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E Holmgren

Publications and source records attributed to E Holmgren.

At least 19 recordsLinked to original sources
PubMed2006

On-line strategies for determining trace levels of nitroaromatic explosives and related compounds in water.

We report the development and tests of several systems for the simultaneous determination of 18 energetic compounds and related congeners in untreated water samples. In these systems a Restricted Access Material trap or liquid-chromatography precolumn (with a C(18) or porous graphitic carbon, PGC, stationary phase) followed by a PGC analytical column are used for sample clean-up, enrichment and separation of the trace level analytes, which are then analyzed by mass spectrometry (MS). The relative merits of two MS ionization interfaces (atmospheric pressure chemical ionization, APCI, and atmospheric pressure photoionization, APPI) were also compared for the MS identification and quantification of these analytes. APCI was found to be superior in cases where both alternatives are applicable. A major drawback when applying APPI is that no signal is obtained for the cyclic nitramines and nitrate esters. Using APCI, a wide spectrum of unstable compounds can be determined in a single analysis, and the feasibility of using large volume samples (up to 100 mL) in combination with the sensitivity of the MS detection system provide method detection limits ranging from 2.5 pg/mL (for 2,4-dinitrotoluene and 2,6-diamino-6-nitrotoluene) to 563 pg/mL (for pentaerythritol tetranitrate, PETN), with repeatability ranging from 2 to 7%. Other chemometric parameters such as robustness, selectivity, repeatability, and intermediate precision were also evaluated in the validation of the extraction methods for use in water analysis. Tests with untreated groundwater and drinking water samples, spiked with 20 ng of the analytes, yielded results similar to those obtained with high purity water samples.

Analytic Sample Preparation Methods↗
PubMed2005

Minimally important differences were estimated for the Functional Assessment of Cancer Therapy-Colorectal (FACT-C) instrument using a combination of distribution- and anchor-based approaches.

OBJECTIVE: To estimate minimally important differences (MIDs) on the Functional Assessment of Cancer Therapy-Colorectal (FACT-C) instrument using anchor- and distribution-based methods. STUDY DESIGN AND SETTING: Preliminary MIDs were generated for FACT-C scores based on published results for two samples (n = 60 and n = 63) from the FACT-C validation study. Preliminary MIDs were confirmed using data from a Phase II randomized controlled clinical trial (n = 104) and a population-based observational study (n = 568). MIDs were estimated for the colorectal cancer subscale (CCS); the FACT-C Trial Outcome Index (TOI-C), which is the sum of the CCS, physical well-being, and functional well-being subscales; and the FACT-C total score. Both cross-sectional and longitudinal analyses were used. RESULTS: MIDs were stable across the different patient samples. The recommended MIDs ranged from 2 to 3 points for the CCS, 4 to 6 points for the TOI-C, and 5 to 8 points for the FACT-C total score. CONCLUSIONS: MIDs can enhance the interpretability of FACT-C scores, and they can be used to provide a basis for sample size estimation and to determine clinical benefit in combination with other measures of efficacy. General guidelines for estimating MIDs for other FACT instruments are suggested.

Adult↗
PubMed2001

Cooperative effects by the initiation codon and its flanking regions on translation initiation.

The purine-rich Shine-Dalgarno (SD) sequence located a few bases upstream of the mRNA initiation codon supports translation initiation by complementary binding to the anti-SD in the 16S rRNA, close to its 3' end. AUG is the canonical initiation codon but the weaker UUG and GUG codons are also used for a minority of genes. The codon sequence of the downstream region (DR), including the +2 codon immediately following the initiation codon, is also important for initiation efficiency. We have studied the interplay between these three initiation determinants on gene expression in growing Escherichia coli. One optimal SD sequence (SD(+)) and one lacking any apparent complementarity to the anti-SD in 16S rRNA (SD(-)) were analyzed. The SD(+) and DR sequences affected initiation in a synergistic manner and large differences in the effects were found. The gene expression level associated with the most efficient of these DRs together with SD(-) was comparable to that of other DRs together with SD(+). The otherwise weak initiation codon UUG, but not GUG, was comparable with AUG in strength, if placed in the context of two of the DRs. The +2 codon was one, but not the only, determinant for this unexpectedly high efficiency of UUG.

Base Sequence↗
PubMed2001

Phase I safety and pharmacokinetic study of recombinant human anti-vascular endothelial growth factor in patients with advanced cancer.

PURPOSE: We investigated the safety and pharmacokinetics of a recombinant human monoclonal antibody to vascular endothelial growth factor (rhuMAb VEGF) in patients with cancer. PATIENTS AND METHODS: Cohorts of patients with metastatic cancer having failed prior therapy entered a phase I trial of rhuMAb VEGF administered by a 90-minute intravenous infusion at doses from 0.1 to 10.0 mg/kg on days 0, 28, 35, and 42. Patients underwent pharmacokinetic sampling on day 0 and had serum samples obtained during the subsequent 28 days. Response assessment was carried out on days 49 and 72. RESULTS: Twenty-five patients with a median Eastern Cooperative Oncology Group performance status of 0 were accrued. There were no grade III or IV adverse events definitely related to the antibody. There were three episodes of tumor-related bleeding. Infusions of rhuMAb VEGF were well tolerated without significant toxicity. Grades I and II adverse events possibly or probably related to study drug included asthenia, headache, and nausea. Pharmacokinetics revealed a linear profile with a half-life of 21 days. There were no objective responses, though 12 patients experienced stable disease over the duration of the study. CONCLUSION: rhuMAb VEGF was safely administered without dose-limiting toxicity at doses ranging up to 10 mg/kg. Multiple doses of rhuMAb VEGF were well tolerated, and pharmacokinetic studies indicate that doses of > or = 0.3 mg/kg have a half-life similar to that of other humanized antibodies. Subsequent trials will explore rhuMAb VEGF alone and in combination chemotherapy.

Adult↗
PubMed2001

Phase Ib trial of intravenous recombinant humanized monoclonal antibody to vascular endothelial growth factor in combination with chemotherapy in patients with advanced cancer: pharmacologic and long-term safety data.

PURPOSE: Tumor angiogenesis mediated by vascular endothelial growth factor (VEGF) is inhibited by the recombinant humanized (rhu) monoclonal antibody (MAb) rhuMAbVEGF, which has synergy with chemotherapy in animal models. The present study was designed to assess the safety and pharmacokinetics of weekly intravenous (IV) rhuMAbVEGF with one of three standard chemotherapy regimens. PATIENTS AND METHODS: Twelve adult patients were enrolled four on each combination. rhuMAbVEGF, 3 mg/kg IV, was administered weekly for 8 weeks with (1) doxorubicin 50 mg/m(2) every 4 weeks; (2) carboplatin at area under the curve of 6 plus paclitaxel 175 mg/m(2) every 4 weeks; and (3) fluorouracil (5-FU) 500 mg/m(2) with leucovorin 20 mg/m(2) weekly, weeks 1 to 6 every 8 weeks. RESULTS: The median number of rhuMAbVEGF doses delivered was eight (range, four to eight doses). Grade 3 toxicities were diarrhea (one 5-FU patient), thrombocytopenia (two patients on carboplatin plus paclitaxel), and leukopenia (one patient on carboplatin plus paclitaxel). These toxicities were likely attributable to the chemotherapy component of the regimen. The mean (+/- SD) peak serum level of rhuMAbVEGF was 167 +/- 46 microg/mL, and the mean terminal half-life was 13 days. Total (free plus bound) serum VEGF levels increased from 51 +/- 39 pg/mL (day 0) to 211 +/- 112 (day 49) pg/mL. Three responding patients continued treatment with rhuMAbVEGF and chemotherapy, receiving the equivalent of 36, 20, and 40 total rhuMAbVEGF doses with no cumulative or late toxicities. CONCLUSION: rhuMAbVEGF can be safely combined with chemotherapy at doses associated with VEGF blockade and without apparent synergistic toxicity. Its contribution to the treatment of advanced solid tumors should be evaluated in randomized treatment trials.

Adult↗
PubMed2000

Fusion protein approach to improve the crystal quality of cytochrome bo(3) ubiquinol oxidase from Escherichia coli.

Crystals of cytochrome bo(3) ubiquinol oxidase from E. coli diffract X-rays to 3.5 A and the structure determination is in progress. The limiting factor to the elucidation of the structural detail is the quality of the crystals; the diffraction spots from the crystals are diffused which leads to difficulties in processing the data beyond 4.0 A. Weak protein-protein contacts within the crystal lattice is assumed to be the cause of this problem. To improve these contacts, we have introduced protein Z to the C-terminal end of the subunit IV of cytochrome bo(3) and expressed both proteins as a single fusion. We have successfully obtained crystals of this fusion protein. The spot shape problem has clearly been solved in the crystals of the fusion protein although further optimization is necessary to obtain higher resolution. We also discuss the potential applications of this approach to the crystallization of membrane proteins in general.

Bacterial Proteins↗
PubMed1996

Structure of a combination of Functional Independence Measure and Instrumental Activity Measure items in community-living persons: a study of individuals with cerebral palsy and spina bifida.

OBJECTIVE: To analyze the structure of a combination of physical items from the Functional Independence Measure (FIM) and seven instrumental activity items using ratings of dependence and perceived difficulty. DESIGN: Disability in terms of dependence and subject's perceived difficulty was studied in patients with cerebral palsy (CP) and spina bifida (SB). Rasch analysis was used to construct calibrated linear measure and to identify suitable models with respect to rating steps. SETTING: Interviews were performed at home with patients from an outpatient university rehabilitation unit for young disabled persons. PATIENTS: Fifty-three CP and 20 SB patients (including 5 with other early acquired spinal cord lesions), 20 to 39 years of age, participated and represented 62% and 80%, respectively, of available patients. All had fulfilled an elementary school program. RESULTS: The best scoring model using Rasch analysis was achieved using 5 levels for dependence and 4 levels for perceived difficulty. Hierarchic orders for all items are presented. The FIM items Bowel and Bladder showed different characteristics in the two groups of patients, especially for perceived difficulty, and were excluded in the joint calibrations. There was close overall agreement between the ratings of dependence and perceived difficulty. Person measure values from the Rasch analyses were separated between wheelchair users and walkers. CONCLUSION: The combination of physical items from FIM and instrumental activity measure (IAM) are useful for disability assessment in community-living persons and should be further studied in other impairment groups.

Activities of Daily Living↗
PubMed1994

Translocation and processing of various human parathyroid hormone peptides in Escherichia coli are differentially affected by protein-A-signal-sequence mutations.

Two staphylococcal protein-A signal sequences were constructed and tested for function in Escherichia coli, after being linked to human parathyroid hormone (hPTH) cDNAS representing the intact form (1-84 amino acids) and two N-terminal (1-37 and 1-7 amino acids) peptides. One signal sequence was identical to the wild type, and the other signal contained a deletion of 12 bp at the 3' end. The truncated hPTH cDNAs were fused at their 3' ends to IgG-binding domains (ZZ) derived from protein A in order to facilitate purification and characterization. The expression plasmid pSPTH, containing the wild-type signal sequence, secreted efficiently the intact recombinant hPTH (1-84) into the medium. Plasmids containing the truncated hPTH genes after the wild-type signal, gave rise to hPTH-ZZ hybrid proteins which were correctly processed at the N-terminal, but the major fractions appeared in the periplasmic compartment. In contrast, the plasmid pS'PTH which harboured the 4-amino-acid signal deletion did not promote a uniform secretion of intact hPTH (1-84) to the medium, but released a non-processed form both into the periplasmic compartment and to the medium. The related plasmids pS'PTH37ZZ and pS'PTH7ZZ with the mutated signal sequence gave rise to small or trace amounts of unprocessed forms of fusion proteins in the medium and periplasm, thus the secretion competence was markedly reduced. Thus, for correct N-terminal processing, we conclude that the amino acid sequence in the signal adjacent to the expressed protein, is a key determinant. However, release into the medium or periplasmic space appeared to be dependent also on protein folding, irrespective of signal-sequence cleavage. Furthermore, we observed that the peptides with the wild-type signal sequence and correct N-terminal processing, were the only forms that showed internal cleavage of hPTH. Uncleaved signals may contribute to folding characteristics of the ensuing protein and e.g., prevent internal proteolysis.

Amino Acid Sequence↗
PubMed1993

Co-existing structures of an mRNA stability determinant. The 5' region of the Escherichia coli and Serratia marcescens ompA mRNA.

The structure of untranslated regions of mRNA is thought to play a key role in the degradation of mRNAs by specific RNases. As a model system, in vitro transcripts of the stability determining 5' non-coding region of bacterial ompA mRNA were investigated by calculation of secondary structure models and by experiments applying the temperature-gradient gel electrophoresis (TGGE). For the theoretical prediction of secondary structures an algorithm was used, which yields the structure of lowest free energy as well as a large set of suboptimal structures. Three structures were predicted to co-exist in similar concentrations under native conditions. They denature in a low temperature transition leading to a unique structure which denatures in a high temperature transition. The prediction of three structures and two transitions could be confirmed experimentally by TGGE. Due to the use of transcripts of different length the conformational transitions could be attributed to distinct parts of the molecules. A pseudoknot structural motif was predicted theoretically, but could not be confirmed experimentally. Comparing ompA transcripts of E. coli and S. marcescens, a conservation of structural features could be shown in spite of a sequence homology of only 63%. Regarding the sequential folding of the transcript after synthesis, a metastable structure is formed first and is converted slowly into structures of lower free energy. The biological implications for in vivo degradation are discussed.

Bacterial Outer Membrane Proteins↗
PubMed1992

An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1-3)insulin-like growth factor I.

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1-3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1-3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1-3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.

Amino Acid Sequence↗
PubMed1992

A method for the evaluation of the efficiency of signal sequences for secretion and correct N-terminal processing of human parathyroid hormone produced in Escherichia coli.

Expression plasmids have been constructed for evaluation of different signal sequences for secretion and correct amino terminal processing of foreign proteins expressed in Escherichia coli. cDNA representing the N-terminal region (1-37) of human parathyroid hormone was inserted between DNA coding for two different forms of the signal sequence and two IgG binding domains (ZZ) derived from Staphylococcal protein A. The expression products were secreted to the periplasm and even to the growth medium and were easily purified by affinity chromatography using the ZZ part as a specific handle. Further analyses showed that the expression products were correctly processed to the mature protein hPTH(1-37)ZZ in a construct where the wild type signal sequence of Staphylococcus protein A was used. When a mutated signal sequence which lacks the normal cleavage site was employed, the fusion protein was not cleaved. Since signal sequences seem to be processed in the correct way in this system, we conclude that the general design of this type of expression vector is well suited for studying the N-terminal processing and secretion of heterologous proteins in E. coli.

Amino Acid Sequence↗
PubMed1992

Fusions to the 5' end of a gene encoding a two-domain analogue of staphylococcal protein A.

A novel gene fusion system has been constructed for fusions to the 5' end of gene zz, encoding a two-domain analogue of staphylococcal protein A designated ZZ. Four different genes were fused to the 5' end of zz, and their gene products were analyzed. One of the genes encodes a protein located intracellularly in Escherichia coli and the other three genes encode gene products destined for secretion across the cytoplasmic membrane by the presence of an amino terminal signal sequence. After production in E. coli, the fusion proteins were purified in a single step by IgG-affinity chromatography. The purified ZZ fusions could be used directly for amino terminal sequencing to confirm the start of translation of the intracellular product and the processing of the signal peptide of the translocated products. This is the first example of ZZ fusions to the C-terminus of gene products. To simplify the general use of fusions to the 5' end of zz, a new plasmid vector was constructed containing a multi restriction enzyme cloning linker and the lacZ' gene which enables screening for production in alpha-complementing supE strains of E. coli on indicator plates.

Amino Acid Sequence↗
PubMed1991

A recombinant Escherichia coli heat-stable enterotoxin (STa) fusion protein eliciting anti-STa neutralizing antibodies.

A recombinant fusion protein consisting of native Escherichia coli heat-stable enterotoxin (STa) and a dimer of a synthetic IgG-binding fragment (ZZ), derived from Staphylococcus aureus protein A was produced in E. coli. The fusion protein (ZZSTa) was secreted in large quantities into the growth medium and recovered by affinity chromatography on IgG-Sepharose. Rabbits immunized with the fusion protein responded by producing high serum levels of anti-STa antibodies that also effectively neutralized STa toxicity in infant mice. The fusion peptide ZZSTa had a substantially decreased toxicity as compared with native STa. A polymeric form of ZZSTa separated by size fractionation was about 100 times less toxic than the monomeric fusion protein, yet both forms had the same capacity to induce neutralizing antibodies. This suggests that modified non-toxic forms of ZZSTa with retained immunogenicity may be produced and tested for their usefulness as functional components in a vaccine against diarrhoea caused by enterotoxigenic E. coli.

Antibodies, Bacterial↗
PubMed1991

Characterization of an extended form of recombinant human insulin-like growth factor II.

To investigate the biological role of variants of human insulin-like growth factor II (IGF-II), an extended form designated IGF-IIE21, with a molecular mass of 9.8 kDa, was produced in Escherichia coli as a stable and soluble secreted fusion protein. After site-specific cleavage of the affinity purified fusion protein, followed by purification using ion exchange and reversed phase chromatography, it could be demonstrated that IGF-IIE21 and IGF-II have similar or identical activities according to radioimmunoassay and radioreceptor assay. However, IGF-IIE21 showed only 1% growth promotion activity as compared with IGF-II in a clonal expansion assay using human K562 cells which lacks IGF-I receptors. These results suggest that this extended variant of IGF-II can bind to the receptor but has limited growth promoting activity.

Amino Acid Sequence↗
PubMed1991

Synthesis and secretion of a fibrinolytically active tissue-type plasminogen activator variant in Escherichia coli.

A gene encoding a variant (lacking amino acids 6-173) of human tissue-type plasminogen activator (t-PA), consisting only of the second kringle domain (K2) and the serine protease domain (P), was fused to a DNA segment coding for the signal peptide of staphylococcal protein A and a synthetic gene coding for a protein with ability to bind immunoglobulin G (IgG). The fusion protein which was synthesized in Escherichia coli and secreted into the growth medium, was found to be fibrinolytically active. Purification of the fusion protein was performed in a single step by affinity chromatography with immobilized IgG. Enzymatically active K2P was liberated from the fusion protein by cleavage at a unique Asn-Gly dipeptide sequence using hydroxylamine. These results demonstrate that a variant of human t-PA can be synthesized and secreted by E. coli as a fibrinolytically active fusion protein, which upon specific cleavage yields an active variant t-PA of the expected size.

Amino Acid Sequence↗
PubMed1991

Thrombin and H64A subtilisin cleavage of fusion proteins for preparation of human recombinant parathyroid hormone.

Human parathyroid hormone, hPTH, an 84 amino acid polypeptide, was produced intracellularly in Escherichia coli as a fusion protein, linked to the C-terminus of a 15 kD IgG-binding protein. Approximately 100 mg fusion protein was obtained per liter fermentation medium. To test the efficiency of two alternative enzymatic cleavage methods, two fusion proteins differing only in the linker region were constructed. Cleavage of a Phe-Phe-Pro-Arg linker was obtained with bovine thrombin and cleavage of a Phe-Ala-His-Tyr linker with recombinant H64A subtilisin. Both enzymes yielded the correct N-terminus and cleaved their respective linkers quantitatively, although additional internal cleavage sites in hPTH were detected and characterized. The linker cleavage conditions were optimized and hPTH was purified to homogeneity. Thrombin cleavage resulted in a final yield of 5 mg hPTH/L, while H64A subtilisin cleavage was more specific and gave 8 mg/L. The purified recombinant product was identical to native hPTH and exhibited full biological activity in an adenylate cyclase assay.

Amino Acid Sequence↗