as substrate is at 10--20 mM Mg2+. The enzymes uses both ribosomal RNA and poly(A) [but not poly(U)] as primers. The purified enzyme is free of poly(A) hydrolase activity.     

Pro-inflammatory effects of hydrogen sulphide on substance P in caerulein-induced acute pancreatitis

Hydrogen sulphide (H(2)S), a novel gasotransmitter, has been recognized to play an important role in inflammation. Cystathionine-gamma-lyase (CSE) is a major H(2)S synthesizing enzyme in the cardiovascular system and DL-propargylglycine (PAG) is an irreversible inhibitor of CSE. Substance P (SP), a product of preprotachykinin-A (PPT-A) gene, is a well-known pro-inflammatory mediator which acts principally through the neurokinin-1 receptor (NK-1R). We have shown an association between H(2)S and SP in pulmonary inflammation as well as a pro-inflammatory role of H(2)S and SP in acute pancreatitis. The present study was aimed to investigate the interplay between pro-inflammatory effects of H(2)S and SP in a murine model of caerulein-induced acute pancreatitis. Acute pancreatitis was induced in mice by 10 hourly intraperitoneal injections of caerulein (50 (g/kg). PAG (100 mg/kg, i.p.) was administered either 1 hr before (prophylactic) or 1 hr after (therapeutic) the first caerulein injection. PAG, given prophylactically as well as therapeutically, significantly reduced plasma H(2)S levels and pancreatic H(2)S synthesizing activities as well as SP concentrations in plasma, pancreas and lung compared with caerulein-induced acute pancreatitis. Furthermore, prophylactic as well as therapeutic administration of PAG significantly reduced PPT-A mRNA expression and NK-1R mRNA expression in both pancreas and lung when compared with caerulein-induced acute pancreatitis. These results suggest that the pro-inflammatory effects of H(2)S may be mediated by SP-NK-1R pathway in acute pancreatitis.