Biomedical subjects

E Holmgren

Publications and source records attributed to E Holmgren.

At least 37 records · Page 2Linked to original sources

PubMed1991

Purification and characterization of recombinant human insulin-like growth factor II (IGF-II) expressed as a secreted fusion protein in Escherichia coli.

Human insulin-like growth factor II (IGF-II) was produced in an Escherichia coli ompT strain as a 22.5-kDa fusion protein. IGF-II was fused to the carboxy-terminal of a synthetic 15-kDa IgG-binding protein, originating from staphylococcal protein A, via a unique methionine linker. During fermentation, the fusion protein was exported to the growth medium at levels exceeding 900 mg/liter and subsequently affinity purified on IgG Sepharose followed by ion exchange on S Sepharose. After chemical cleavage with CNBr, yielding an authentic IGF-II molecule, the recombinant IGF-II was purified to homogeneity by a two step procedure involving ion-exchange and reverse-phase HPLC. A substantial fraction of the secreted protein was found to be biologically active, eliminating the need for complex refolding procedures. The yield of highly purified and biologically active IGF-II was 5-7 mg/liter of fermenter broth. The IGF-II produced by this method displayed biochemical, immunological, receptor binding, and biological activity properties equal to those of native IGF-II isolated from human serum.

Amino Acids↗

PubMed1990

Human immunodeficiency viral protease is catalytically active as a fusion protein: characterization of the fusion and native enzymes produced in Escherichia coli.

Processing of the gag and pol gene precursor proteins of retroviruses is essential for the production of mature infectious virions. The processing is directed by a viral protease that itself is part of these precursors and is presumed to cleave itself autocatalytically. To facilitate study of this process, the protease was produced as a fusion protein in Escherichia coli. In this construct, the 10,793-Da protease was preceeded by two copies of a modified IgG binding domain derived from protein A. The IgG binding domain was linked to the protease by an Asp-Pro peptide bond which could not be cleaved by the viral protease. A dimer of the 25,400-Da fusion protein was catalytically active, specifically cleaving a substrate peptide at the correct Tyr-Pro bond. Thus, the fusion protein could serve as a model of the viral gag-pol polyprotein. The finding that the fusion protein was catalytically active supports the suggestion that a gag-pol dimer can initiate a proteolytic cascade after budding of the immature virus. The fusion protein also provided a source of authentic protease. The protease was released from the fusion construct by incubation with formic acid, cleaving the Asp-Pro linkage which had been inserted between the IgG binding domain and the protease.

Amino Acid Sequence↗

PubMed1990

Three human interferon-alpha 2 subvariants disclose structural and functional differences.

The human interferon-alpha 2 subvariants 2a, 2b and 2c differ by only one or two amino acids at positions 23 and/or 34 of the mature protein. In this study, the coding regions of the three interferon-alpha 2 subvariants were derived from the cDNA of interferon-alpha 2c by site-directed in vitro mutagenesis. The interferon-alpha subvariants were synthesized using the same Escherichia coli strain for production and were subsequently purified. Comparative studies revealed that they differ significantly in their biological and antigenic properties. Therefore, amino acid positions 23 and 34 seem to be crucial for structure/function of human interferon-alpha. Furthermore, the study points to the importance of defining, whether such minor structural variants of naturally occurring polypeptides represent functional variants.

Amino Acids↗

PubMed1990

Expression and characterization of a recombinant human parathyroid hormone secreted by Escherichia coli employing the staphylococcal protein A promoter and signal sequence.

Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids (hPTH(1-84)). Employing the promoter and signal sequence of Staphylococcus aureus-protein A we have expressed hPTH in Escherichia coli. The expressed proteins are excreted to the growth medium, allowing for rapid and easy purification of the desired products. By amino acid sequence analysis and mass spectrometry, we have shown that the major excreted product is correctly processed human identical hPTH(1-84). The purified recombinant hPTH(1-84) stimulates adenylate cyclase activity in rat osteosarcoma cell membranes to exactly the same extent as synthetic parathyroid hormone standards, indicating that the recombinant product has full biological activity.

Adenylyl Cyclases↗

PubMed1990

Enhanced immunogenicity of recombinant peptide fusions containing multiple copies of a heterologous T helper epitope.

We have examined the immune response against the nonimmunogenic heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli using recombinant fusion proteins containing the STa-peptide linked to an IgG-binding analogue of protein A and varying numbers of the T helper epitope 323-339 from ovalbumin (ova). By immunization of inbred strains of mice with a series of STa fusion proteins, containing up to four copies of ova tandemly multiplied, we demonstrated that the anti-STa antibody response is controlled by ova-specific T helper cells in a genetically restricted manner. In the responding mouse strains (2 out of 3 tested), the level of antibody production was increased by addition of multiple ova epitopes, the anti-STa response being considerably higher to fusion proteins containing four than one or two ova epitopes.

Animals↗

PubMed1990

Differential stability of recombinant human insulin-like growth factor II in Escherichia coli and Staphylococcus aureus.

Recombinant human insulin-like growth factor II (IGF-II), produced as a soluble extracellular fusion protein, was shown to be proteolytically degraded in Escherichia coli. In contrast, the fusion protein secreted from Staphylococcus aureus was stable and the full length product could be recovered by affinity chromatography. After site specific cleavage of the fusion protein, soluble IGF-II with biological activity was obtained without refolding procedures. These results demonstrate that a eukaryotic protein unstable in E. coli can be stabilized by expression in a Gram positive host. The full-length fusion protein from S. aureus was used to characterize the protease responsible for the degradation in E. coli. Biochemical and genetic analysis suggests a specific degradation by the outer membrane protease (OmpT).

Amino Acid Sequence↗

PubMed1989

Dual affinity fusion approach and its use to express recombinant human insulin-like growth factor II.

A dual affinity fusion concept has been developed in which the gene encoding the desired product is fused between two flanking heterologous genes encoding IgG- and albumin-binding domains. Using sequential IgG and serum albumin affinity chromatography, a full-length tripartite fusion protein is obtained. This approach was used to recover a full-length fusion product in Escherichia coli containing the human insulin-like growth factor II (IGF-II). Surprisingly, the recombinant IGF-II showed increased stability against proteolytic degradation in E. coli when produced as a dual affinity fusion protein, as compared to an N-terminal fusion protein. After site-specific cleavage of the tripartite fusion protein, IGF-II molecules with immunological and receptor binding activity were obtained without renaturation steps. The results demonstrate that proteins can fold into biologically active structures, even if provided with large flanking heterologous protein domains. The concept was further used to characterize the specific degradation of recombinant IGF-II in this heterologous host.

Amino Acid Sequence↗

PubMed1989

Rhizobium meliloti anthranilate synthase gene: cloning, sequence, and expression in Escherichia coli.

We determined the DNA sequence of the Rhizobium meliloti gene encoding anthranilate synthase, the first enzyme of the tryptophan pathway. Sequences similar to those seen for the two subunits of the enzyme as found in all other procaryotic species studied are present in a single open reading frame of 729 codons. This apparent gene fusion joins the C terminus of the large subunit (TrpE) to the N terminus of the small subunit (TrpG) through a short connecting segment. We designate the fused gene trpE(G). The gene is flanked by a typical rho-independent terminator at the 3' end and a complex regulatory region at the 5' end resembling those of operons under transcriptional attenuation control. The location of the promoter was determined by S1 nuclease protection, using Rhizobium mRNA. Although this promoter was inactive in Escherichia coli, mutations eliciting activity were easily obtained. One of these was a C----T change at position -9 in the -10 region. The +1 position of the mRNA is the first base of the initiation codon of the leader peptide, implying that unlike trpE(G), which has a normal Shine-Dalgarno sequence, the leader peptide gene lacks a ribosome-binding site.

Amino Acid Sequence↗

PubMed1988

The Reaction Level Scale (RLS85). Manual and guidelines.

The Reaction Level Scale (RLS85) is a "coma scale" for the direct assessment of overall reaction level in patients with acute brain disorders. It is devised for reliable use even in the management of patients who are difficult to assess, such as intubated patients and patients with swollen eyelids. We here present the manual of the RLS85 and the guidelines for its use. The underlying concepts as well as limitations are outlined. Condensed information of known reliability and validity is presented. A training scheme for presumed observers (doctors, nurses and assistant nurses) is outlined. It is suggested that users of the RLS85 refer to these guidelines and in scientific reports clearly state any deviations from this present manual in order to facilitate valid comparisons between different studies and different groups of patients.

Acute Disease↗

PubMed1988

Assessment of responsiveness in acute cerebral disorders. A multicentre study on the reaction level scale (RLS 85).

A new scale for assessment of overall responsiveness, the Reaction Level Scale (RLS 85), which has been shown to have better reliability than the Glasgow Coma Scale (GCS), has been tested in four neurosurgical departments regarding inter-observer agreement and coverage i.e. the proportion of patients that could be assessed by the scale. In a carefully designed study 51 observers pairwise performed 164 tests on 88 patients. Reliability was studied by the Kappa method, which is defined as inter-observer agreement corrected for agreement by chance. The inter-observer agreement measured as overall Kappa was good (K = 0.69 +/- 0.05) and there were no significant differences between the departments, professional categories or aetiologies. Regarding the separate RLS 85 levels the Kappa values were above 0.65, except for withdrawing (K = 0.51) and flexor responses (K = 0.55). There was good inter-observer agreement on coma (K = 0.71). In conclusion, the RLS 85 proved to be easily learnt, it showed full coverage without pseudoscoring, and it was used in a consistent way by doctors, nurses and assistant nurses of four different neurosurgical departments in two Scandinavian countries.

Adolescent↗

PubMed1988

Current reporting of responsiveness in acute cerebral disorders. A survey of the neurosurgical literature.

One hundred sixty-six papers published in seven neurosurgical journals from 1983 through 1985 have been surveyed to determine the methods used for assessment of overall patient responsiveness in acute cerebral disorders (coma grading). Fifty-one different coma scales or modifications were found. The Glasgow Coma Scale (GCS) sum score (that is, the sum of the scores of the individual eye, verbal, and motor scales) dominated (54%), and was used in 73 (76%) of 96 of the head-injury studies; in 56 (77%) of these 73 studies it was the single method of grading neurological status. The GCS sum score was used in 16 (23%) of 70 studies in patients with other etiologies. The Hunt and Hess scale was used in 26 (57%) of 46 reports of patients with subarachnoid hemorrhage. In 31 (55%) of the 56 studies of head injuries using the GCS alone, it was not obvious if the 12- or 13-grade scale was used. In 13 studies (23%) no reference to methodological investigations was made. In 44 papers (79%) the handling of untestable features, such as intubation or swollen eyes, was not reported. In the 56 studies using the GCS alone, coma was defined in many different ways and in 22 studies the definition of coma was not specified. In 63% of reports, the GCS sum score scale was combined in one to five groups of scores and this was done in 32 different ways. No information was available to describe the procedure of data aggregation or the reliability of the 13-grade GCS sum score. The lack of standardization makes it unnecessarily difficult to perform valid comparisons between different series of patients. Since the GCS sum score is the most widely used scale, it is suggested that the reporting of the GCS sum score should be standardized regarding pseudoscoring, coma definition, and use of combined scores. Further studies on the reliability of the GCS sum score are needed.

Acute Disease↗

PubMed1988

A comparison of the Glasgow Coma Scale and the Reaction Level Scale (RLS85).

The Glasgow Coma Scale (GCS) and the Reaction Level Scale (RLS85) were compared for rating neurosurgical patients in regard to ranking order of deficit severity, interobserver variability, and coverage for relevant factors. Four physicians, four registered nurses, and four assistant nurses performed 72 pairwise ratings on 47 neurosurgical patients. The rank correlation between the GCS sum score and the RLS85 was -0.94, suggesting the same ranking order of severity and indicating that the underlying concepts of somnolence, delirium, and motor responses in coma are evaluated in the same way. By the sign test, the RLS85 was shown to have better interobserver agreement than the GCS sum score and the eye-motor-verbal (EMV) profile. The interobserver grading disagreements in both scales were distributed over the entire range of responsiveness, and for the GCS sum score they were slanted to combined segments 9 to 15. The RLS85 showed full coverage of relevant factors, while 43 (60%) of the 72 test occasions in the GCS sum score and the EMV profiles showed untestable features, most often because of patient intubation. The pseudoscore (that is, the choice of value given to untestable features) affects interobserver agreement as well as the estimated overall patient responsiveness in the GCS sum score. Assessment by the order of applying the scales showed a significant effect on the GCS eye-opening scale (p = 0.01) and the GCS sum score (p = 0.03), indicating a sensitivity to environmental stimuli unrelated to the patient's status. This study demonstrates that basically the same information as that found in the separate eye, motor, and verbal scales of the GCS can be combined directly into the RLS85, which has better interobserver agreement and better coverage than the GCS sum score.

Coma↗

PubMed1987

Expression of human insulin-like growth factor I in bacteria: use of optimized gene fusion vectors to facilitate protein purification.

Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.

Cloning, Molecular↗

PubMed1987

Fibrinopeptide A and fibrinogen fragment B beta 15-42 and their relation to the operative trauma and post-operative thromboembolism in neurosurgical patients.

In an earlier study on post-operative thromboembolism in neurosurgery the incidence of deep vein thromboses (DVT) diagnosed by the fibrinogen uptake test and phlebography was reduced to the same extent by two different prophylactic methods (low dose heparin or calf muscle stimulation + dextran). However, patients with lower limb paresis due to a brain lesion experienced relatively often a less successful prophylaxis compared to patients with spinal lesions. There are few reports on successful clinical methods for haematological screening of post-operative DVT. The aim of this study was to examine possible haematological indicators for post-operative thromboembolism and secondarily to elucidate whether there exist some special coagulatory or fibrinolytic characteristics in patients who had been operated upon for brain lesions. We have studied two specific coagulatory factors (FPA reflecting thrombin generation and B beta 15-42 reflecting plasmin activity) in connection with neurosurgical operations. Patients in the above-mentioned study on post-operative DVT operated upon for malignant cerebral tumours or intracranial vascular disease exhibited post-operatively higher values for FPA compared to other neurosurgical diagnoses. B beta 15-42 was higher in the malignant tumour group and almost significantly higher in the intracranial vascular group (p less than 0.065). These differences could not be ascribed to the occurrence of DVT. Another 15 patients divided into a minor and a major lesion group were investigated with determination of both parameters pre- and post-operatively. Concerning FPA an increase was noticed post-operatively compared to pre-operatively in the major lesion group.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Diseases↗

PubMed1987

A gene fusion system for generating antibodies against short peptides.

A novel method to obtain specific antibodies against short peptides is described, involving synthesis of the corresponding oligodeoxynucleotides followed by cloning into a new set of fusion vectors, pEZZ8 and pEZZ18, based on two synthetic IgG-binding domains (ZZ) of Staphylococcus aureus protein A. The soluble gene fusion product thus obtained, can be collected from the culture medium of Escherichia coli and rapidly recovered in a one-step procedure by IgG affinity chromatography. The system was used to express a fusion protein consisting of the two Z fragments and the C-terminal part [amino acids (aa) 57-70] of human insulin-like growth factor I (IGF-I). This 16-kDa protein was purified by affinity chromatography on IgG Sepharose and antibodies were raised in rabbits. The fusion protein elicited peptide-specific antibodies, as measured by solid-phase radioimmuno assay and Western blotting, reactive with both synthetic C-terminal peptide and the native human IGF-I protein. The results suggests that the gene fusion system can be used for efficient antibody production against short peptides encoded by synthetic oligodeoxynucleotides.

Amino Acid Sequence↗

PubMed1986

Production of specific antibodies against protein A fusion proteins.

The gene for Staphylococcal protein A was fused to the coding sequence of bacterial beta-galactosidase, alkaline phosphatase and human insulin-like growth factor I (IGF-I). The fusion proteins, expressed in bacteria, were purified by affinity chromatography on IgG-Sepharose and antibodies were raised in rabbits. All three fusion proteins elicited specific antibodies against both the inserted protein sequences and the protein A moiety. In the case of IGF-I, the protein A moiety in the fusion protein may act as an adjuvant since native IGF-I alone is a poor immunogen. The results suggest that the protein A fusion system can be used for efficient antibody production against peptides or proteins expressed from cloned or synthetic genes. To facilitate such gene fusions a set of optimized vectors have been constructed.

Alkaline Phosphatase↗

PubMed1986

Post-operative thromboembolism in neurosurgery. A study on the prophylactic effect of calf muscle stimulation plus dextran compared to low-dose heparin.

This study compares the safety and effectiveness of two methods for the prophylaxis of post-operative thromboembolism in neurosurgical patients: A: low-dose heparin (5,000 IU X 2 s.c.) started preoperatively and continued daily for one week post-operatively, and B: per-operative electrical calf muscle stimulation with groups of impulses plus post-operative dextran infusions every other day for one week. Neurosurgical patients aged 40 years or more with normal laboratory coagulation values and operated under general anaesthesia were included. The 125:I-fibrinogen uptake test (FUT) was used for screening and phlebography for verification of deep venous thrombosis (DVT). 122 patients entered the study and 104 completed the prophylactic protocol, 58 in group A and 46 in group B. The two groups were comparable according to pre-operative data and distribution of diagnoses. 89 patients completed screening for post-operative DVT. We found an overall incidence of 5/49 (10 percent) DVT in group A and 5/40 (13 percent) in group B, compared to a frequency of 32-50 percent for controls without prophylaxis reported in the literature, In spite of prophylaxis our patients with intracranial neoplasms and intracranial vascular disease showed a relatively higher incidence of DVT, 4/23 (17 percent) and 4/14 (29 percent) respectively, compared to patients with spinal diagnoses 2/25 (8 percent). In combination with cranial diagnoses paretic lower limbs meant an apparent risk factor, 4/7 (57 percent). However, paretic limbs appearing in cases with spinal disorders did not predetermine an unsuccessful prophylaxis, 2/14 (14 percent). Blood loss, transfusion requirements and post-operative complications did not differ significantly between the two prophylactic groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Central Nervous System Diseases↗

PubMed1986

On a free autologous graft of the greater omentum for spinal CSF drainage.

A pedicular flap of the greater omentum can after transposition through the posterior abdominal wall to the lumbar subarachnoid space resorb cerebrospinal fluid (CSF), according to earlier studies. The present study investigates the basic prerequisites in rats and cats for a simplification of the method, utilizing a free omental graft transplanted to the lumbar or cervical region, in contact with neighbouring sacrospinal muscles. It is concluded from long term studies that the free omental graft "survives" and preserves its histological characteristics by revascularization between muscle and omentum, indicating a useful resorptive function.

Animals↗