Structure-function studies of an IGF-I analogue that can be chemically cleaved to a two-chain mini-IGF-I

The structure and biological activities of two disulphide isomersof a C-region deletion mutant of insulin-like growth factor-I(IGF-I) which has an Asn–Gly link engineered at the junctionof the A- and B-regions were studied before and after chemicalcleavage. Circular dichroism (CD) spectra and binding affinityto IGF binding protein 3 (IGFBP3) indicated that the treatmentwith hydroxylamine did not disrupt the overall tertiary foldof the hormones. Cleavage restored some binding affinity forthe IGF-I receptor in both isomers and weakly restored the abilityto stimulate incorporation of tritiated thymidine into DNA inNIH 3T3 fibroblasts transfected with the human IGF-I receptor.Cleavage also restored metabolic capacity, as measured by theability of the isomers to promote lipogenesis in isolated ratadipocytes through the insulin receptor. These results are consistentwith the theory that binding of IGF-I to the IGF-I receptorrequires a conformational change similar to that involved ininsulin binding the insulin receptor. The weak affinity forthe IGF-I receptor after cleavage is consistent with the beliefthat residues in the C-region interact with the IGF-I receptor.This structural difference between insulin and IGF-I gives eacha higher binding affinity for its own receptor.     

A model for the C5a receptor and for its interaction with the ligand

A printer's error was introduced into the title of this articlewhen it was in press. The correct title is as follows. A model for the C5a receptor and for its interaction with theligand^* An omission was also made on the second line of the first columnon page 769. This and the subsequent four lines should readas follows. pairing of Cys86(B) with Cys109(C) or Cys285(G), but stronglysuggest that with either Cys293 or Cys294(G). A choice can bemade with arguments given below. Pairing with Cys285(G), forinstance, would require a vertical relative shift of two helixturns which would immerse polar side chains in the membrane.     

Emulating concurrency in a circuit card assembly system

Typical component-placement systems for populating surface mount technology printed circuit boards now exhibit a high degree of concurrency in their functional operations. This concurrency ideally yields high burst-rate estimates of throughput. However, if the concurrency is not properly understood and exploited, the burst rate is severely degraded, as exhibited by process rates observed in the actual production environment. This discernment requires an experimental characterization of the system's functional operation, which must also reflect the peculiarities of the controller. Such an experimental analysis is an essential precursor to performance-optimization procedures of numerically controlled flexible manufacturing systems. This article describes our analysis of an extremely complex workcell with a high degree of concurrency. Due to its enveloping complexity, the methodological framework for the analysis should be applicable to a broad class of concurrent systems. Empirically verifying the characterization required the development of an emulator that quantitatively defines the system to be modeled. As such, it is a numerical, off-line design and analysis tool. It has been utilized to obtain the process rate for particular products, preevaluate proposed engineering changes, interactively construct setups and sequences, and obtain parameters required for line-balancing procedures.     

Mars Science Laboratory Curiosity Rover Megaripple Crossings up to Sol 710 in Gale Crater

After landing in Gale Crater on August 6, 2012, the Mars Science Laboratory Curiosity rover traveled across regolith‐covered, rock‐strewn plains that transitioned into terrains that have been variably eroded, with valleys partially filled with windblown sands, and intervening plateaus capped by well‐cemented sandstones that have been fractured and shaped by wind into outcrops with numerous sharp rock surfaces. Wheel punctures and tears caused by sharp rocks while traversing the plateaus led to directing the rover to traverse in valleys where sands would cushion wheel loads. This required driving across a megaripple (windblown, sand‐sized deposit covered by coarser grains) that straddles a narrow gap and several extensive megaripple deposits that accumulated in low portions of valleys. Traverses across megaripple deposits led to mobility difficulties, with sinkage values up to approximately 30% of the 0.50 m wheel diameter, resultant high compaction resistances, and rover‐based slip up to 77%. Analysis of imaging and engineering data collected during traverses across megaripples for the first 710 sols (Mars days) of the mission, laboratory‐based single‐wheel soil experiments, full‐scale rover tests at the Dumont Dunes, Mojave Desert, California, and numerical simulations show that a combination of material properties and megaripple geometries explain the high wheel sinkage and slip events. Extensive megaripple deposits have subsequently been avoided and instead traverses have been implemented across terrains covered with regolith or thin windblown sand covers and megaripples separated by bedrock exposures.     

Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis

Benzoylformate decarboxylase (BFD) from Pseudomonas putida wassubjected to directed molecular evolution to generate mutantswith increased carboligase activity which is a side reactionof the enzyme. After a single round of random mutagenesis mutantswere isolated which exhibited a 5-fold increased carboligaseactivity in aqueous buffer compared to the wild-type enzymewith a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone.From the same library, mutants with enhanced carboligase activityin water-miscible organic solvents have been isolated. The selectedmutants have been characterized by sequencing, revealing thatall mutants carry a mutation at Leu476, which is close to theactive site but does not directly interact with the active center.BFD-L476Q has a 5-fold higher carboligase activity than thewild-type enzyme. L476 was subjected to saturation mutagenesisyielding eight different mutants with up to 5-fold increasedcarboligase activity. Surprisingly, all L476 mutants catalyzethe formation of 2-hydroxy-1-phenyl-propanone with significantlyhigher enantioselectivity than the wild-type enzyme althoughenantioselectivity was not a selection parameter. Leu476 potentiallyplays the role of a gatekeeper of the active site of BFD, possiblyby controlling the release of the product. The biocatalyst couldbe significantly improved for its side reaction, the C–Cbond formation and for application under conditions that arenot optimized in nature.     

A novel and efficient xanthenic dye–organometallic ion‐pair complex for photoinitiating polymerization

The photochemistry of the complex formed between a Rose Bengal dianion (RB) and a ferrocenium salt [Fc(+)] is described in this article. Stoichiometric analysis of the crystal showed that the Fc(+):RB ratio is 2:1. The dissociation percentage of complex RB(Fc)₂ in several solvents was evaluated using fluorescence studies. In nonpolar media, in which most of the RB(Fc)₂ exists as contact ion pairs, the photodegradation rate of the complex was found to be unaffected by the presence of oxygen and led to photoproducts capable of initiating a radical polymerization reaction. Because of this, the initiation step showed low oxygen inhibition. In fact, in a visible‐light photoinitiating system such as one composed of xanthene dye (Rose Bengal), ferrocenium salt, amine, and hydroperoxide, substituting the complex RB(Fc)₂ for the sensitizing dye RB led to an increase in efficiency of the polymerization (especially under aerated conditions) as compared to that obtained in the presence of benzoyl phosphine oxide derivatives. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 2368–2376, 2001     

Engineering the C-region of human insulin-like growth factor-1: implications for receptor binding

Recombinant wild-type human IGF-1 and a C-region mutant in whichresidues 28–37 have been replaced by a 4-glycine bridge(4-Gly IGF-1) were secreted and purified from yeast. An IGF-1analogue in which residues 29–41 of the C-region havebeen deleted (mini IGF-1) was created by site-directed mutagenesisand also expressed. All three proteins adopted the insulin-foldas determined by circular dichroism. The significantly raisedexpression levels of mini IGF-1 allowed the recording of two-dimensionalNMR spectra. The affinity of 4-Gly IGF-1 for the IGF-1 receptorwas {small tilde}100-fold lower than that of wild-type IGF-1and the affinity for the insulin receptor was {small tilde}10-foldlower. Mini IGF-1 showed no affinity for either receptor. Notonly does the C-region of IGF-1 contribute directly to thefree energy of binding to the IGF-1 receptor, but also the absenceof flexibility in this region eliminates binding altogether.As postulated for the binding of insulin to its own receptor,it is proposed that binding of IGF-1 to the IGF-1 receptor alsoinvolves a conformational change in which the C-terminal B-regionresidues detach from the body of the molecule to expose theunderlying A-region residues.     

A model for the C5a receptor and for T its interaction with the ligand

A model of the C5a receptor was built based on the assumptionthat the seven membrane-spanning helices of known inward/outwarddirection are in an arrangement roughly similar to that in bacteriorhodopsin.Guidelines for the positioning of the helices were cysteinepairing, ‘ridges into grooves’ interdigitation ofside chains and aromatic cluster formation. The chain segmentsprotruding from the membrane are too short for folding intoan independent ectodomain. The only longer segment (179–202)is tied down in its centre onto the membrane by a disulphidebridge and, thereby, made into two short loops as well. Ideasof the interaction of the C5a receptor with its ligand werederived mainly from the search for accommodation of the functionallyessential arginine residues 40 and 74 of C5a. Asp82 is the onlycharged residue in a pocket {small tilde}20 A below the receptorsurface and is conserved in the rhodopsin superfamily. It commendsitself for binding Arg74 which is the tip of the flexible C-terminalchain of C5a, and rules out Arg40 in the structurally well-definedpart of the molecule. The latter may bind to Glul80 at the bottomof a more shallow pocket which happens to resemble the substrate-bindingsite of trypsin.