On the Persistence of Researchers in Technological Development

This paper examines several factors that might influence a researcher'slikelihood of continuing with a particular avenue of researchand development. Using the literature as a source of data tomeasure researcher contribution spans in the development ofcochlear implants, specific hypotheses regarding the determinantsof persistence are develped and empircally tested. The resultsnot only provide the basis for developing a theory of researcherpersitence, but also provide a means to track the emergenceof technologies and shape their evolution.     

Characterization of the UDP-N-acetylgalactosamine binding domain of bovine polypeptide alphaN-acetylgalactosaminyltransferase T1

UDP-GalNAc:polypeptide alphaN-acetylgalactosaminyltransferases (ppGaNTases) transfer GalNAc from UDP-GalNAc to Ser or Thr. Structural features underlying their enzymatic activity and their specificity are still unidentified. In order to get some insight into the donor substrate recognition, we used a molecular modelling approach on a portion of the catalytic site of the bovine ppGaNTase-T1. Fold recognition methods identified as appropriate templates the bovine alpha1,3galactosyltransferase and the human alpha1,3N-acetylgalactosaminyltransferase. A model of the ppGaNTase-T1 nucleotide-sugar binding site was built into which the UDP-GalNAc and the Mn2+ cation were docked. UDP-GalNAc fits best in a conformation where the GalNAc is folded back under the phosphates and is maintained in that special conformation through hydrogen bonds with R193. The ribose is found in van der Waals contacts with F124 and L189. The uracil is involved in a stacking interaction with W129 and forms a hydrogen bond with N126. The Mn2+ is found in coordination both with the phosphates of UDP and the DXH motif of the enzyme. Amino acids in contact with UDP-GalNAc in the model have been mutated and the corresponding soluble forms of the enzyme expressed in yeast. Their kinetic constants confirm the importance of these amino acids in donor substrate interactions.     

Independent self-assembly of cadmium-binding {alpha}-fragment of metallothionein in Escherichia coli without participation of {beta}-fragment

We examined the independent self-assembly of the <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">- and ß-fragmentsof human metallothionein (MT) into cadmiumbinding conformationin an Escherichia coli expression system, in addition to wild-typeMT expression. The expressed <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-fragment formed independentlythe structure of a metal-binding cluster without the aid ofthe ß-fragment. The <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-fragment and wild-type MT expressedin E.coli were purified and analyzed for their biochemical andspectroscopic properties. The apparent cadmium binding of the<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-fragment was approximately 12-fold greater than that for thewild-type MT, whereas in other respects the studied biochemicalproperties were similar. In contrast, we were unable to obtainany independently expressed ß-fragment as the cadmium-bindingform in this study. Possible explanations for this phenomenonare discussed.     

High-yield expression in Escherichia coli of soluble human alpha-hemoglobin complexed with its molecular chaperone

The alpha-subunits of human hemoglobin (Hb) have been more difficult to express than beta-chains owing to the high instability of alpha-chains. Here, we describe the production in Escherichia coli of a soluble recombinant alpha-Hb with human alpha-hemoglobin-stabilizing protein (AHSP), its molecular chaperone. To succeed in this expression, we have constructed a vector pGEX-alpha-AHSP which contains two cassettes arranged in tandem in the same orientation permitting to express alpha-hemoglobin and human AHSP. While the GST-alpha-Hb alone was expressed in E.coli as insoluble protein, even after adding lysate containing recombinant AHSP, the expression vector pGEX-alpha-AHSP permits the co-expression of soluble GST-alpha-Hb and GST-AHSP. The alpha-Hb, produced at a high yield of 12 to 20 mg per liter of culture, was then purified as a complex with its chaperone. Biochemical and biophysical properties of recombinant AHSP/recombinant alpha-Hb complex were similar to those of recombinant AHSP/native alpha-Hb complex as assessed by UV/visible and CO or O(2) binding properties. This co-expression technique can be use to study the interaction between a molecular chaperone and its target protein and, more generally, this system would be particularly interesting for the study of partner proteins when one or both proteins are individually unstable.     

Cooperative deformation of a de novo designed protein

A de novo protein design has been made to understand the uniquepacking of natural proteins that have a ß/<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-barrelfold. A carefully designed 207 amino acid sequence was synthesizedusing an Escherichia coli expression system and the structuraland thermodynamic characteristics of the purified protein werestudied. At neutral pH the protein is soluble and monomeric,with large amounts of secondary structure and a hydrophobiccore, although the broad resonance peaks of its NMR spectrumsuggest that the designed protein does not have a unique structurewith tightly packed side chains. In an H–D exchange experiment,no amido protons of the designed protein exchanged slowly withdeuterons. At acidic pH, thermal unfolding was observedwitha remarkable change in the excess heat capacity measured directlyby a differential scanning microcalorimeter. The enthalpy andentropy differences at 110°C, extrapolated from analyzedthermodynamic parameters, are <IMG SRC="http://peds.oxfordjournals.org/math/bsim.gif" ALT="{bsim}" BORDER="0">1/3 of the common values for naturalproteins. These measurements indicate that the folding is significantlycooperative as expected, but that the protein is still looselypacked.     

Second-generation octarellins: two new de novo ({beta}/{alpha})8 polypeptides designed for investigating the influence of {beta}-residue packing on the {alpha}/{beta}-barrel structure stability

The sequence of octarellin I, the first de novo (ß/<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">)₈polypeptide, was revised according to several criteria, amongothers the symmetry of the sequence, ß-residue volumeand hydrophobicity, and charge distribution. These considerationsand the overall conclusions drawn from the first design ledto two new sequences, corresponding to octarellins II and III.Octarellin II retains perfect 8-fold symmetry. Octarellin IIIhas the same sequence as octarellin II, except for the ß-strandswhich exhibit a 4-fold symmetry. The two proteins were producedin Escherichia coli. Infrared and CD spectral analyses of octarellinsII and III reveal a high secondary structure content. Non-denaturinggel electrophoresis, molecular sieve chromatography and analyticalultracentrifugation suggest that both of these second-generationartificial polypeptides exist as a mixture of a monomer anda dimer form. Octarellins II and III are at least 10 times moresoluble than octarellin I. Ureainduced unfolding followed byfluorescence emission suggests that the tryptophan residues,designed to be buried in the (ß/<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">)₈, are indeed packedin the hydrophobic core of both proteins. However, octarellinIII displays a higher stability towards urea denaturation, indicatingthat introducing 4-fold symmetry into the ß-barrelmight be important for stability of the overall folding.     

Limitations of yeast surface display in engineering proteins of high thermostability

Engineering proteins that can fold to unique structures remains a challenge. Protein stability has previously been engineered via the observed correlation between thermal stability and eukaryotic secretion level. To explore the limits of an expression-based approach, variants of the highly thermostable three-helix bundle protein alpha3D were studied using yeast surface display. A library of alpha3D mutants was created to explore the possible correlation of protein stability and fold with expression level. Five efficiently expressed mutants were then purified and further studied biochemically. Despite their differences in stability, most mutants expressed at levels comparable with that of wild-type alpha3D. Two other related sequences (alpha3A and alpha3B) that form collapsed, stable molten globules but lack a uniquely folded structure were similarly expressed at high levels by yeast display. Together these observations suggest that the quality control system in yeast is unable to discriminate between well-folded proteins of high stability and molten globules. The present study, therefore, suggests that an optimization of the surface display efficiency on yeast may yield proteins that are thermally and chemically stable yet are poorly folded.     

Relativized Degree Spectra

We present a relativized version of the notion of a degree spectrumof a structure with respect to finitely many abstract structures.We study the connection to the notion of joint spectrum. Weprove that some properties of the degree spectrum as a minimalpair theorem and the existence of quasi-minimal degrees aretrue for the relative spectrum.     

调制系统中带通滤波器的设计与应用

本文介绍了调制系统中LC带通滤波器的设计与应用，通过函数的选取，可实现带内信号线性相位传输，同时实现较高的带外幅频抑制度，有效降低窄脉冲调制接收机输出旁瓣电平和提高噪抑制，度就LC滤波器与SAW带通滤波器进行了比较。