Biomedical subjects

M Hansson

Publications and source records attributed to M Hansson.

At least 55 records · Page 3Linked to original sources

PubMed1999

PBSC harvests individually optimized by using pre-collection CD34(+) values and on-line flow cytometric analysis of the mononuclear cell enrichment.

BACKGROUND: We have investigated how the amount of blood processed during collection of PBSC affects the yield of CD34 cells. METHODS: We first established a method of significantly increasing the enrichment of mononuclear cells (MNC), CD34(+) cells and granulocyte-macrophage progenitors (GM-CFU) by on-line flow cytometric (FCM) analysis of the leukocyte populations in the collect line. A total of 166 PBSC collections from 94 patients, were devided into five groups according to the blood volume processed: < 12 L of processed blood, 12-13L, 13-14L, 14-15L and > 15L. RESULTS: When the yield of CD34(+) cells war compared between these groups, a positive correlation was seen (r=0.97) between the processed blood volume and the yield, expressed as a ratio between total number of CD34(+) cells in the harvest and the CD34(+) cell concentration in blood. This correlation can be used to estimate the volume that must be processed to exceed a specific target number of CD34(+) cells. The implications of these results on the need for one, two or more leukapheresis procedures in order to collect a sufficient amount of stem and progenitor cells for a given patient are discussed, i n relation to clinical logistics and the benefits for the patients. DISCUSSION: PBSC harvest can be improved by individually-adjusted leukapheresis according to on-line FCM analysis and pre-harvest levels of CD34(+) cells and the processed blood volume can be used to predict the CD34(+) yield.

Antigens, CD34↗

PubMed1999

Histamine protects T cells and natural killer cells against oxidative stress.

Oxidative stress inflicted by monocytes/macrophages (MO) is recognized as an important immunosuppressive mechanism in human neoplastic disease. We report that two types of lymphocytes of relevance for protection against malignant cells, T cells and natural killer (NK) cells, became anergic to the T cell and NK cell activator interleukin-2 (IL-2) after exposure to MO-derived reactive oxygen metabolites and subsequently acquired features characteristic of apoptosis. The MO-induced anergy and apoptosis in T cells and NK cells were reversed by histamine, an inhibitor of reactive oxygen metabolite synthesis in MO. We propose that strategies to circumvent oxidative inhibition of lymphocytes may be of benefit in immunotherapy of neoplastic disease.

Antigens, CD↗

PubMed1999

Crystallization and preliminary X-ray analysis of the Rhodobacter capsulatus magnesium chelatase BchI subunit.

The Rhodobacter capsulatus BchI protein is one of three subunits of Mg chelatase, the enzyme which catalyzes the first committed step of chlorophyll and bacteriochlorophyll biosynthesis. The BchI protein was produced with an inducible T7 RNA polymerase expression system in Escherichia coli. The protein was purified from the soluble cell-extract fraction and crystallized from polyethylene glycol solution. The crystals diffract to a minimum Bragg spacing of 2.1 A. The space group is P63 with unit-cell dimensions a = b = 90.6, c = 84.1 A.

Base Sequence↗

PubMed1999

Insulin-regulated mitochondrial gene expression is associated with glucose flux in human skeletal muscle.

To identify abnormally expressed genes contributing to muscle insulin resistance in type 2 diabetes, we screened the mRNA populations from normal and diabetic human skeletal muscle using cDNA differential display and isolated abnormally expressed cDNA clones of mitochondrial-encoded NADH dehydrogenase 1 (ND1), cytochrome oxidase 1, tRNA(leu), and displacement loop. We then measured mRNA expression of these mitochondrial genes using a relative quantitative polymerase chain reaction method in biopsies taken before and after an insulin clamp in 12 monozygotic twin pairs discordant for type 2 diabetes and 12 matched control subjects and in muscle biopsies taken after an insulin clamp from 13 subjects with type 2 diabetes, 15 subjects with impaired glucose tolerance, and 14 subjects with normal glucose tolerance. Insulin infusion increased mRNA expression of ND1 from 1.02 +/- 0.04 to 2.55 +/- 0.30 relative units (P < 0.001) and of cytochrome oxidase 1 from 0.80 +/- 0.01 to 1.24 +/- 0.10 relative units (P < 0.001). The ND1 response to insulin correlated with glucose uptake (r = 0.46, P = 0.002). Although the rate of insulin-mediated glucose uptake was decreased in the diabetic versus the nondiabetic twins (5.2 +/- 0.7 vs. 8.5 +/- 0.8 mg x kg(-1) fat-free mass x min(-1), P < 0.01), insulin-stimulated ND1 expression was not significantly different between them (2.4 +/- 0.5 vs. 2.7 +/- 0.5 relative units). Neither was there any significant intrapair correlation of ND1 expression between the monozygotic twins (r = -0.15, NS). We conclude that insulin upregulates mitochondrial-encoded gene expression in skeletal muscle. Given the positive correlation between ND1 expression and glucose uptake and the lack of intrapair correlation between monozygotic twins, mitochondrial gene expression may represent an adaptation to intracellular glucose flux rather than an inherited trait.

Aged↗

PubMed1998

Binding sites for VIP in the reorganizing mucosa of the irradiated bowel.

Rats were given radiotherapy (total dose 30 Gy) over the abdomen. Seven days later specimens of the duodenum were prepared for in vitro receptor autoradiography using the radioligand [125I]VIP. The autoradiograms were quantitatively analyzed using a computer system. Histological examination revealed that a very marked reorganization of the mucosa had occurred in response to irradiation. Using receptor autoradiography, we found [125I]VIP-specific binding sites in the reorganizing mucosa, except where denudation had occurred. Such binding sites also occurred in the smooth muscle layer of the duodenal wall. The observations suggest that VIP has profound effects in radiation-induced enteropathy.

Animals↗

PubMed1998

Increased immunoexpression of atrial natriuretic peptide in the heart conduction system of the rat after cardiac sympathectomy.

The present investigation was designed to elucidate which role the sympathetic nerves play in the immunoexpression of atrial natriuretic peptide in the cardiac conduction system of the rat. In order to destroy the cardiac sympathetic nerve terminals, both surgical and chemical sympathectomy were performed. By use of immunohistochemical and radioimmunoassay techniques, the immunoreactivity and level of atrial natriuretic peptide in the conduction system and in the cardiac myocardium were determined. In contrast to the low degree of immunoreaction for atrial natriuretic peptide seen in control rats, the sympathectomized rats exhibited pronounced immunoreactivity for atrial natriuretic peptide in the atrioventricular bundle and bundle branches, which normally have high numbers of sympathetic nerve fibres. On the other hand, in the peripheral parts of the conduction system, where there are ordinarily few sympathetic nerve fibres, the degree of immunoreaction was unchanged. The quantitative measurements also showed that the entire ventricles, including the conduction system, contained increased levels of atrial natriuretic peptide in the treated hearts. The present study shows that destruction of the sympathetic nervous system leads to an increased level of atrial natriuretic peptide in the Purkinje fibres of bundle branches, which thus seem to have a dormant capacity for synthesis of this peptide. The results provide new evidence about the change in atrial natriuretic peptide levels that occurs when sympathetic innervation is altered.

Animals↗

PubMed1998

A system for tracking changes in the mid-latency evoked potential during anesthesia.

This paper describes a method to measure changes in the mid-latency auditory evoked potential (MLAEP) during anesthesia. It is claimed that the position of the Nb-trough of the MLAEP indicates the level of consciousness. The component shows graded changes corresponding to the dose of anesthetic and it exhibits stable reproducible properties between different subjects. We propose a system that reduces the disturbances in an averaged MLAEP using fewer realizations than in the standard averaging procedure. The resulting smoothing error is reduced if the number of stimulus is decreased. Unfortunately, the variance of the waveform estimate is, thereby, increased. An improved method must be used in order to estimate the Nb-trough within a prescribed time interval of one minute. The procedure is based on inherent properties of the MLAEP and the noise. A simulation and examples of the performance on real data recorded during surgery are shown.

Algorithms↗

PubMed1997

ATPases and phosphate exchange activities in magnesium chelatase subunits of Rhodobacter sphaeroides.

Three separate proteins, BchD, BchH, and BchI, together with ATP, insert magnesium into protoporphyrin IX. An analysis of ATP utilization by the subunits revealed the following: BchH catalyzed ATP hydrolysis at the rate of 0.9 nmol per min per mg of protein. BchI and BchD, tested individually, had no ATPase activity but, when combined, hydrolyzed ATP at the rate of 117.9 nmol/min per mg of protein. Magnesium ions were required for the ATPase activities of both BchH and BchI+D, and these activities were inhibited 50% by 2 mM o-phenanthroline. BchI additionally catalyzed a phosphate exchange reaction from ATP and ADP. We conclude that ATP hydrolysis by BchI+D is required for an activation step in the magnesium chelatase reaction, whereas ATPase activity of BchH and the phosphate exchange activity of BchI participate in subsequent reactions leading to the insertion of Mg2+ into protoporphyrin IX.

Adenosine Diphosphate↗

PubMed1997

Crystal structure of ferrochelatase: the terminal enzyme in heme biosynthesis.

BACKGROUND: The metallation of closed ring tetrapyrroles resulting in the formation of hemes, chlorophylls and vitamin B12 is catalyzed by specific enzymes called chelatases. Ferrochelatase catalyzes the terminal step in heme biosynthesis by inserting ferrous ion into protoporphyrin IX by a mechanism that is poorly understood. Mutations in the human gene for ferrochelatase can result in the disease erythropoietic protoporphyria, and a further understanding of the mechanism of this enzyme is therefore of clinical interest. No three-dimensional structure of a tetrapyrrole metallation enzyme has been available until now. RESULTS: The three-dimensional structure of Bacillus subtilis ferrochelatase has been determined at 1.9 A resolution by the method of multiple isomorphous replacement. The structural model contains 308 of the 310 amino acid residues of the protein and 198 solvent molecules. The polypeptide is folded into two similar domains each with a four-stranded parallel beta sheet flanked by alpha helices. Structural elements from both domains build up a cleft, which contains several amino acid residues that are invariant in ferrochelatases from different organisms. In crystals soaked with gold and cadmium salt solutions, the metal ion was found to be coordinated to the conserved residue His 183, which is located in the cleft. This histidine residue has previously been suggested to be involved in ferrous ion binding. CONCLUSIONS: Ferrochelatase seems to have a structurally conserved core region that is common to the enzyme from bacteria, plants and mammals. We propose that porphyrin binds in the identified cleft; this cleft also includes the metal-binding site of the enzyme. It is likely that the structure of the cleft region will have different conformations upon substrate binding and release.

Amino Acid Sequence↗

PubMed1997

Isolated Bacillus subtilis HemY has coproporphyrinogen III to coproporphyrin III oxidase activity.

Oxidation of coproporphyrinogen III to coproporphyrin III is found in extracts of Escherichia coli cells containing the Bacillus subtilis HemY protein (M. Hansson and L. Hederstedt, J. Bacteriol. 176, 5962-5970). We have analysed whether this activity is due to the heterologous expression system, since it in vivo would lead to disruption of the heme biosynthetic pathway. B. subtilis hemY was fused in its 3'-end to a polynucleotide encoding six histidine residues and expressed from plasmids in both E. coli and B. subtilis. The His6-tagged HemY protein extracted from membranes using non-ionic detergent was purified by Ni2+ affinity chromatography. Isolated HemY fusion protein synthesised in E. coli and B. subtilis oxidised coproporphyrinogen III to coproporphyrin III. No direct formation of protoporphyrin IX from coproporphyrinogen III could be detected. Our results suggest that the coproporphyrinogen III to coproporphyrin III activity of HemY is either avoided in B. subtilis in vivo or that coproporphyrin III is a heme biosynthetic intermediate in this bacterium.

Amino Acid Sequence↗

PubMed1997

Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD.

Magnesium chelatase catalyses the insertion of Mg2+ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg2+ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg2+. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272,000 x g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A260:A280 ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal RNA, respectively.

Chlorophyll↗

PubMed1997

Polychlorinated dibenzo-p-dioxin and dibenzofuran levels and patterns in polyvinylchloride and chloralkali industry workers.

The production of chlorine and vinyl chloride (VCM) is associated with the formation of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). The objective of the present study was to determine whether the levels and congener patterns of PCDDs and PCDFs in the blood of workers involved in the production of such substances could be related to their occupational environment. PCDD and PCDF levels in blood plasma from VCM and chloralkali workers and in referents were determined by means of high-resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS) techniques and the results were evaluated through principal component analysis (PCA). The overall levels of PCDDs and PCDFs were low, but the relative congener distribution detected in the workers differed from that found in the referents. 1,2,3,4,6,7,8-HpCDF, 1,2,3,4,7,8- and 1,2,3,6,7,8-HxCDFs are congeners related to work in VCM and chloralkali industries. Exposure to PCDDs and PCDFs in these industrial environments changes the congener-distribution pattern in the blood of workers as compared with referents. A combination of isomer-specific analysis of PCDDs and PCDFs in blood plasma and PCA is suitable for the differentiation between nonoccupational and occupational exposure and provides a means of assessing workers' exposure situation in different occupational settings.

Animals↗

PubMed1997

Occurrence of binding sites for [125I] ANP in the myocardium but not in Purkinje fibers of the bovine heart.

Atrial natriuretic peptide has frequently been detected in the cardiac conduction system and has been shown to regulate some intracellular effects in Purkinje fibers. To determine if atrial natriuretic peptide works as an autocrine and/or paracrine hormone on cardiac Purkinje fibers, we examined the different parts of the conduction system in the bovine heart by use of in vitro receptor autoradiography. In no parts of the bovine conduction system were specific binding sites for [125I] atrial natriuretic peptide observed, whereas the ventricular myocardium exhibited a large number of [125I] atrial natriuretic peptide binding sites. This is the first morphologic study showing the presence of [125I] atrial natriuretic peptide binding sites in the ventricular myocardium and their absence in the conduction system. The present observations together with results obtained in studies using other methods strongly suggest that natriuretic peptide receptors are localized on ventricular myocytes.

Animals↗

PubMed1997

Natriuretic peptide immunoreactivity in nerve structures and Purkinje fibres of human, pig and sheep hearts.

Atrial natriuretic peptide is a well-described peptide in cardiac Purkinje fibres and has been shown to interfere with the autonomic regulation in the heart of various species, including man. Recently, we detected immunoreactivity for the peptide in intracardial ganglionic cells and nerve fibre varicosities of bovine hearts, by the use of a modified immunostaining technique that induced an improved detection of natriuretic peptides. These findings raised the question as to whether natriuretic peptides are detectable in these tissues in man and other species. The conduction system from human, pig and sheep hearts was dissected processed with antisera against atrial natriuretic peptide and the closely related brain natriuretic peptide. Immunostaining for the brain natriuretic peptide was detected in some Purkinje fibres in all of these species. Interestingly, in pig, sheep and human hearts, some ganglionic cells and nerve fibres showed atrial natriuretic peptide immunoreactivity, particularly in the soma of human ganglionic cells. This is the first study showing immunoreactivity for the atrial natriuretic peptide in nerve structures and for the brain natriuretic peptide in Purkinje fibres of the human heart. The results give a morphological correlate for the documented effects of atrial natriuretic peptide on the heart autonomic nervous system and for the presumable effects of brain natriuretic peptide in the conduction system of man.

Adult↗

PubMed1997

Efficient harvest of in vivo IL-2-activated CD3+ lymphocytes for adoptive immunotherapy by selective leukapheresis (lymphocytapheresis).

Autologous activated lymphocytes are an alternative to tumor-infiltrating lymphocytes for adoptive immunotherapy. We developed a method of selective lymphocytapheresis that harvests large numbers of interleukin-2 (IL-2)-activated autologous T lymphocytes. Five patients with metastatic malignant melanoma received 2.4 x 10(6) IU/m2 IL-2 sc. once a day 5 days a week for 3 weeks before lymphocytapheresis. Four patients went through lymphocytapheresis without IL-2 pretreatment. After IL-2 pretreatment, activated memory T cells increased significantly. Increasing CD3+ cells paralleled the significant enhancement of the cytotoxic activity against an HLA-A2-matched allogeneic melanoma cell line during the 3 weeks of IL-2 pretreatment. Lymphocytapheresis was performed 72 h after the last IL-2 injection to obtain the maximum recovery of activated lymphocytes at the peak of the rebound phenomenon. IL-2 pretreatment resulted in many more lymphocytes in the harvest than without pretreatment. The percentage, number, and lytic units of CD3+ cells harvested by the differential apheresis were significantly higher than were present in peripheral blood before lymphocytapheresis. These results show that pretreatment of melanoma patients with low-dose IL-2 before lymphocytapheresis allows the selective harvest of large numbers of activated T lymphocytes for adoptive immunotherapy.

Adult↗

PubMed1997

Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus.

The heterologous surface expression of the cholera toxin B subunit (CTB) from Vibro cholerae in two staphylococcal species, Staphylococcus xylosus and Staphylococcus carnosus, has been investigated. The gene encoding native CTB (103 amino acids) was introduced into gene constructs encoding chimeric receptors designed to be translocated and anchored on the outer cell surface of the staphylococci. Since functionality of CTB is correlated with its ability to form pentamers and the capacity of the pentameric CTB to bind the GM1 ganglioside, both the surface accessibility and the functionality of the surface-displayed CTB receptors were evaluated. It could be concluded that the chimeric receptors were targeted to the cell wall of the staphylococci, since they could be released by lysostaphin treatment and, after subsequent affinity purification, identified as full-length products by immunoblotting. Surface accessibility of the chimeric receptors was demonstrated by a colorimetric assay and by immunofluorescence staining with a CTB-reactive rabbit antiserum. Pentamerization was investigated by using a monoclonal antibody described to be specific for pentameric CTB, and the functionality of the receptors was tested in a binding assay with digoxigenin-labelled GM1. It was concluded that functional CTB was present on both types of staphylococci, and for S. carnosus, the reactivity to the pentamer-specific monoclonal antibody and in the GM1 binding assay was indeed significant. The implications of the results for the design of live bacterial vaccine delivery systems intended for administration by the mucosal route are discussed.

Antibodies, Bacterial↗

PubMed1997

Structure prediction and fold recognition for the ferrochelatase family of proteins.

An alpha/beta barrel is predicted for the three-dimensional (3D) structure of Bacillus subtilis ferrochelatase. To arrive at this structure, the THREADER program was used to find possible homologous 3D structures and to predict the secondary structure for the ferrochelatase sequence. The secondary structure was fit by hand to the selected homologous 3D structure then the MODELLER program was used to predict the fold of ferrochelatase. Molecular biological information about the conserved residues of ferrochelatase was used as the criteria to help select the homologous 3D structure used to predict the fold of ferrochelatase. Based on the predicted structure possible, ligands binding to the iron and protoporphyrin IX are discussed. The structure has been deposited in the Brookhaven database as ID 1FJI.

Algorithms↗

PubMed1996

Induction of apoptosis in NK cells by monocyte-derived reactive oxygen metabolites.

Human NK cells (with CD3-/56+ phenotype) acquired features characteristic of apoptosis after incubation with autologous monocytes, as revealed by apoptotic nuclear morphology, degradation of DNA into oligonucleosomal fragments, and reduced nuclear interchalation of propidium iodide. In contrast, T cells (CD3+/56-) remained non-apoptotic. The monocyte-induced apoptosis in NK cells was prevented by catalase, a scavenger of hydrogen peroxide; whereas superoxide dismutase (a scavenger of superoxide anion), hydroxyl radical scavengers such as mannitol and deferoxamine, or the hypochlorus acid scavenger taurine did not prevent apoptosis. Sodium azide, a myeloperoxidase inhibitor, substantially reduced the monocyte-induced apoptosis in NK cells. Exogenous hydrogen peroxide, at concentrations exceeding 1 microns, induced apoptosis in both NK and T cells. Apoptosis induced by hydrogen peroxide occurred independently of synthesis of protein or mRNA and was blocked by the endonuclease inhibitor aurin tricarboxylic acid. Furthermore, oxidatively induced apoptosis in NK cells was inhibited by herbimycin A, indicating that apoptosis was dependent on protein kinases. Two to five times more hydrogen peroxide was required to induce apoptosis in T cells compared with NK cells. Similarly, NK cells were considerably more susceptible to apoptosis induced by the topoisomerase II inhibitor etoposide or by gamma-irradiation than were T cells. We conclude that monocyte-derived reactive oxygen metabolites kill NK cells by apoptosis and that NK cells are unusually sensitive to oxidatively as well as non-oxidatively induced apoptosis.

Apoptosis↗