A Mathematical Analysis of Clustering-Free Local SAR Compression Algorithms for MRI Safety in Parallel Transmission.

Parallel transmission (pTX) is a versatile solution to enable UHF MRI of the human body, where radiofrequency (RF) field inhomogeneity appears very challenging. Today, state of the art monitoring of the local SAR in pTX consists in evaluating the RF power deposition on specific SAR matrices called Virtual Observation Points (VOPs). It essentially relies on accurate electromagnetic simulations able to return the local SAR distribution inside the body in response to any applied pTX RF waveform. In order to reduce the number of SAR matrices to a value compatible with real time SAR monitoring ( << 103) , a VOP set is obtained by partitioning the SAR model into clusters, and associating a so- called dominant SAR matrix to every cluster. More recently, a clustering-free compression method was proposed, allowing for a significant reduction in the number of SAR matrices. The concept and derivation however assumed static RF shims and their extension to dynamic pTX is not straightforward, thereby casting doubt on the strict validity of the compression approach for these more complicated RF waveforms. In this work, we provide the mathematical framework to tackle this problem and find a rigorous justification of this criterion in the light of convex optimization theory. Our analysis led us to a variant of the clustering-free compression approach exploiting convex optimization. This new compression algorithm offers computational gains for large SAR models and for high-channel count pTX RF coils.

Similarity of the Escherichia coli proteome upon completion of different biopharmaceutical fermentation processes.

A comprehensive view of the physiological state of Escherichia coli cells at the completion of fermentation processes for biopharmaceutical production was attained via two-dimensional gel electrophoretic analysis of cellular proteins. For high cell density fermentations in which phosphate is depleted to induce recombinant protein expression from the alkaline phosphatase promoter, proteome analysis confirms that phosphate limitation occurs. Known phosphate starvation inducible proteins are observed at high levels; these include the periplasmic phosphate binding protein and the periplasmic phosphonate binding protein. The phn (EcoK) locus of these E. coli K-12 strains remains cryptic, as demonstrated by failure to grow with phosphonate as the sole phosphorus source. Proteome analysis also provided evidence that cells utilize alternative carbon and energy sources during these fermentation processes. To address regulatory issues in the biopharmaceutical industry, comparative electrophoretic analyses were conducted on a qualitative basis for four different fermentation processes. Using this approach, the protein profiles for these processes were found to be highly similar, with the vast majority (85-90%) of proteins detected in all profiles. The observed similarity in proteomes suggests that multiproduct host cell protein immunoassays are a feasible means of quantifying host-derived polypeptides from a variety of biopharmaceutical fermentation processes.

Tethering of CpxP to the inner membrane prevents spheroplast induction of the cpx envelope stress response.

The Cpx envelope stress response of Escherichia coli is controlled by a two-component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors. We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tethering an MBP-CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment. Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy. Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon. Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activation of the sigmaE stress response. Because the sigmaE response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process.

Scanning electron microscopy study of the effect of tetracycline HCl on smear layer removal and fibrin network formation.

Scanning electron microscopy (SEM) was used to evaluate root surface characteristics of human teeth affected with periodontitis following periodontal instrumentation and topical application of tetracycline HCl (TTC-HCl; pH 1.6; 4 min). Specimens were randomly assigned to periodontal instrumentation alone (control 1); periodontal instrumentation plus TTC-HCl (test 1); periodontal instrumentation plus trypsin solution after extraction (control 2); and periodontal instrumentation plus TTC-HCl plus trypsin solution after extraction (test 2). Tetracycline solution was applied with a cotton pellet. Twenty-two single root periodontitis affected human teeth scheduled for extraction were selected. Mucoperiosteal flaps were raised, root surfaces were mechanically and chemically treated, flaps were repositioned and maintained in place for 20 min. Teeth were extracted, rinsed and placed in cold phosphate buffer solution (PBS) and control 2 and test 2 groups were treated with trypsin solution. Specimens were examined using SEM. Smear layer was successfully removed, exposing dentinal tubules; however, fibrin network formation in situ was not improved by application of TTC-HCl.

Overexpression of Escherichia coli oxidoreductases increases recombinant insulin-like growth factor-I accumulation.

Transient overexpression of either DsbA or DsbC can double the yield of periplasmic insulin-like growth factor (IGF)-I in Escherichia coli to 8.5 g/liter. Strikingly, most of the overexpressed DsbA or DsbC is found in the reduced form, implying that enhanced disulfide isomerization is responsible for the substantial increase in IGF-I yield. All of the accumulated IGF-I has had the signal sequence removed, underscoring the secretion capacity of this organism as well as its utility for efficient production of polypeptide with the correct amino terminus. The overexpressed IGF-I constitutes approximately 30% of the total cell protein. Overproduction of active site mutants of DsbA instead of the wild-type gene do not produce this increase in yield. With wild-type levels of DsbA and DsbC, most of the secreted IGF-I is found in disulfide-linked aggregates, although 10% is soluble and about 5% is correctly folded. Contrary to expectations, overexpression of the disulfide oxidoreductases decreased the soluble fraction. Because the aggregated protein can be efficiently solubilized and refolded, the increased yield is a significant benefit for the production of IGF-I.

In vitro and in vivo redox states of the Escherichia coli periplasmic oxidoreductases DsbA and DsbC.

DsbC is a periplasmic protein of Escherichia coli that was previously identified by a genetic selection that rescued sensitivity to dithiothreitol in Tn10 mutagenized cells. The Erwinia chrysanthemi dsbC gene was identified in a previous genetic screen to restore motility in a dsbA null strain. In order to analyze the biochemical role of E. coli DsbC, the protein was overexpressed, purified, and compared with DsbA in terms of disulfide isomerization, thiol oxidation, and in vivo redox state. In vitro, DsbC and DsbA have an equivalent kcat for disulfide isomerization with the model substrate, misfolded insulin-like growth factor-1. However, DsbA is a more effective oxidant than DsbC of protein dithiols. In vivo, DsbA is found exclusively in the oxidized state in wild-type strains grown in rich media. On the other hand, in vivo DsbC has one pair of cysteines oxidized and one pair reduced. DsbD is required to maintain this reduced pair of cysteines, confirming previous genetic results. A dsbC deletion strain showed decreases in the production of some, but not all, heterologous proteins containing multiple disulfide bonds. Notably, those proteins affected by the dsbC deletion do not have the cysteines paired consecutively.

Exploring the microtubule-binding region of bovine microtubule-associated protein-2 (MAP-2): cDNA sequencing, bacterial expression, and site-directed mutagenesis.

A 1.1 kilobase fragment of bovine microtubule-associated protein-2 (MAP-2) cDNA coding for bovine MAP-2 microtubule-binding region (MTBR) was sequenced. Relative to mouse, rat, and human MAP-2, we observed striking preservation of primary structure, even beyond the sequence and spacing of the three nonidentical peptide repeats responsible for microtubule-binding interactions. For further analysis of microtubule-MAP interactions using site-directed mutagenesis, we developed a bacterial expression system coding for the MT-binding fragment of MAP-2 starting at the thrombin cleavage site (position 1629) and continuing to the C-terminus. This MT-binding fragment was purified to homogeneity by taking advantage of the unusual heat-stability and isoelectric properties of this cytomatrix component. We found that the MT-binding domain readily promoted tubulin polymerization, and the critical tubulin concentration was reduced in the presence of this recombinant protein. Because a second repeated sequence analogue can promote tubulin polymerization as well as displace the MT-binding region of MAP-2, this study was designed to learn more about the importance of each repeated sequence in MT binding. Accordingly, we mutated the first and third sequences to resemble the second repeated sequence, thereby generating the mutants designed m12-m2-m3, m1-m32, and m12-m2-m32. These recombinant proteins bound with an affinity comparable to or slightly better than equal concentrations of wild-type MT-binding fragment. Likewise, when the first or third sequence was replaced by an exact copy of the second octadecapeptide repeat, there was little, if any, increase in binding affinity, as reflected in the ability of mutant MT-binding fragments to promote tubulin polymerization.(ABSTRACT TRUNCATED AT 250 WORDS)

Subunit dynamics in Escherichia coli preprotein translocase.

SecY, SecE, and band 1 copurify as the SecY/E integral membrane domain of Escherichia coli preprotein translocase. To measure the in vivo association of these polypeptides and assay possible exchange, plasmid-borne secY and secE genes were placed under control of the ara regulon and fused to DNA encoding the influenza hemagglutinin epitope. Cells were incubated with [35S]methionine, grown for a "chase" period, and then induced with arabinose to express epitope-tagged, nonradioactive SecY and SecE. Both the wild-type and epitope-tagged polypeptides assembled into functional, heterotrimeric SecY/E complex. However, immunoprecipitation with antibody to the epitope tag did not cross-precipitate radiolabeled SecY or SecE. Thus, these subunits normally associate stably in vivo.

Protein folding activities of Escherichia coli protein disulfide isomerase.

DsbA is an Escherichia coli periplasmic protein that mediates disulfide bond formation in newly secreted proteins in vivo. Addition of thiol reagents to purified dsbA reduces its disulfide bond and yields disulfide isomerase activity after removal of the thiol reagent. DsbA can catalyze the conversion of a stable misfolded protein, misfolded IGF-I (mis-IGF-I), to its correctly folded conformation under physiological conditions. This conversion is the result of breaking and re-forming two disulfide bonds. The uncatalyzed rate of this reaction is undetectable. Kinetic analysis of the reaction yielded a Km of 43 microM and a kcat of 0.2 min-1. The oxidized form of dsbA stimulates the oxidative folding of completely reduced IGF-I at pH 7.0. Thus, dsbA has two possible functions depending on its redox state. The reduced form of the protein is a disulfide isomerase while the oxidized protein can assist formation of disulfide bonds in reduced substrates under physiological conditions.

Translocation can drive the unfolding of a preprotein domain.

Precursor proteins are believed to have secondary and tertiary structure prior to translocation across the Escherichia coli plasma membrane. We now find that preprotein unfolding during translocation can be driven by the translocation event itself. At certain stages, translocation and unfolding can occur without exogenous energy input. To examine this unfolding reaction, we have prepared proOmpA-Dhfr, a fusion protein of the well studied cytosolic enzyme dihydrofolate reductase (Dhfr) connected to the C-terminus of proOmpA, the precursor form of outer membrane protein A. At an intermediate stage of its in vitro translocation, the N-terminal proOmpA domain has crossed the membrane while the folded Dhfr portion, stabilized by its ligands NADPH and methotrexate, has not. When the ligands are removed from this intermediate, translocation occurs by a two-step process. First, 20-30 amino acid residues of the fusion protein translocate concomitant with unfolding of the Dhfr domain. This reaction requires neither ATP, delta mu H+ nor the SecA subunit of translocase. Strikingly, this translocation accelerates the net unfolding of the Dhfr domain. In a second step, SecA and ATP hydrolysis drive the rapid completion of translocation. Thus energy derived from translocation can drive the unfolding of a substantial protein domain.

The SecA and SecY subunits of translocase are the nearest neighbors of a translocating preprotein, shielding it from phospholipids.

To study the environment of a preprotein as it crosses the plasma membrane of Escherichia coli, unique cysteinyl residues were introduced into proOmpA and the genes for these mutant preproteins were fused to the gene of dihydrofolate reductase (Dhfr). A photoactivable, radiolabeled and reducible cross-linker was then attached to the unique cysteinyl residue of each purified protein. Partially translocated polypeptides were generated and arrested in their membrane transit by the folded structure of the dihydrofolate reductase domain. After photolysis to label their nearest neighbors and reduction of the disulfide bond between proOmpA-Dhfr and the cross-linker, radiolabeled cross-linker was selectively recovered with the SecA and SecY subunits of preprotein translocase. Strikingly, neither the SecE nor Band 1 subunits were cross-linked to any of the constructs and the membrane phospholipids were almost entirely shielded from cross-linking. The fact that SecY and SecA are the only membrane proteins cross-linked to the translocating chains suggests that they may form an entirely proteinaceous pathway through which secreted proteins pass during membrane transit.

Peptides corresponding to the second repeated sequence in MAP-2 inhibit binding of microtubule-associated proteins to microtubules.

Bovine brain high molecular weight microtubule-associated proteins (MAPs) can be displaced from assembled tubules by peptides corresponding to the second of three nonidentical repeated sequences in mouse MAP-2. The octadecapeptide m2 (VTSKCGSLKNIRHRPGGG) can release MAP-1b from MAP-containing microtubules, and the extended second-sequence peptide m2' (VTSKCGSLKNIRHRPGGGRVK) displaces MAP-1a and MAP-1b as well as MAP-2a and MAP-2b. Peptides m2 and m2' stimulate tubulin polymerization in the absence of MAPs or microtubule-stabilizing agents, and m2' acts as a competitive inhibitor of radiolabeled MAP-2 binding. The dissociation constant for MAP-2 binding to taxol-stabilized tubules was 3.4 microM in the absence of m2' and 14 microM in the presence of 1.5 mM of the m2' peptide. We estimate that the inhibition constant for peptide m2' is about 0.5 mM, about 100 times lower than for the Km of MAP-2. These observations suggest that the second repeated sequence in MAP-2 may represent an important recognition site for MAP binding to microtubules and that other structural features within MAP-2 may reinforce the strength of MAP-microtubule interactions.

The microtubule-binding fragment of microtubule-associated protein-2: location of the protease-accessible site and identification of an assembly-promoting peptide.

Thrombin cleavage of bovine brain microtubule-associated protein (MAP-2) yields two stable limit polypeptide fragments (28,000 and 240,000 Mr). The smaller cleavage product contains the microtubule-binding domain and is derived from the carboxyl terminus of MAP-2 while the 240,000 Mr fragment is derived from the amino terminus. The amino terminal sequence of the smaller cleavage product is homologous with the microtubule-binding fragment of tau in sequence and in a similar location relative to three imperfect octadecapeptide repeats implicated in microtubule binding. Peptides corresponding to the cleavage site and the three repeats of MAP-2 were synthesized. Only the second octadecapeptide repeat (VTSKCGSLKNIRHRPGGG) was capable of stimulating microtubule nucleation and elongation. Microtubules formed in the presence of this peptide displayed normal morphology and retained the inhibition properties of calcium ion, podophyllotoxin, and colchicine. Our result indicates that a region comprising only approximately 1% of the MAP-2 sequence can promote microtubule assembly.

The 28,000 Mr microtubule-binding domain of microtubule-associated protein-2 also contains a neurofilament-binding site.

We have developed a thrombin proteolytic cleavage procedure to obtain higher yields of the Mr 28,000 microtubule-binding and Mr 240,000 microtubule-projection components of MAP-2. The former is a highly basic component, whereas the latter and intact MAP-2 are acidic polypeptides. Most notably, our studies reveal that this Mr 28,000 fragment binds to neurofilaments, but the Mr 240,000 projection domain fails to interact. These data indicate that microtubules and neurofilaments share a common binding site on high-molecular-weight MAP-2.