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Biomedical subjects

J Lund

Publications and source records attributed to J Lund.

At least 73 records · Page 4Linked to original sources
PubMed1996

Structural basis for calcium binding by uteroglobins.

Uteroglobins, i.e. proteins with similar three-dimensional structure and ligand binding specificity to uteroglobin from rabbit uterus, have been found in rat, mouse and human lung. We have recently demonstrated the binding of calcium by human uteroglobin, and we have therefore tried to find potential binding sites for metals in the three-dimensional structure of uteroglobin by the use of two different computational procedures. A putative binding site for calcium in uteroglobin was identified by means of a hydrophobic contrast function. The spatial disposition of atoms that could ligand calcium in the putative calcium-binding site appears similar to that of the primary calcium-binding site of secretory phospholipase A2 enzymes, consisting of the carboxyl group of an aspartic acid residue and a loop providing three backbone carbonyl oxygens. From inspection of their primary sequences and three-dimensional structures, it became clear that this putative calcium-binding motif is conserved among uteroglobins from different species. The potential significance of the predicted site was investigated by site-directed point mutagenesis of human uteroglobin in which Asp46 was replaced by Asn or Lys. In both mutants, the ruthenium red and 45Ca2+ binding was significantly reduced. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis under non-reducing conditions indicated that the mutant proteins had the expected molecular masses and that their ability to dimerize was not disturbed by these mutations. Valence calculations also identified the putative calcium-binding site, but only after optimization of its conformation by the use of molecular dynamics with a restrained calcium ion. Our results support the notion that Asp46 of uteroglobins acts as a "cap" residue in a calcium-binding site structurally similar to the primary calcium binding sites of phospholipases A2.

Amino Acid Sequence↗
PubMed1996

The effect of topical lignocaine on intubating conditions after propofol-alfentanil induction.

BACKGROUND: Recent studies have found satisfactory conditions for intubation of the trachea without using muscle relaxants using an intravenous technique combining propofol and alfentanil. In this study we evaluate intubating conditions with this method and either lignocaine applied topically in the larynx and trachea or placebo. METHODS: Sixty adult patients of ASA class I were premedicated with diazepam 15-20 mg and randomly allocated to one of two groups. For induction of anaesthesia both groups were given propofol 2.5 mg/kg and alfentanil 30 micrograms/kg. One group received 4 ml of lignocaine 40 mg/ml (< or = 3 mg/kg) topically into the larynx and trachea (group L), the other group an equal amount of isotonic saline (group S) in a double-blind design. Intubation conditions were assessed as excellent, good, moderately good, poor or impossible, scored on the basis of jaw relaxation, ease of insertion of the tube and coughing on intubation. RESULTS: The total score for group L was significantly better than the score for group S (P < 0.0001) with significant differences with respect to ease of intubation and coughing after intubation. CONCLUSIONS: Induction of anaesthesia with propofol 2.5 mg/kg and alfentanil 30 micrograms/kg combined with 4 ml of lignocainespray 40 mg/ml into the larynx and trachea offered consistent and satisfactory intubation conditions. We thus recommend this method for tracheal intubation, where the use of muscle relaxants is not indicated.

Administration, Topical↗
PubMed1996

Clara cell secretory protein: a determinant of PCB bioaccumulation in mammals.

Clara cell secretory protein (CCSP) is a product of nonciliated cells of the conducting airway epithelium. The normal physiological function of CCSP is unknown. However, the ability of CCSP to bind small lipophilic molecules, such as steroid hormones and certain pollutants, has led to speculation that this protein may mediate the biological accumulation of potentially harmful polychlorinated biphenyl (PCB) metabolites within the lung. To investigate the contribution of CCSP in the in vivo accumulation of methylsulfonyl-PCB, a line of mice was established that were homozygous for a null allele of the CCSP gene. CCSP-deficient mice were healthy and fertile, with no gross physiological or pathological abnormalities Parenteral challenge with the PCB metabolite 4-methylsulfonyl-2,2',4',5,5'-pentachlorobiphenyl (MeSO2-PCB) demonstrated that CCSP-deficient mice no longer accumulate this class of pollutants within lung and kidney tissues. These data demonstrate that CCSP is the determinant for MeSO2-PCB accumulation within mice and support the notion that bioconcentration of MeSO2-PCB pollutants occurs at sites of CCSP localization, such as the respiratory and reproductive tracts of humans.

Animals↗
PubMed1995

Calcium-dependent binding of uteroglobin (PCB-BP/CCSP) to negatively charged phospholipid liposomes.

To investigate interactions between the polychlorinated biphenyl-binding protein uteroglobin and phospholipids, we used a liposome-pelletting assay. PCB-BP/uteroglobin bound to liposomes made from negatively charged phospholipids (PtdSer and PtdIns) in the presence of 5 mM calcium. No binding to liposomes made from phospholipids without net charge (PtdChol and PtdEtn) was observed, nor could we detect binding in the absence of calcium or when magnesium was substituted for calcium. This suggests that PCB-BP/uteroglobin can bind to phospholipids in vivo and may have a role in the phospholipid homeostasis of the airway and/or secretory pathway of the Clara cell.

Calcium↗
PubMed1995

Novel involvement of a mitochondrial steroid hydroxylase (P450c11) in xenobiotic metabolism.

Adrenocortical mitochondrial cytochrome P450 isozymes of the Cyp11 family normally synthesize steroids with a very strict substrate specificity. However, for the first time, P450c11 was additionally shown to metabolize and bioactivate the adrenotoxic environmental pollutant 3-methylsulfonyl-2,2-bis(4-chlorophenyl)-1,1-dichloroethene (MeSO2-DDE). This conclusion is based on a striking correlation between inductions of MeSO2-DDE and deoxycorticosterone metabolism by forskolin in the adrenocortical cell lines Y1 and Kin-8, inhibition of P450c11-dependent activities in Y1 cells by MeSO2-DDE, and metabolism of MeSO2-DDE by non-steroidogenic COS cells after transfection with a cDNA encoding P450c11. The interaction between xenobiotics and glucocorticoid synthesis should focus more attention to xenobiotic-induced hormonal disturbances.

Adrenal Cortex↗
PubMed1995

Adrenocorticotrophic-hormone-dependent regulation of a mu-class glutathione transferase in mouse adrenocortical cells.

Three different forms of glutathione transferase (GST) have been resolved in the two mouse adrenal tumour cell lines Y1 and Kin 8. Two of these belong to the mu and pi classes respectively. The third form is so far unidentified. In the Y1 cells, the levels of the mu form (mGTmu1) and the unidentified form, are both down-regulated in the presence of adrenocorticotrophic hormone (ACTH) while the pi form is unaffected. The Kin 8 cell line is derived from Y1 cells and harbours a defect in the cyclic AMP (cAMP)-dependent protein kinase, making it refractory to cAMP-dependent regulation of several enzymes. The GST levels in this cell line were unaffected by ACTH. Also, the steady-state levels of mGTmu1 mRNA were much lower in Y1 cells treated with forskolin (which activates adenylate cyclase) compared with control cells, but there was no difference in mGTmu1 mRNA levels between control and forskolin-treated Kin 8 cells. This indicates that the ACTH-dependent regulation of the mu class GST is pre-translational and that a functional cAMP-dependent protein kinase is required for the regulation. We have further shown that the difference in mRNA steady-state levels between control and forskolin-treated Y1 cells is abolished when transcription is inhibited by actinomycin D. In light of the stability of mGTmu1 mRNA, it would appear most likely that actinomycin D inhibits the transcription of short-lived factors which regulate the turn-over of mGTmu1 transcripts in response to changes in intracellular cAMP levels.

Adrenal Cortex↗
PubMed1995

Recognition sites on human IgG for Fc gamma receptors: the role of glycosylation.

Human IgG subclass proteins exhibit more than 95% primary amino acid sequence homology in their Fc regions, but each has a unique profile for recognition by the 3 human Fc gamma receptors. The Fc gamma Rs are themselves highly homologous members of the immunoglobulin supergene family. Consistent with these data we have proposed that Fc gamma RI, Fc gamma RII and Fc gamma RIII recognise overlapping non-identical interaction sites in the lower hinge region of the CH2 domain of the IgG molecule. Evidence in support was provided by protein engineering effecting single amino acid replacements in the proposed site. Alternatively, we have demonstrated that the primary amino acid sequence alone is not sufficient for IgG molecules to fold with the generation of Fc gamma R interaction sites and that glycosylation of Asn 297 of the CH2 domain is essential. We have further defined a 'core' oligosaccharide structure that provides for the generation of Fc gamma R interaction sites which suggests that the addition of outer-arm sugar residues does not affect this primary activity; although in vivo it could influence other essential biological activities. These findings have opened up a new approach to engineering antibody function--by protein engineering of amino acid residues that form contacts with the oligosaccharide moiety. In the present report we demonstrate that replacement of contact residues for galactose on the alpha(1-6) arm does not affect Fc gamma RI and Fc gamma RII recognition while replacement of Asp 265, a contact for a 'core' N-acetylglucosamine residue, results in a loss of Fc gamma RI and Fc gamma RII recognition.

Binding Sites, Antibody↗
PubMed1995

Solution structure of a mammalian PCB-binding protein in complex with a PCB.

Metabolites of polychlorinated biphenyls (PCBs) bind with high affinity to uteroglobin, a small homodimeric protein that also binds progesterone. We present the solution structure of the reduced form of rat uteroglobin in complex with a PCB methylsulphone, (MeSO2)2-TCB. The structure reveals the molecular basis for the accumulation of (MeSO2)2-TCB by uteroglobin. The structure also shows how ligand binding and release might be controlled by reduction/oxidation of two intermolecular disulphide bonds. Breakage of these bonds induces a local unfolding of the N- and C-termini and a separation of helices creating a channel into the binding site. These effects make the ligand binding cavity readily accessible to entry of the ligand.

Amino Acid Sequence↗
PubMed1995

Oligosaccharide-protein interactions in IgG can modulate recognition by Fc gamma receptors.

X-ray crystal structures of IgG-Fc provide evidence of extensive noncovalent interactions between the protein and carbohydrate moieties, and glycosylation, at Asn-297 within the Fc, has been shown to be important for effector functions mediated through Fc gamma receptors expressed on leukocytes. We have applied protein engineering in an attempt to define protein/carbohydrate interactions essential to wild-type biological activity. We demonstrate that replacement of Lys-246, Asp-249, and Glu-258, which make contacts with GlcNac and Gal on the outer alpha[1-->6] arm, do not affect recognition of human chimeric IgG3 by human Fc gamma RI and Fc gamma RII. However, replacement of Asp-265, which make contacts with the primary GlcNac sugar residue and is covalently attached to Asn-297, resulted in loss of recognition of both Fc gamma RI and Fc gamma RII. Similarly, replacement of Asp-265 in mouse IgG2b resulted in loss of recognition by mouse Fc gamma RII. These results suggest that noncovalent contacts of Asp-265 with the primary GlcNac residue are important for maintenance of recognition of IgG by Fc gamma receptors whereas contacts with GlcNac and Gal on the alpha [1-->6] arm do not have a measurable effect. This conclusion was supported by experiments in which galactose-deficient and fully galactosylated forms of a human IgG4-Fc fragment were shown to be equivalent in their ability to inhibit superoxide generation by IgG4 stimulated U937 cells.

Animals↗
PubMed1995

Mutually exclusive interactions of two nuclear orphan receptors determine activity of a cyclic adenosine 3',5'-monophosphate-responsive sequence in the bovine CYP17 gene.

ACTH-dependent transcriptional activation of the bovine CYP17 gene (the gene encoding cytochrome P450 steroid 17 alpha-hydroxylase) involves two cAMP-responsive sequences (CRS1 and CRS2) located in the promoter region. Here we demonstrate that two nuclear orphan receptors, chicken ovalbumin upstream promoter transcription factor (COUP-TF) and steroidogenic factor-1 (SF-1), bind to the part of the CRS2 element that contains the repeated sequences AAGTCA and AGGTCA spaced by six nucleotides (repCRS2). Overexpression of COUP-TF and SF-1 in both steroidogenic and nonsteroidogenic cells demonstrated that SF-1 is an activator of repCRS2-dependent transcription of reporter genes. Furthermore, the SF-1-dependent transcription could be further stimulated by activation of the cAMP-dependent protein kinase. In contrast, COUP-TF alone had no effect on repCRS2-dependent reporter gene activity. Mutations that interfere with the binding of SF-1 to repCRS2 in vitro abolished the cAMP-induced activities mediated by the element in transfected Y1 cells. The mutational analysis of repCRS2 further indicated that the binding sites for the two receptors overlap, and electrophoretic mobility shift assays demonstrated that the receptors bound in a mutually exclusive manner. Overexpression of both SF-1 and COUP-TFI simultaneously demonstrated that COUP-TFI inhibited SF-1-dependent activation of reporter genes. Transient transfection experiments with a construct containing a -100/+19 base pair fragment from the bovine CYP17 gene demonstrated that SF-1 and COUP-TF had similar effects on the intact promoter as on the repCRS2/reporter gene constructs. Our data suggest that the two orphan receptors bind in a mutually exclusive manner to repCRS2 and that SF-1 is involved in the activation and COUP-TF in the repression of repCRS2-dependent transcription.

Animals↗
PubMed1995

Activation of effector functions by immune complexes of mouse IgG2a with isotype-specific autoantibodies.

Analysis of five monoclonal autoantibodies, rheumatoid factors produced by hybridomas generated from spleen cells of BALB/c mice repeatedly infected with A/PR/8/34 human influenza A virus, revealed that they recognized distinct but spatially related epitopes. The differing isoallotypic specificity of the IgM and IgA monoclonal antibodies correlated with the presence of Ile258 and Ala305, respectively. Although these data suggest that the epitopes recognized are within the CH2 domain, all antibodies failed to inhibit IgG antigen reactivity with Staphylococcus aureus protein A (SpA), C1q, mouse C3, human Fc gamma RI or mouse Fc gamma RII, activities known to be predominantly determined by CH2 domain structures. Reactivity of the IgA antibody, Z34, with IgG2b allowed further specificity studies using a panel of 26 mutant IgG2b proteins, each having single amino acid replacements over the surface of the CH2 domain. The only substitution that affected Z34 reactivity was Asn/Ala297, which destroyed the glycosylation sequon, resulting in secretion of an aglycosylated IgG molecule. The epitope recognized by Z34 therefore seems to be located outside of the Fc gamma R and C1q binding sites, but to be dependent on the presence of carbohydrate for expression. In contrast to the binding studies, complement activation by aggregated IgG2a, through classical or alternative pathways, was inhibited by the presence of autoantibodies. The functional significance of isotype-specific autoantibody in immune regulation is discussed.

Animals↗
PubMed1994

[Mortality among long-term psychiatric patients in Denmark 1972-1988].

By use of the computerized, nationwide psychiatric case register in Denmark, three cohorts of psychiatric patients hospitalized for at least one year during either 1972, 1977 or 1982, were identified and followed until April 1, 1988, regarding time of death. A decline in overall excess mortality compared with the general population was seen from the 1972-cohort to the 1982-cohort irrespective of sex, age group and diagnosis. This may be due to real improved survival, but could also reflect different mechanisms of selection. To analyse cause of death, 298 long-stay patients at Psychiatric hospital in Arhus were followed for 5.5 years. Lung disease caused the highest excess mortality. The authors emphasize that risk of death in long-stay patients below 50 years of age is still fourfold compared to the general population and point to the necessity of improving medical care.

Cohort Studies↗
PubMed1994

Heterologous expression of human uteroglobin/polychlorinated biphenyl-binding protein. Determination of ligand binding parameters and mechanism of phospholipase A2 inhibition in vitro.

High level expression of a human polychlorinated biphenyl-binding protein (hPCB-BP; also termed uteroglobin or CC10) was achieved in Escherichia coli. The recombinant protein (rhPCB-BP) constituted approximately 1% of total bacterial lysate proteins as judged from in vitro ligand binding assays using 4,4'-bis([3H]methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl. rhPCB-BP was purified to homogeneity in its native dimeric form. Saturation analysis experiments indicated a Kd of approximately 69 nM for the binding of 4,4'-bis([3H]methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl to rhPCB-BP. The average number of binding sites (Bmax) calculated from such experiments on purified rhPCB-BP was 49 nmol/mg of protein and is close to the theoretical value of 1 mol of ligand associating with 1 mol of dimeric protein. Purified rhPCB-BP was also found to cause a dose-dependent inhibition of the enzyme porcine pancreatic phospholipase A2 (PLA2) in vitro. Increasing the concentrations of calcium abolished the inhibition of PLA2 by rhPCB-BP, suggesting that the protein functions in vitro by sequestering Ca2+, an essential PLA2 cofactor. This notion was further supported by direct evidence that 45Ca2+ binds to rhPCB-BP. 1 mol of dimeric protein was also found to bind 2 mol of ruthenium red, an organic dye that detects Ca(2+)-binding proteins, with a Kd of 3 microM. This binding was inhibited by Ca2+, with an IC50 of 7 mM. Finally, it was demonstrated that the addition of a high affinity ligand for the protein had no effect on its ability to inhibit PLA2 under conditions of limiting concentrations of calcium, and the addition of Ca2+ did not affect the binding characteristics of the PCB ligand, suggesting that these two properties of the protein are independent. Our results strongly support the notion that ligand binding is a conserved feature of the homologous uteroglobin/PCB-BP/cc10 proteins in different species, whereas our results question the suggested role of these proteins as specific inhibitors of PLA2.

Calcium↗
PubMed1994

Brain damage following low flow cardiopulmonary bypass in pigs.

Reduction of pump flow during cardiopulmonary bypass (CPB) reduces the formation of microemboli and trauma to the blood components, reduces both rewarming of the heart and the noncoronary collateral flow, and improves surgical exposure. Recent studies indicate that a reduction in pump flow, even at normothermia, does not increase the incidence of postoperative cerebral dysfunction. We examined the cerebral consequences of 2 h of normothermic CPB in pigs carried out at pump flows of either 70 ml/kg per min or 50 ml/kg per min, and compared the results with those of a nonperfused control group. We measured the regional cerebral glucose metabolism and the regional capillary diffusion capacity simultaneously in ten different brain regions. Brain morphology, the blood-brain barrier permeability to serum proteins and the regional cerebral water content were also determined in the same animals. Glucose metabolism decreased significantly in both CPB groups (P < 0.001), and significant differences were found between the capillary diffusion capacities of the three groups (P < 0.05), with decreases in eight out of ten brain regions examined in the 50 ml/kg per min group. The results indicate that a reduction of pump flows from 70 ml/kg per min to 50 ml/kg per min is deleterious to the brain, and that a pump flow of 70 ml/kg per min itself has an injurious effect, when normothermic CPB is carried out for 2 h without the use of vasoactive drugs to maintain the blood pressure. Mean arterial blood pressure (MAP) rather than pump flow seemed to determine the adequacy of the cerebral perfusion.

Animals↗