Synthesis and Characterization of Bulk Nanostructured Thermoelectric Ca3Co4O9.

Nanostructuring has been proposed as an effective strategy for the reduction of the phonon contribution to the thermal conductivity, resulting in an increase in the figure of merit of thermoelectric materials. However, obtaining bulk samples presenting high relative density and nanometric grain size can be quite challenging, particularly in the case of ceramic phases. Only few examples have been reported and none in the case of Ca3Co4O9. In this work, we used a sol­gel synthesis coupled with ball milling to prepare powders of Ca3Co4O9 presenting a grain size as small as 4 nm. These nanopowders were then sintered at different temperature and pressures using the High-Pressure Field-Assisted Sintering Technique (HP-FAST). Relative densities up to 95 vol% where obtained while maintaining a nanometric grain size. The microstructural and electrical properties of the sintered samples have been characterized. The results show that in this oxide a reduction to the nanometric grain size produces a drastic reduction in the electrical conductivity, which cannot be compensated by the reduction in the thermal conductivity. The Seebeck effect, on the other hand, appears to be affected only marginally by the grain size and porosity.

Influence of day of pregnancy on rat placental, uterine, and ovarian prostaglandin synthesis and metabolism.

Synthesis and metabolism of prostaglandins in reproductive tissues of the gravid rat were studied from the time of post-implantation to just prior to parturition. Rat placental prostaglandin synthesis is low on day 8 of pregnancy, sharply increases on day 11, falls on day 14, and remains at a low level for the remainder of gestation. In the tissue PGE2 synthesis is 6 times greater than that of PGF2alpha on day 11. Prostaglandin metabolism in the placenta was high on day 11, low on days 8 and 14, and elevated on days 16, 18, and 21 of pregnancy. PGE1 metabolism was 8 times greater than that of PGF2alpha. Uterine prostaglandin synthesis was low until day 16, and then increased until the end of pregnancy. PGE2 synthesis was very low in this tissue in comparison to PGF2alpha synthesis. Prostaglandin metabolism in the uterus was relatively low until day 16 and then sharply increased for the remainder of gestation. This increase in metabolism was not directly proportional to uterine growth. PGE1 metabolism was 5 times higher than PGF2alpha metabolism in this organ. Ovarian prostaglandin synthesis was very low in comparison to that of the other reproductive organs. Prostaglandin metabolism in this tissue decreased from day 8 through day 18 of pregnancy. PGE1 metabolism in the ovary was twice that of PGF2alpha. These studies demonstrate patterns for synthesis and metabolism of prostaglandin in each tissue studied which may indicate inter-relationships with the physiological requirements of pregnancy.

Crystals of soluble interleukin-1 receptor complexed with its natural antagonist reveal a 1:1 receptor-ligand complex.

Interleukin-1 is a cytokine involved in the acute phase response against infection and injury. We obtained crystals of a complex of soluble, recombinant human interleukin-1 receptor and recombinant human interleukin-1 receptor antagonist, a naturally occurring antagonist. The crystals are suitable for X-ray analysis and diffract to 2.7 A resolution. Solvent content calculations indicate that the crystals contain one receptor and one antagonist molecule per asymmetric unit. Other receptor to antagonist ratios are highly unlikely. These results suggest that the interleukin-1 antagonist binds a single receptor molecule and does not cause receptor aggregation.

A monoclonal antibody that recognizes the receptor binding region of human urokinase plasminogen activator.

An anti-urokinase monoclonal antibody 5B4 (MAB 5B4) was obtained by fusing the murine myeloma cell line X63-Ag8.653 with the spleen cells from a female BALB/c mouse immunized with high-molecular-weight urokinase (HMW-uPA). MAB 5B4 is an IgG1 that binds selectively to the single-chain form of uPA (sc-uPA), to HMW-uPA and to the 17,000 Mr aminoterminal fragment of the A-chain (ATF) but not to the low-molecular-weight urokinase (LMW-uPA) nor to the reduced form of HMW-uPA. This strongly suggests that MAB 5B4 recognizes a conformational determinant on the A-chain. The antibody has an affinity constant for uPA-Sepharose of 1.42 X 10(7) M-1, calculated from equilibrium binding data, and can be used for one step purification of HMW-uPA by immunoaffinity chromatography. MAB 5B4 and the previously obtained antibody 105IF10 recognize the A-chain: the epitopes, however, are distinct as shown by double-antibody-sandwich enzyme immunoassay. Finally MAB 5B4 inhibits the binding of ATF to the uPA receptor of different human cells, whereas 105IF10 does not. Thus this antibody represents a potentially, useful tool for the study of uPA receptor physiology.

Purification and characterization of single-chain urokinase-type plasminogen activator (pro-urokinase) from human A431 cells.

A single-chain urokinase-type plasminogen activator (A431sc-uPA) was purified approximately 18,000-fold from A431 human epidermoid carcinoma cell supernatants by monoclonal antibody immunoaffinity chromatography on 5B4-agarose and ion-exchange FPLC (overall yield 63%). More than 100 micrograms of A431sc-uPA can be recovered per liter of supernatant. The product is homogeneous by SDS-PAGE and reverse phase FPLC analysis while two main isoelectric forms of pI 9.05 and pI 9.20 were observed by IEF. SDS-PAGE in reducing and non-reducing conditions, Western blot analysis and zymography showed that A431sc-uPA is a single-chain protein of about 50,000 Mr immunologically related to urokinase (uPA) and distinct from tissue plasminogen activator (tPA). The N-terminal aminoacid sequence of A431sc-uPA (27 residues) is identical to that of human kidney single-chain uPA. A431sc-uPA does not incorporate 3H-diisopropylfluorophosphate and is virtually inactive on the synthetic substrate S-2444. Plasmin treatment converts A431sc-uPA into a two-chain active form with a fibrinolytic specific activity of 123,000 I. U./mg.

The differential glycosylation of human pro-urokinase from various recombinant mammalian cell lines does not affect activity and binding to PAI-1.

Human chromosomal DNA encoding single-chain urokinase-type Plasminogen Activator (scu-PA, or pro-urokinase) was inserted in an expression plasmid and transfected in human A431, mouse LB6 and CHO cells. LB6 cells were also transfected with a Bovine Papilloma Virus derivative containing the scu-PA gene. Human scu-PA was purified from cell supernatants of recombinant clones and characterized for structure and function. All recombinant scu-PAs are undistinguishable from human urine-derived scu-PA for peptide backbone, but possess a higher sugar content, as revealed by SDS-PAGE analysis after digestion with glycopeptidase F. This difference is partly due to an increased sialic acid content, as shown by analysis of neuraminidase-treated scu-PAs. No difference was found, however, among recombinant and natural scu-PAs in the kinetics of conversion into two-chain active forms (tcu-PAs) by human plasmin, and in the KM and kcat values of tcu-PA activity on the chromogenic substrate S-2444 and on human plasminogen. Also, recombinant and non-recombinant tcu-PAs displayed similar dose-response curves for binding to the endothelial inhibitor PAI-1. In conclusion, the glycosylation pattern of u-PA does not affect its interaction with the plasma proteins directly involved in its fibrinolytic function.

Epitope mapping of the anti-urokinase monoclonal antibody 5B4 by isolated domains of urokinase.

The amino terminal fragment (ATF) of urokinase-type plasminogen activator (uPA) is a degradation product comprising the entire growth factor-like and kringle domains. It has been previously shown that ATF is able to bind to the u-PA receptor through the growth factor-like domain and that the anti u-PA monoclonal antibody 5B4 (Mab 5B4) binds to ATF preventing u-PA receptor binding. To localize more precisely the epitope recognized by Mab 5B4, ATF was subfragmented by controlled enzymatic proteolysis with V8 protease. Three subfragments of 4,000 Mr (F-4k), 11,000 Mr (F-11k) and 12,000 Mr (F-12k) were purified from the reaction mixture and characterized. SDS-PAGE under reducing and non-reducing conditions, N-terminal aminoacid sequence analysis and C-terminal aminoacid analysis of each fragment indicate that F-4k and F-11k correspond to intact growth factor-like domain and kringle domain (residues 4-43 and 44-135 respectively) while F-12k corresponds to the kringle domain cleaved in the first loop at the glu52-gly53 bond. By Western blot and competitive binding experiments we show that Mab 5B4 recognizes an epitope located on the kringle domain of u-PA and that the binding is strongly reduced when the kringle contains an additional cleavage in its first loop. Since the receptor binding site of u-PA has been previously shown to be located on the growth factor-like domain, Mab 5B4 inhibits the binding of uPA to its cellular receptor likely by steric hindrance. Besides the proven utility in epitope localization of anti u-PA monoclonal antibodies, these u-PA fragments may represent powerful tools for studies of structure-function relationship of u-PA.

Human IL-1 receptor antagonist from Escherichia coli: large-scale microbial growth and protein purification.

Interleukin-1 receptor antagonist (IL-1ra) is a recently discovered cytokine which specifically inhibits IL-1 pro-inflammatory activities in various experimental conditions. In this work, the growth conditions of a recombinant E. coli strain which in laboratory studies expressed human IL-1ra mostly in insoluble form, have been optimized at the level of 6-1 bioreactors and then scaled up to a 50-1 process. As a result, a high amount (0.43 g l-1 of microbial culture) of soluble, active IL-1ra has been directly obtained in the large-scale cell lysate with no need for protein solubilization. Also, an efficient purification procedure has been developed for the soluble protein, based on cation exchange expanded bed adsorption directly followed by anion exchange chromatography. This process, which does not include any intermediate dialysis step or gradient elutions, can be easily scaled up to larger production volumes and is therefore well-suited for manufacturing. As a result of the overall optimization study, more than 12 g of pure IL-1ra have been obtained from a single 50-1 fermentation run, without any denaturation/renaturation process. The final product, whose identity and purity have been checked also by MALDI-TOF and ESI-MS, shows full biological activity both in cellular assays and in in vivo experiments with Cynomolgus monkeys.

A new non-hormonal pregnancy-terminating agent.

DL 111-IT, 3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4 triazole, a compound belonging to a new class of non-hormonal antifertility agents, when given subcutaneously, intramuscularly, intravaginally or orally terminates pregnancy in the rat, the mouse, the hamster and the dog. Time-course and dose-activity studies indicate that its effectiveness is dependent on dose, vehicle, route and time of pregnancy. DL 111-IT has no pre-implantation activity. The most effective time for treatment is the early post-implantation period. The compound has an antifertility effect through a slow and continuing action that results in the degeneration and subsequent resorption or expulsion of conceptuses. As a result, there must be sustained availability of active principle to arrest the pregnancy. Administered parenterally in a proper vehicle (oily) and with a suitable schedule of treatment (x 2-5 days), it demonstrates a very high pregnancy terminating activity (ED50: 0.04-0.7 mg/kg/day). Multiple intravaginal and oral administrations are also effective but the daily doses required are 10-20 and 40-100 times higher than the parenteral ones. Studies of the mechanism of action indicate that the site of action is the utero-placental complex. In fact, in pregnant rats, mice, hamsters and dogs, both plasma progesterone levels and the ineffectiveness of progesterone therapy rule out luteolysis as a basis for the activity. Moreover in hypophysectomized, ovariectomized animals whose pregnancies were maintained with proper hormonal treatments, DL 111-IT terminates pregnancy and adrenalectomy does not prevent its effect, which suggests that pituitary, ovaries and adrenals are not required for the antifertility action.

Targeted screening for elongation factor Tu binding antibiotics.

The development of a screen targeted to antibiotics which bind elongation factor Tu (EF-Tu) is described. The method was based on selection of antimicrobial activities which were antagonized by exogenous EF-Tu. Kirromycin, a known inhibitor of EF-Tu, was positive in this screen. Among 47,000 microorganisms screened, several producers of kirromycin-type antibiotics were detected and the novel antibiotics GE2270 and GE37468 were discovered. These thiopeptide molecules constitute, along with amithiamycin, a novel class of antibiotics acting on EF-Tu.

Binding of the glycopeptide antibiotic teicoplanin to D-alanyl-D-alanine-agarose: the effect of micellar aggregates.

Teicoplanin, as well as the other antibiotics of the vancomycin group, was shown to bind specifically to D-alanyl-D-alanine-agarose (D-Ala-D-Ala-AGA) (A. Corti and G. Cassani, Appl. Biochem. Biotechnol. 11, 101-110 (1985)). This finding is extended, showing that the binding is as a function of concentration and physical form of the antibiotic in solution, i.e., monomers or micellar aggregates. At concentrations below the critical micelle concentration (CMC) teicoplanin binds with an affinity and a capacity similar to the other antibiotics of the same group such as vancomycin and ristocetin A. At concentrations above the CMC three times more teicoplanin is bound to D-Ala-D-Ala-AGA than the other two antibiotics. Equilibrium binding experiments carried out at different pHs with teicoplanin in the monomeric or micellar form indicate that the excess binding of teicoplanin occurs in the presence of micelles. Elaboration of binding data according to Scatchard indicates that the maximum binding capacity of the resin is increased 3.6 times when teicoplanin is in the micellar form. On the contrary, the apparent binding affinity is lower.

Sulphonate buffers affect the recovery of microtubule-associated proteins MAP1 and MAP2: evidence that MAP1A promotes microtubule assembly.

The influence of two commonly used sulphonate buffers, PIPES and MES, on the in vitro assembly of bovine brain microtubule protein was examined. Microtubule assembly was monitored by turbimetry and, after centrifugation, the polymerised protein was analysed by SDS-PAGE and western blotting. Assembly in MES when compared with PIPES resulted in a higher recovery of microtubule proteins at both pH 6.4 and pH 6.9 and in an altered protein composition. The buffer pH affected the total amount of protein polymerised but did not significantly affect the protein composition. At both pH conditions the recovery of HMW-MAPs was markedly increased in MES buffer and this increase was mostly due to an increase in the amount of MAP1.

Sensitivity of elongation factor Tu (EF-Tu) from different bacterial species to the antibiotics efrotomycin, pulvomycin and MDL 62879.

The sensitivity of elongation factor Tu (EF-Tu) from different species of bacteria to the EF-Tu-binding antibiotics efrotomycin, pulvomycin and MDL 62879 was tested by measuring the effect of these antibiotics on cell-free protein synthesis systems. EF-Tu from four different Gram-negative species was sensitive to all three antibiotics. Among Gram-positive bacteria, EF-Tu of Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis was resistant to efrotomycin and less sensitive to pulvomycin than EF-Tu of Gram-negative bacteria. EF-Tus from streptococci were significantly less sensitive than EF-Tus from Gram-negative bacteria to both efrotomycin and pulvomycin. All of the EF-Tus were sensitive to MDL 62879. The same sensitivity pattern emerged from GDP exchange assays, performed with partially purified EF-Tu from different bacterial species and pure Escherichia coli EF-Ts. These results suggest that the site of action of MDL 62879 is more conserved among bacterial species than those of efrotomycin and pulvomycin. Heterogeneity of EF-Tus from different bacterial species was also reflected in differences in their apparent molecular masses estimated by SDS-PAGE. EF-Tus from the Gram-positive species had higher molecular masses than those from all but one of the Gram-negative species.

Charge heterogeneity of recombinant pro-urokinase and urinary urokinase, as revealed by isoelectric focusing in immobilized pH gradients.

When analysing homogeneous preparations of recombinant pro-urokinase and urinary urokinase by isoelectric focusing (IEF) in immobilized pH gradients, an extreme charge heterogeneity was detected (at least ten major and ten minor bands in the pH range 7-10). This extensive polydispersity was not caused by different degrees of glycosylation, or by IEF artefacts, such as binding to carrier ampholytes or carbamylation by urea. A great part of this heterogeneity could be traced back to the existence of a multitude of protein molecules containing Cys residues at different oxidation levels (-SH, -S-S-, even cysteic acid). Owing to the very large number of Cys residues in pro-urokinase (24 out of a total of 411 amino acids) and to the relatively high pI of its native forms (pI 9.5-9.8; the native form is believed to contain all Cys residues as -S-S- bridges), the presence of SH or cysteic acid residues would increase the negative surface charge, as even SH groups would be extensively ionized. In pro-urokinase, part of the heterogeneity was also due to spontaneous degradation to urokinase and possibly also to cleavage into lower-molecular-mass fragments. When all these causes of heterogeneity were removed, the pI spectrum was reduced to only four, about equally intense bands. The cause of this residual heterogeneity is unknown.

Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes.

The purified amino-terminal fragment (ATF) of human urokinase plasminogen activator (residues 1-135), which is not required for activation of plasminogen, binds with high affinity to specific plasma membrane receptors on U937 monocytes. Intact urokinase efficiently competes for 125I-labeled ATF binding; 50% competition occurs with 1 nM urokinase. A large part of receptor-bound urokinase remains on the cell surface for at least 2 hr at 37 degrees C. Differentiation of U937 monocytes into macrophage-like cells specifically increases ATF binding 10- to 20-fold. These results suggest an important role for urokinase in monocyte/macrophage biology: the native enzyme binds to the cells with the amino-terminal domain; the catalytic, carboxyl-terminal domain remains exposed on the cell surface to stimulate localized proteolysis and facilitate cell migration.

Antibiotics MDL 62,879 and kirromycin bind to distinct and independent sites of elongation factor Tu (EF-Tu).

Antibiotic MDL 62,879 inhibits bacterial protein synthesis by acting on elongation factor Tu (EF-Tu). In this study we show that the inhibition of protein synthesis by MDL 62,879 in an Escherichia coli cell-free system was fully reversed by addition of stoichiometric amounts of EF-Tu but not by large excesses of EF-Ts, ribosomes, or aa-tRNA. MDL 62,879 bound tightly to EF-Tu and formed a stable 1:1 MDL 62,879:EF-Tu (M:EF-Tu) complex. We show that binding of MDL 62,879 to EF-Tu strongly affects the interaction of EF-Tu with aa-tRNA and causes rapid dissociation of preformed EF-Tu.aa-tRNA complex, suggesting that inhibition of aa-tRNA binding is due to a conformational change in EF-Tu rather than competition for the aa-tRNA binding site. Indication of a conformational change in EF-Tu induced by MDL 62,879 is further confirmed by proteolytic cleavage experiments: MDL 62,879 binding strongly protects EF-Tu against trypsin cleavage. The observed effects of MDL 62,879 appear to be different from those of the kirromycin class of antibiotics, which also inhibit protein synthesis by binding to EF-Tu, suggesting two distinct binding sites. Indeed, the M:EF-Tu complex was able to bind stoichiometric amounts of kirromycin to form a 1:1:1 M:EF-Tu:kirromycin (M:EF-Tu:K) complex, providing direct evidence that the two antibiotics bind to independent and distinct sites on the EF-Tu molecule. The interaction of the M:EF-Tu:K complex with aa-tRNA and other co-factors suggest that the contemporary binding of the two antibiotics locks EF-Tu into an intermediate conformation in which neither antibiotic exhibits complete dominance.

Inhibition of bacterial protein synthesis by elongation-factor-Tu-binding antibiotics MDL 62,879 and efrotomycin.

MDL 62,879 (formerly GE 2270 A) is a novel antibiotic active against Gram-positive bacteria by inhibiting protein synthesis. MDL 62,879 is not active against Gram-negative bacteria, but inhibits cell-free protein synthesis in extracts from Escherichia coli, and shows a high binding affinity for its elongation factor Tu (EF-Tu). We prepared ribosomes and protein-synthesis elongation factors from three sources: E. coli, Bacillus subtilis, and a strain of B. subtilis selected for resistance to MDL 62,879 (strain G1674). Homologous and heterologous reconstituted systems were used to compare the effects of MDL 62,879 and of efrotomycin, an EF-Tu inhibitor of the kirromycin class, which is inactive against both B. subtilis and E. coli. We showed that in cell-free protein synthesis: (a) E. coli was sensitive to both MDL 62,879 and efrotomycin; (b) B. subtilis was sensitive to MDL 62,879, but not to efrotomycin; (c) B. subtilis G1674 was resistant to both antibiotics. In the E. coli system and in the system from wild-type B. subtilis, inhibition by MDL 62,879 was reversed upon addition of purified EF-Tu from B. subtilis G1674. This demonstrates that the antibiotic acts by inhibition of EF-Tu. In contrast, extracts from B. subtilis failed to restore activity in an efrotomycin-inhibited E. coli system. Dominance or resistance to MDL 62,879 and of sensitivity to efrotomycin in heterologous cell-free protein synthesis confirms that inhibition of EF-Tu by the two antibiotics is mediated by different mechanisms of action.

Purification procedure for bacterial translational initiation factors IF2 and IF3.

In bacteria the initiation of protein synthesis is a complex phenomenon in which specific proteins, termed initiation factors (IFs) IF1, IF2, and IF3, are involved. Notwithstanding the progress made in understanding their functions, the precise molecular mechanisms of action of these factors remain somewhat obscure. One reason for this lack of knowledge is the difficulty involved in purifying sufficient quantities of these proteins. We have developed a new procedure for purification of IFs from recombinant Escherichia coli strains producing high levels of E. coli IF3 and Bacillus stearothermophilus IF2. This new procedure is quicker than previous methods, easily scaled up to large volumes, and can be used, with only minor modifications, for different IFs. This new purification method consists essentially of one chromatographic (FPLC) separation on an ion-exchange resin (S-Sepharose fast-flow or Mono-S HR). Using this procedure we have been able to obtain chromatographically pure and biologically active preparations of both IF2 and IF3.

A new cytokine-receptor binding mode revealed by the crystal structure of the IL-1 receptor with an antagonist.

Inflammation, regardless of whether it is provoked by infection or by tissue damage, starts with the activation of macrophages which initiate a cascade of inflammatory responses by producing the cytokines interleukin-1 (IL-1) and tumour necrosis factor-alpha (ref. 1). Three naturally occurring ligands for the IL-1 receptor (IL1R) exist: the agonists IL-1alpha and IL-1beta and the IL-1-receptor antagonist IL1RA (ref. 2). IL-1 is the only cytokine for which a naturally occurring antagonist is known. Here we describe the crystal structure at 2.7 A resolution of the soluble extracellular part of type-I IL1R complexed with IL1RA. The receptor consists of three immunoglobulin-like domains. Domains 1 and 2 are tightly linked, but domain three is completely separate and connected by a flexible linker. Residues of all three domains contact the antagonist and include the five critical IL1RA residues which were identified by site-directed mutagenesis. A region that is important for biological function in IL-1beta, the 'receptor trigger site' is not in direct contact with the receptor in the IL1RA complex. Modelling studies suggest that this IL-1beta trigger site might induce a movement of domain 3.

Refined crystal structure of the interleukin-1 receptor antagonist. Presence of a disulfide link and a cis-proline.

Interleukin-1 (IL-1) molecules are cytokines involved in the acute-phase response against infection and injury. Three naturally occurring IL-1 molecules are known, two agonists: IL-1 alpha and IL-1 beta, and one antagonist, the IL-1 receptor antagonist (IL-1ra). Although IL-1 action protects the organism by enhancing the response to pathogens, its overproduction can lead to pathology and has been implicated in disease states that include septic shock, rheumatoid arthritis, graft versus host disease and certain leukemias. The crystal structure of IL-1ra has been solved at 0.21-nm resolution by molecular replacement using the IL-1 beta structure as a search model. The crystals contain two independent IL-1ra molecules which are very similar. IL-1ra has the same fold as IL-1 alpha and IL-1 beta. The fold consists of twelve beta-strands which form a six-stranded beta-barrel, closed on one side by three beta-hairpin loops. Cys69 and Cys116 are linked via a disulfide bond and Pro53 has been built in the cis-conformation. Comparison of the IL-1ra structure with the IL-1 alpha and IL-1 beta structures present in the Protein Data Bank shows that a putative receptor interaction region, involving the N-terminus up to the beginning of strand beta 1 and the loops D and G, is very different in the three IL-1 molecules. Other putative interaction regions, as identified with mutagenesis studies, are structurally conserved and rigid, allowing precise and specific interactions with the IL-1 receptor.