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Biomedical subjects

D Speck

Publications and source records attributed to D Speck.

12 recordsLinked to original sources
PubMed1995

Parallel sequence alignment in limited space.

Sequence comparison with affine gap costs is a problem that is readily parallelizable on simple single-instruction, multiple-data stream (SIMD) parallel processors using only constant space per processing element. Unfortunately, the twin problem of sequence alignment, finding the optimal character-by-character correspondence between two sequences, is more complicated. While the innovative O(n2)-time and O(n)-space serial algorithm has been parallelized for multiple-instruction, multiple-data stream (MIMD) computers with only a communication-time slowdown, typically O(log n), it is not suitable for hardware-efficient SIMD parallel processors with only local communication. This paper proposes several methods of computing sequence alignments with limited memory per processing element. The algorithms are also well-suited to serial implementation. The simpler algorithms feature, for an arbitrary integer L, a factor of L slowdown in exchange for reducing space requirements from O(n) to O(L square root of n) per processing element. Using this result, we describe an O(n log n) parallel time algorithm that requires O(log n) space per processing element on O(n) SIMD processing elements with only a mesh or linear interconnection network.

Algorithms↗
PubMed1994

Expression of alpha 1-proteinase inhibitor in Escherichia coli: effects of single amino acid substitutions in the active site loop on aggregate formation.

Overproduction of eukaryotic proteins in microorganisms often leads to the formation of insoluble protein aggregates which accumulate as intracellular inclusion bodies. alpha 1-Proteinase inhibitor (alpha 1-PI) when produced as a cytoplasmic protein in Escherichia coli (E. coli) forms inclusion bodies containing the majority of the inhibitor in an inactive form. Several variants of alpha 1-PI with single amino acid substitutions within their active site loop (amino acids 345-358) were produced in a bioreactor showing that substitution of Met351 with Glu resulted in significantly reduced aggregate formation compared to the other variants and to wild-type protein. In addition, this variant proved to be fully functional as a proteinase inhibitor. Based on these findings and on results of previous structural studies a mechanism for aggregate formation during expression of alpha 1-PI is suggested.

Binding Sites↗
PubMed1994

Recombinant cholera toxin B subunit in Escherichia coli: high-level secretion, purification, and characterization.

The gene coding for cholera toxin subunit B (CT-B) was fused to a modified ompA signal sequence and subsequently cloned into a high expression vector based on the regulatory signals of the arabinose operon of Salmonella typhimurium. Upon induction of gene expression in Escherichia coli, a product of the expected size for CT-B monomer was detected at a level of approximately 60% of total periplasmic protein. At pilot scale, batch cultivation in a 20-liter bioreactor allowed a production level of 1 g/liter of recombinant CT-B (rCT-B), the majority of which was released into the culture medium. The latter phenomenon was dependent on the medium selected for cultivation. A simple and inexpensive purification scheme was developed which enabled the recovery of 81% of rCT-B from the culture supernatant. Comparing amino acid composition, amino-terminal sequence, mass spectrum, pentamerisation, and GM1-binding, rCT-B is indistinguishable from natural CT-B produced by Vibrio cholerae. This rCT-B overproducing E. coli strain represents an interesting alternative to overexpressing systems developed in V. cholerae.

Amino Acid Sequence↗
PubMed1991

Inhibitory activity and conformational transition of alpha 1-proteinase inhibitor variants.

Several variants of alpha 1-proteinase inhibitor (alpha 1-PI) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) with Arg in alpha 1-PI containing an Arg residue in position 358 (yielding [Thr345----Arg, Met358----Arg]alpha 1-PI) results in complete loss of its inhibitory activity against human alpha-thrombin; whereas an exchange of residue Met351 (P8) by Glu [( Met351----Glu, Met358----Arg]alpha 1-PI) does not alter activity. [Thr345----Arg, Met358----Arg]alpha 1-PI is rapidly cleaved by thrombin, while [Met358----Arg]alpha 1-PI and [Met351----Glu, Met358----Arg]alpha 1-PI form stable proteinase-inhibitor complexes. The stability of [Thr345----Arg, Met358----Arg]alpha 1-PI against guanidinium chloride denaturation is significantly enhanced compared to wild-type alpha 1-PI, and does not change after cleavage, resembling ovalbumin, a serpin with no inhibitory activity, from which the Thr345----Arg amino acid exchange had been derived. [Met351----Glu, Met358----Arg]alpha 1-PI and [Met358----Arg]alpha 1-PI resemble the wild-type protein in this respect. The CD spectra of intact and cleaved alpha 1-PI variants do not compare well with the wild-type protein, probably reflecting local structural differences. Insertion of a synthetic peptide, which corresponds to residues Thr345----Met358 of human alpha 1-PI, leads to the formation of binary complexes with all variants having the characteristic features of the binary complex between peptide and wild-type protein.

Amino Acid Sequence↗
PubMed1991

Isolation of Bacillus sphaericus biotin synthesis control mutants: evidence for transcriptional regulation of bio genes.

The genes involved in biotin synthesis have recently been isolated from Bacillus sphaericus [Gloeckler et al., Gene 87 (1990) 63-70]. Sequence analysis revealed that they are organized into two gene clusters, designated bioXWF and bioDAYB. The 5'-noncoding region of the bioD locus fused to the xylE reporter gene was inserted into the Gram-positive pUB110 replicon and the resulting plasmid was introduced into B. sphaericus IFO3525. Transformants expressed the xylE gene only if the biotin concentration in the growth medium remained below 50 ng/ml. After mutagenesis, colonies were screened for their ability to express the chromogenic marker in the presence of an excess of biotin. Most of these mutants escaped biotin repression of xylE gene expression. Classical genetic analysis showed they formed two main categories: chromosomal mutations, pleiotropically acting in trans on both bioXWF and bioDAYB 5'-noncoding regions, in which a 15-bp region common to both promoters represented a hot-spot for the second class of plasmid-associated mutations. These results, completed by the identification of transcription start points for the bioD and bioX genes, strongly suggest that this 15-bp sequence overlaps the site of a biotin-mediated negative regulation circuit controlling the transcription of the bio genes.

Amino Acid Sequence↗
PubMed1991

Purification and biochemical characterization of recombinant alpha 1-antitrypsin variants expressed in Escherichia coli.

Site-directed variants of alpha 1-antitrypsin (alpha 1AT) expressed in a recombinant strain of Escherichia coli have been isolated with an overall process yield of 50% following tangential flow ultrafiltration, anion-exchange, immobilized metal affinity, and hydrophobic interaction chromatography. The primary structure of the purified variants including the integrity of the N- and C-termini has been verified by electrospray mass spectrometry of the intact molecules (44 kDa) for two of the variants (alpha 1AT Leu-358 and alpha 1AT Ala-357, Arg-358). Complementary classical peptide mapping and automated amino acid sequencing have verified 75% of the primary sequence of alpha 1AT Ala-357, Arg-358. Isoelectric focusing in an immobilized pH gradient revealed some microheterogeneity which proved to be reproducible from one purification batch to another. The isolated variants of alpha 1AT did not show any signs of proteolytic degradation during the purification process and proved to be fully active against their target proteases. The described process also allowed the complete removal of endotoxins from the preparations, opening the possibility to evaluate these novel protease inhibitors for their in vivo efficacy in different animal models of human disease.

Amino Acid Sequence↗
PubMed1991

Biotin formation by recombinant strains of Escherichia coli: influence of the host physiology.

Strains of Escherichia coli were transformed with different plasmids bearing the gene clusters bioXWF and bioDAYB isolated from the Gram positive bacterium Bacillus sphaericus. These genes encode for the enzymes involved in the metabolic pathway which synthesizes biotin from the precursor pimelic acid. Transformed E. coli strains were grown in bioreactors to reach a biomass of 18 g l-1 cell dry weight in 1 litre batch culture with substrate feeding and approximately 50 g l-1 in 10 l fed batch culture. Improved yields of total vitamers and biotin formed in these processes were achieved after a comparative analysis of different culture conditions, medium compositions, host strains and expression systems. Production of 27 mg l-1 of biotin and 200 mg l-1 of vitamers was achieved in 1 litre batch culture. Using a 10-1 fed batch process, biotin and vitamer concentrations reached maximum values of 45 mg l-1 and 350 mg l-1, respectively.

Bacillus↗
PubMed1990

Cloning and characterization of the Bacillus sphaericus genes controlling the bioconversion of pimelate into dethiobiotin.

Using 8.8 kb of genetic information from Bacillus sphaericus, it was possible to confer to Escherichia coli bio- strains, including delta bioA-D, bioC-, bioH-, the ability to convert exogenous pimelate into biotin. The bio genes were borne on two recombinant plasmids with inserts of 4.3 kb and 4.5 kb, which had been isolated from a genomic bank of HindIII-digested B. sphaericus DNA, by phenotypic complementation of various E. coli bio mutants. The B. sphaericus bioD and bioA genes were unambiguously identified within the 4.3-kb insert and shown to be closely linked to bioY (coding for a protein with a presently unknown function) and to bioB [Ohsawa et al., Gene 80 (1989) 39-48]. These genes are clustered in the order bioDAYB. The 4.5-kb fragment contains genetic information for three different proteins, the products of bioX, bioW and bioF. Complementation studies using an E. coli bioF mutant and a B. subtilis bio112TG3 strain, revealed that the third ORF of this cluster encodes 7-keto-8-aminopelargonic acid synthetase. A combination of bioW and bioF allows an efficient complementation of E. coli bioC and bioH mutants, provided that pimelate is added to the biotin-depleted growth medium. No function could be identified for the product of bioX. The gene order of this cluster is bioXWF. By sequence analysis, the two cloned DNA fragments were shown to bear overlapping open reading frames and secondary structures at their 3' ends, typical of transcription terminators.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus↗
PubMed1989

Cloning of the biotin synthetase gene from Bacillus sphaericus and expression in Escherichia coli and Bacilli.

Biotin synthetase (BS) catalyses the biotransformation of dethiobiotin (DTB) to biotin. Here we report the cloning, characterization and expression of the gene encoding BS of Bacillus sphaericus. A recombinant plasmid pSB01, containing an 8.2-kb DNA fragment from B. sphaericus, was isolated by phenotypic complementation of an Escherichia coli bioB strain. Nucleotide sequence analysis of this fragment and N-terminal sequence determination of the recombinant protein product revealed that the bioB gene of B. sphaericus consists of a 996-bp open reading frame which is closely associated with at least one other gene. E. coli cells transformed with a bioB expression vector performed efficient bioconversion of DTB to biotin under defined culture conditions. Biotin production from transformed Bacillus subtilis and B. sphaericus recombinant strains was also demonstrated. Comparison of the amino acid sequences of BS from E. coli and B. sphaericus revealed extensive similarity.

Amino Acid Sequence↗
PubMed1983

Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene.

A method to isolate fragments of DNA that promote gene expression in Bacillus subtilis is described. The system is based on production of catechol 2,3-dioxygenase [CatO2ase; catechol:oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.2] encoded by the Pseudomonas putida TOL plasmid gene xylE. The gene was transferred to aB. subtilis/Escherichia coli plasmid vector to construct pTG402. Although xylE is functionally expressed in E. coli, CatO2ase is not detected in B. subtilis unless a fragment of DNA capable of promoting gene expression is ligated into a cleavage site on pTG402 upstream from xylE. Fragments of chromosomal DNA from B. subtilis, Bacillus licheniformis, Bacillus pumilus, and E. coli are shown to promote xylE gene expression in B. subtilis. The special feature of the system is the method of detection: colonies of cells that express xylE become yellow within seconds after selection plates are sprayed with catechol, a colorless substrate that is converted by CatO2ase to the yellow product, 2-hydroxymuconic semialdehyde. The complete nucleotide sequence of xylE is presented. Strong complementarity between the ribosome binding site and 16S rRNA suggests that xylE mRNA translation in B. subtilis may commence at the same site as that recognized by P. putida. Identity of CatO2ase produced in B. subtilis, E. coli, and P. putida support the hypothesis. Our sensitive color assay offers an approach to develop plasmid gene expression vectors for a wide variety of host organisms.

Bacillus subtilis↗
PubMed1977

[Extracorporeal perfusion with baboon liver in the treatment of hepatic coma (author's transl)].

In 11 patients with hepatic coma (stage IV and V according to Abouna) extracorporeal haemoperfusion using the Scribner shunt (radial or profunda femoris artery) was performed over 12 to 27 hours with 22 baboon and one human livers. Eight patients emerged from coma, six of them showed sufficient regeneration of the diseased liver. Four patients were discharged as cured, one patient died of acute pancreatic necrosis, a further one due to bleeding from an old gastric ulcer. In the 2 remaining patients the coma recurred within 48 hours. Tree patients never came round from coma. After perfusion no antibodies against baboon proteins were demonstrable in the patients. Thus there is very little danger of an anaphylactic reaction when perfusion is repeated. The titre of preformed cytotoxic antibodies against baboon cells in patients' serum rises only after 1-2 weeks and decreases again after 4 weeks. In our experience extracorporeal liver perfusion with baboon or human livers is the most promising method for treatment of hepatic coma.

Adult↗