Search PubMed⌕ Search

Biomedical subjects

M Ekberg

Publications and source records attributed to M Ekberg.

At least 19 recordsLinked to original sources
PubMed2006

Recombinant protein expression and solubility screening in Escherichia coli: a comparative study.

Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His(6)-tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore ;expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization.

Algorithms↗
PubMed1998

Preserved catalytic activity in an engineered ribonucleotide reductase R2 protein with a nonphysiological radical transfer pathway. The importance of hydrogen bond connections between the participating residues.

A hydrogen-bonded catalytic radical transfer pathway in Escherichia coli ribonucleotide reductase (RNR) is evident from the three-dimensional structures of the R1 and R2 proteins, phylogenetic studies, and site-directed mutagenesis experiments. Current knowledge of electron transfer processes is difficult to apply to the very long radical transfer pathway in RNR. To explore the importance of the hydrogen bonds between the participating residues, we converted the protein R2 residue Asp237, one of the conserved residues along the radical transfer route, to an asparagine and a glutamate residue in two separate mutant proteins. In this study, we show that the D237E mutant is catalytically active and has hydrogen bond connections similar to that of the wild type protein. This is the first reported mutant protein that affects the radical transfer pathway while catalytic activity is preserved. The D237N mutant is catalytically inactive, and its tyrosyl radical is unstable, although the mutant can form a diferric-oxo iron center and a R1-R2 complex. The data strongly support our hypothesis that an absolute requirement for radical transfer during catalysis in ribonucleotide reductase is an intact hydrogen-bonded pathway between the radical site in protein R2 and the substrate binding site in R1. Our data thus strongly favor the idea that the electron transfer mechanism in RNR is coupled with proton transfer, i.e. a radical transfer mechanism.

Amino Acid Substitution↗
PubMed1998

Beta-cell apoptosis in an accelerated model of autoimmune diabetes.

BACKGROUND: The non-obese diabetic (NOD) mouse is a model of human type 1 diabetes in which autoreactive T cells mediate destruction of pancreatic islet beta cells. Although known to be triggered by cytotoxic T cells, apoptosis has not been unequivocally localized to beta cells in spontaneously diabetic NOD mice. We created a model of accelerated beta-cell destruction mediated by T cells from spontaneously diabetic NOD mice to facilitate the direct detection of apoptosis in beta cells. MATERIALS AND METHODS: NOD.scid (severe combined immunodeficiency) mice were crossed with bm1 mice transgenically expressing the costimulatory molecule B7-1 (CD80) in their beta cells, to generate B7-1 NOD.scid mice. Apoptosis in islet cells was measured as DNA strand breakage by the TdT-mediated-dUTP-nick end labeling (TUNEL) technique. RESULTS: Adoptive transfer of splenocytes from spontaneously diabetic NOD mice into B7-1 NOD.scid mice caused diabetes in recipients within 12-16 days. Mononuclear cell infiltration and apoptosis were significantly greater in the islets of B7-1 NOD.scid mice than in nontransgenic NOD.scid mice. Dual immunolabeling for TUNEL and either B-7 or insulin, or the T cell markers CD4 and CD8, and colocalization by confocal microscopy clearly demonstrated apoptosis in beta cells as well in a relatively larger number of infiltrating T cells. The clearance time of apoptotic beta cells was estimated to be less than 6 min. CONCLUSIONS: B7-1 transgenic beta cells undergo apoptosis during their accelerated destruction in response to NOD mouse effector T cells. Rapid clearance implies that beta cells undergoing apoptosis would be detected only rarely during more protracted disease in spontaneously diabetic NOD mice.

Adoptive Transfer↗
PubMed1997

Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding.

BACKGROUND: Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis, catalyzing all de novo synthesis of deoxyribonucleotides. The enzyme comprises two dimers, termed R1 and R2, and contains the redox active cysteine residues, Cys462 and Cys225. The reduction of ribonucleotides to deoxyribonucleotides involves the transfer of free radicals. The pathway for the radical has previously been suggested from crystallographic results, and is supported by site-directed mutagenesis studies. Most RNRs are allosterically regulated through two different nucleotide-binding sites: one site controls general activity and the other controls substrate specificity. Our aim has been to crystallographically demonstrate substrate binding and to locate the two effector-binding sites. RESULTS: We report here the first crystal structure of RNR R1 in a reduced form. The structure shows that upon reduction of the redox active cysteines, the sulfur atom of Cys462 becomes deeply buried. The more accessible Cys225 moves to the former position of Cys462 making room for the substrate. In addition, the structures of R1 in complexes with effector, effector analog and effector plus substrate provide information about these binding sites. The substrate GDP binds in a cleft between two domains with its beta-phosphate bound to the N termini of two helices; the ribose forms hydrogen bonds to conserved residues. Binding of dTTP at the allosteric substrate specificity site stabilizes three loops close to the dimer interface and the active site, whereas the general allosteric binding site is positioned far from the active site. CONCLUSIONS: Binding of substrate at the active site of the enzyme is structurally regulated in two ways: binding of the correct substrate is regulated by the binding of allosteric effectors and binding of the actual substrate occurs primarily when the active-site cysteines are reduced. One of the loops stabilized upon binding of dTTP participates in the formation of the substrate-binding site through direct interaction with the nucleotide base. The general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.

Allosteric Regulation↗
PubMed1996

Two conserved tyrosine residues in protein R1 participate in an intermolecular electron transfer in ribonucleotide reductase.

The enzyme ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, which are each inactive alone. R1 contains the active site and R2 contains a stable tyrosyl radical essential for catalysis. The reduction of ribonucleotides is radical-based, and a long range electron transfer chain between the active site in R1 and the radical in R2 has been suggested. To find evidence for such an electron transfer chain in Escherichia coli ribonucleotide reductase, we converted two conserved tyrosines in R1 into phenylalanines by site-directed mutagenesis. The mutant proteins were shown to be enzymatically inactive. In addition, the mechanism-based inhibitor 2'-azido-2'-deoxy-CDP was incapable of scavenging the R2 radical, and no azido-CDP-derived radical intermediate was formed. We also show that the loss of enzymatic activity was not due to impaired R1-R2 complex formation or substrate binding. Based on these results, we predict that the two tyrosines, Tyr-730 and Tyr-731, are part of a hydrogen-bonded network that constitutes an electron transfer pathway in ribonucleotide reductase. It is demonstrated that there is no electron delocalization over these tyrosines in the resting wild-type complex.

Base Sequence↗
PubMed1990

Chemotherapeutic drug sensitivity testing of human leukemia cells in vitro using a semiautomated fluorometric assay.

A simple and reproducible semiautomated fluorometric method for drug sensitivity testing of leukemic cells in microculture is described. The assay is based on hydrolysis of nonfluorescent fluorescein diacetate (FDA) to a strongly fluorescent product (fluorescein) by cells with intact plasma membranes after 72 hr of culture and was in the present study applied to acute lymphocytic leukemia (ALL) cell lines and specimens from patients with lymphocytic and myelocytic leukemia. FDA fluorescence was linearly related to viable cell number within a wide range of cell densities (3-4 logs) as well as in the presence of different added proportions of dead cells. The assay reliably detects high and low grade resistance to vincristine (vcr) and daunorubicin, respectively, as well as the subsequent reversal of vcr resistance by cyclosporin A and the calcium channel blocker verapamil. Using ALL cell lines, drug sensitivity was in good correspondence with data obtained by the microculture tetrazolium assay. Furthermore, drug sensitivity data of fresh leukemia cells from patients with leukemia were readily obtained. The results indicate that the presently described method is applicable for simple and reliable chemosensitivity testing of leukemia cell lines as well as tumor specimens from patients with leukemia.

Cell Count↗
PubMed1988

Coarctation of the abdominal aorta in elderly patients. Case report and review of the literature.

Coarctation of the abdominal aorta as a congenital disease is infrequent, the diagnosis mostly being made at an early age because of renovascular hypertension. Patients who reach the age of 40 more often tend to have the problems located distally to the renal arteries. A 66-year-old female is described, who developed an aortic occlusion, renovascular hypertension and uremia. She was cured with a bifurcation graft and a patch angioplasty of a stenotic renal artery and nephrectomy of the contralateral kidney with an occluded artery without refilling. A literature survey is made of patients older than 40 years.

Aged↗
PubMed1988

Lipoproteins and metabolic control in hypertensive type II diabetics treated with clonidine.

Twenty patients with type II diabetes mellitus and hypertension (WHO stages I and II) participated in a 3-month double-blind cross-over study to evaluate the effects of clonidine (75-300 micrograms daily) on blood pressure, glycemic control and plasma lipoproteins. Already after 1 month's treatment with clonidine the systolic and diastolic blood pressures had decreased, from 168/103 to 161/98 mmHg (p less than 0.01). Fasting blood glucose and HbA1c concentrations were unaffected by 3 months' treatment. Similarly, plasma lipid and lipoprotein concentrations remained unchanged throughout the study (i.e. mean high and low density lipoprotein cholesterol concentrations were 0.89 and 3.87 mmol/l on placebo vs. 0.90 and 3.98 mmol/l on clonidine). Adverse effects were mild and tolerable, and consisted mainly of dryness of the mouth. We conclude that clonidine lowers the blood pressure in patients with type II diabetes without any adverse effects on glycemic control or plasma lipoproteins.

Blood Glucose↗
PubMed1978

Combination of fibromuscular hyperplasia, renal aplasia, hypoplasia or dysplasia and otosclerosis occurring in the same individual or the same family.

We have found 3 reports of fibromuscular hyperplasia (FMH) of the renal arteries with hypertension occurring in 2 siblings as well as a few instances of renal agenesis and unilateral renal aplasia occurring in the same family. In this paper we report on FMH of the renal arteries in 2 women, one giving birth to a child with renal agenesis, the other to a child with a focally dysplastic hypoplasia of the kidneys. A third family is reported, heavily loaded with hypertension and otosclerosis, in which 2 siblings with FMH and otosclerosis were found. Another 4 patients without known heredity for hypertension, but with FMH combined with renal or urinary tract anomalies, are also reported. The findings are discussed, particularly in relation to the findings in a large material of chronic non-obstructive pyelonephritis, where in those with well maintained renal function, hypertension below the age of 40 was found predominantly in females with a positive family history of hypertension and signs highly suggestive of infected hypoplasia or dysplasia of the kidney.

Abnormalities, Multiple↗
PubMed1978

Pharmacokinetic and therapeutic studies of pivmecillinam in patients with normal and impaired renal function.

Pivmecillinam which is the oral form of mecillinam was evaluated by treatment of 26 patients presenting various types of urinary tract infections and by prophylactic treatment of 12 patients. Pivmecillinam given in a daily dosage of 1.2 g for periods of 1 to 56 weeks was well tolerated in all of the patients including those with impaired renal function without any dose reduction. The original bacterial strain in the urine was eradicated in 100% of the cases. Two patients had a superinfection and 4 had a recurrence during a 3-month follow-up period. The oral absorption of pivmecillinam was investigated in 15 patients with normal or slightly reduced renal function and in 5 patients on maintenance hemodialysis. High serum levels were achieved within 1 to 2 h after administration. Patients with reduced renal function showed retarded elimination rates, suggesting that the dose should be adjusted in this category of patients.

Acute Kidney Injury↗