BBA - Molecular Basis of Disease (v.1500, #3)

Nitrovasodilators, high extracellular Mg2+, and some other relaxing agents can cause smooth muscle relaxation without reductions in myosin regulatory light chain (MRLC) phosphorylation. Relaxations without MRLC dephosphorylation suggest that other regulatory systems, beyond MRLC phosphorylation, are present in smooth muscle. We tested whether changes in caldesmon phosphorylation, heat shock protein 20 (HSP20) phosphorylation, or intracellular pH (pHi) could be responsible for relaxation without MRLC dephosphorylation. In unstimulated tissues, caldesmon was phosphorylated 1.02±0.10 mol Pi/mol caldesmon (mean±1 S.E.M.), HSP20 was phosphorylated 0.005±0.003 mol Pi/mol HSP20, and estimated pHi was 7.21±0.07. Histamine stimulation induced a contraction, an intracellular acidosis, but did not significantly change caldesmon or HSP20 phosphorylation. Addition of nitroglycerin induced a relaxation, significantly increased HSP20 phosphorylation to 0.18±0.02 mol Pi/mol HSP20, did not significantly change caldesmon phosphorylation, and pHi returned to near unstimulated values. Increase in extracellular Mg2+ to 10 mM induced a relaxation, but did not significantly change HSP20 or caldesmon phosphorylation. These data suggest that changes in caldesmon phosphorylation, HSP20 phosphorylation, or pHi cannot be the sole explanation for relaxation without MRLC dephosphorylation. However, it is possible that HSP20 phosphorylation may be involved in nitroglycerin-induced relaxation without MRLC dephosphorylation.
Keywords: Caldesmon; Calcium ion concentration; Intracellular pH; Vascular smooth muscle;

Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines by T.H. Scott-Taylor; R. Pettengell; I. Clarke; G. Stuhler; M.C. La Barthe; P. Walden; A.G. Dalgleish (265-279).
Hybrid cells created by fusion of antigen presenting and tumour cells have been shown to induce potent protective and curative anti-tumour immunity in rodent cancer models. The application of hybrid cell vaccines for human tumour therapy and the timely intervention in disease control are limited by the requirement to derive sufficient autologous cells to preserve homologous tumour antigen presentation. In this study, the efficiency of various methods of electrofusion in generating hybrid human cells have been investigated with a variety of human haemopoietic, breast and prostate cell lines. Cell fusion using an electrical pulse is enhanced by a variety of stimuli to align cells electrically or bring cells into contact. Centrifugation of cells after an exponential pulse from a Gene Pulser electroporation apparatus provided the highest yield of mixed cell hybrids by FACS analysis. An extensive fusogenic condition generated in human cells after an electrical pulse contradicts the presumption that prior cell contact is necessary for cell fusion. Alignment of cells in a concurrent direct current charge and osmotic expansion of cells in polyethylene glycol also generated high levels of cell fusion. Waxing of one electrode of the electroporation cuvette served to polarise the fusion chamber and increase cell fusion 5-fold. Optimisation of a direct current charge in combination with a fusogenic pulse in which fusion of a range of human cells approached or exceeded 30% of the total pulsed cells. The yield of hybrid prostate and breast cancer cells with dendritic cells was similar to the homologous cell fusion efficiencies indicating that dendritic cells were highly amenable to fusion with human tumour cells under similar electrical parameters. Elimination of unfused cells by density gradient and culture is possible to further increase the quantity of hybrid cells. The generation and purification of quantities of hybrid cells sufficient for human vaccination raises the possibility of rapid, autologous tumour antigen presenting vaccines for trial with common human tumours.
Keywords: Electrofusion; Hybrid cell; Cancer vaccine; Human dendritic cell fusion;

A role for the A3 adenosine receptor in determining tissue levels of cAMP and blood pressure: studies in knock-out mice by Zhihui Zhao; Konstantinos Makaritsis; Cynthia E Francis; Haralambos Gavras; Katya Ravid (280-290).
Adenosine administration has been reported to lower blood pressure by activating specific membrane receptors. The rat and human heart and aorta have been previously found to express both A2-type adenosine receptors, which activate adenylyl cyclase, and A3 adenosine receptors (A3AR), which inhibit adenylyl cyclase. In the current study, we used A3 adenosine receptor (A3AR) knock-out mice to examine the hypothesis that the relative levels of the A2-type adenosine receptors and A3AR determine the steady-state levels of cAMP in the cells and may affect blood pressure. We found that the A3AR knock-out mice express normal levels of the A1- and A2-type adenosine receptors. In situ hybridization demonstrated that the level of A3AR is high in the vascular smooth muscle layer of aortas derived from wild-type mice, but is not detectable in the knock-out mice. The steady-state level of cAMP is elevated in the aorta and heart of knock-out mice, as compared to wild-type mice, but is not altered in platelets, where A3AR is not expressed naturally. A3AR knock-out mice possess a blood pressure comparable to this in wild-type mice. However, when challenged with adenosine, the knock-out mice display a further increase in cAMP levels in the heart and vascular smooth muscle and a significant decrease in blood pressure, as compared to wild-type mice. In contrast, the effect of adenosine on ADP-induced platelet aggregation is similar in both types of mice. These studies indicate that the A3AR affects the steady-state level of cAMP in the tissues where it is naturally expressed, and that it influences the blood pressure in response to adenosine.

A subclass of patients with classic infantile autism have uric acid excretion which is >2 S.D.s above the normal mean. These hyperuricosuric autistic individuals may comprise approx. 20% of the autistic population. In order to determine the metabolic basis for urate overexcretion in these patients, de novo purine synthesis was measured in the cultured skin fibroblasts of these patients by quantification of the radiolabeled purine compounds produced by incubation with radiolabeled sodium formate. For comparison, de novo purine synthesis in normal controls, in normouricosuric autistic patients, and cells from patients with other disorders in which excessive uric acid excretion is seen was also measured. These experiments showed that de novo purine synthesis is increased approx. 4-fold in the hyperuricosuric autistic patients. This increase was less than that found in other hyperuricosuric disorders. No unusual radiolabeled compounds (such as adenylosuccinate) were detected in these experiments, and no gross deficiencies of radiolabeled nucleotides were seen. However, the ratio of adenine to guanine nucleotides produced by de novo synthesis was found to be lower in the cells of the hyperuricosuric autistic patients than in the normal controls or the cells from patients with other disorders. These results indicate that the hyperuricosuric subclass of autistic patients have increased de novo purine synthesis, and that the increase is approximately that expected for the degree of urate overexcretion when compared to other hyperuricosuric disorders. No particular enzyme defect was suggested by either gross deficiency of a radiolabeled compound or the appearance of an unusual radiolabeled compound, and no potentially neurotoxic metabolites were seen. Although an enzyme defect responsible for the accelerated purine synthesis was not identified, the abnormal ratio of adenine to guanine nucleotides suggests a defect in purine nucleotide interconversion.
Keywords: Autism; Hyperuricosuria; Purine metabolism; Uric acid;

Alterations of Na+ homeostasis in hepatocyte reoxygenation injury by Rita Carini; Maria Grazia De Cesaris; Roberta Splendore; Marco Bagnati; Giorgio Bellomo; Emanuele Albano (297-305).
Reperfusion injury represents an important cause of primary graft non-function during liver transplantation. However, the mechanism responsible for cellular damage during reoxygenation has not yet been completely understood. We have investigated whether changes in intracellular Na+ distribution might contribute to cause hepatocyte damage during reoxygenation buffer after 24 h of cold storage. Hepatocyte reoxygenation resulted in a rapid increase in cellular Na+ content that was associated with cytotoxicity. Na+ accumulation and hepatocyte death were prevented by the omission of Na+ from the incubation medium, but not by the addition of antioxidants. Blocking Na+/H+ exchanger and Na+/HCO3 co-transporter by, respectively, 5-(N,N-dimethyl)-amiloride or omitting HCO3 from the reoxygenation medium significantly decreased Na+ overload and cytotoxicity. Stimulation of ATP re-synthesis by the addition of fructose also lowered Na+ accumulation and cell death during reoxygenation. A significant protection against Na+-mediated reoxygenation injury was evident in hepatocytes maintained in an acidic buffer (pH 6.5) or in the presence of glycine. The cytoprotective action of glycine or of the acidic buffer was reverted by promoting Na+ influx with the Na+/H+ ionophore monensin. Altogether, these results suggest that Na+ accumulation during the early phases of reoxygenation might contribute to liver graft reperfusion injury.
Keywords: Sodium; Reperfusion injury; Liver transplantation; Glycine; Acidic pH; Cold storage;

Q-band EPR investigations of neuromelanin in control and Parkinson’s disease patients by Leonardo Lopiano; Mario Chiesa; Giuseppe Digilio; Sabrina Giraudo; Bruno Bergamasco; Elena Torre; Mauro Fasano (306-312).
New insights into the understanding of the changes induced in the iron domain of neuromelanin (NM) upon development of Parkinson’s disease (PD) have been gained by electron paramagnetic spectroscopy (EPR). The results of this study are compared with a previously reported variable temperature analysis of X-band EPR spectra of a NM specimen obtained from control brain tissues. The availability of high sensitivity instruments operating in the Q-band (34.4 GHz) allows us to deal with the low amounts of NM available from PD brains. The organization of iron in NM is in the form of polynuclear superparamagnetic/antiferromagnetic aggregates, but the lack of one or more signals in the EPR spectra of NM from PD suggests that the development of the pathology causes NM to decrease its ability to bind iron. Furthermore, the detection of the Mn(II) signal in the Q-band spectra is exploited as an additional internal probe to assess minor structural differences in iron domains of PD and control NM specimens.
Keywords: Parkinson’s disease; Substantia nigra; Neuromelanin; Electron paramagnetic spectroscopy; Iron storage;

The separation of trophoblast cells from the maternal circulation could provide a valuable diagnostic tool for prenatal diagnosis of genetic abnormalities. This has been attempted using antibody methods, but due to non-specificity of the antibodies, maternal cell contamination remains a problem. We have investigated the potential of dielectrophoretic separation methods as a means of isolating trophoblast cells from mixed peripheral blood mononuclear cells. To determine the potential of this method the dielectric properties of trophoblast cells and mixed peripheral blood mononuclear cells were measured using dielectrophoretic crossover and single cell electrorotation methods. Both dielectrophoretic crossover data and electrorotation data gave an average specific membrane capacitance of the peripheral blood mononuclear cells of 11.5 mF m−2. Trophoblast cells prepared using three different methods had a higher average specific membrane capacitance in the range 13–18 mF m−2. The differences in capacitance between the cell types could be exploited as the basis of an AC electrokinetic-based system for the separation of trophoblast cells from peripheral blood mononuclear cells.
Keywords: Trophoblast; Dielectrophoresis; Electrorotation; Prenatal diagnosis; Cell separation;

The mouse N-acetylgalactosamine-6-sulfate sulfatase (Galns) gene: cDNA isolation, genomic characterization, chromosomal assignment and analysis of the 5′-flanking region by Adriana Maria Montaño; Atsushi Yamagishi; Shunji Tomatsu; Seiji Fukuda; Neal G. Copeland; Kenji E. Orii; Koji Isogai; Naoto Yamada; Zen-Ichiro Kato; Nancy A. Jenkins; Debra J. Gilbert; Kazuko Sukegawa; Tadao Orii; Naomi Kondo (323-334).
Deficiency of lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) leads to mucopolysaccharidosis IV A (MPS IV A), for which there is no definitive treatment so far. Although a number of mutations of the GALNS gene of MPS IV A patients have been described, pathogenesis of the disorder still remains elusive. In order to facilitate in vivo studies using model animals for MPS IV A, we isolated and performed molecular characterization of the mouse homolog of human GALNS. The 2.3-kb cDNA contains a 1560-bp open reading frame encoding 520 amino acid residues. The coding region has 84% similarity to the human GALNS cDNA at amino acid level. The mouse Galns gene was mapped by interspecific backcross analysis to the distal region of chromosome 8 where it co-segregates with Aprt. Northern blot analysis showed a wide expression of a single-copy gene, being higher especially in liver and kidney. The Galns gene was isolated from S129vJ genomic library and its genomic organization was characterized. The mouse Galns gene was about 50-kb long and organized into 14 exons and 13 introns. All intron–exon splice junctions conformed to the GT/AG consensus sequence except exon 8/intron 8 junction. Primer extension shows multiple transcription initiation sites between −44 and −75 although major transcription initiation site was observed at −90 bp from the ATG codon. The 5′-flanking region lacks canonical TATA and CAAT box sequences, but is G+C rich with 10 GC boxes (potential Sp1 binding sites), characteristic of a housekeeping gene promoter.
Keywords: Mucopolysaccharidosis IV A; Morquio disease; Lysosomal storage disease; Mouse; Galns gene;

We studied the effect of trans-4-hydroxy-2-nonenal on the wild-type human adenylosuccinate lyase and on the enzyme from a patient compound-heterozygous for two missense mutations (P75A/D397Y; McKusick 103050.0003/103050.0004). Both the enzymes were inhibited by 10–50 μM trans-4-hydroxy-2-nonenal in a concentration-dependent manner by means of a mixed-type co-operative mechanism. A significantly stronger inhibition was noticed in the presence of the defective enzyme. Nonanal and trans-2,3-nonenal inhibited the enzymes to a less extent and at about 10-times higher concentrations. Hydroxylamine reversed the inhibition by trans-4-hydroxy-2-nonenal, trans-2,3-nonenal or nonanal in the case of the wild-type enzyme, but it was ineffective to reverse the inhibition by trans-4-hydroxy-2-nonenal on the defective enzyme. Dithiothreitol slightly decreased the inhibition exerted by trans-4-hydroxy-2-nonenal on both the wild-type and the defective adenylosuccinate lyase, while it did not produce practically any change in the presence of trans-2,3-nonenal or nonanal.
Keywords: Adenylosuccinate lyase; 4-Hydroxy-2-nonenal; Psychomotor retardation; Lipid peroxidation; Inherited diseases;

Iron overload in paediatrics undergoing cardiopulmonary bypass by Sharon Mumby; Rajiv R. Chaturvedi; Joe Brierley; Christopher Lincoln; Andy Petros; Andrew N. Redington; John M.C. Gutteridge (342-348).
Pathological changes in iron status are known to occur during bypass and will be superimposed upon physiological abnormalities in iron distribution, characteristic of the neonatal period. We have sought to define the severity of iron overload in these patients. Plasma samples from 65 paediatric patients undergoing cardiopulmonary bypass (CPB) were analysed for non-haem iron, total iron binding capacity, transferrin and bleomycin-detectable iron. Patients were divided into four age groups for analysis. Within each age group, patients who were in iron overload at any time point were statistically compared to those who were not. The most significant changes in iron chemistry were seen in the plasma of neonates, with 25% in a state of plasma iron overload. 18.5% of infants and 14.3% of children at 1–5 years were also in iron overload at some time point during CPB. No children over 5 years, however, went into iron overload. Increased iron saturation of transferrin eliminates its ability to bind reactive forms of iron and to act as an antioxidant. When transferrin is fully saturated with iron, reactive forms of iron are present in the plasma which can stimulate iron-driven oxidative reactions. Our data suggest that paediatric patients are at greater risk of iron overload during CPB, and that some form of iron chelation therapy may be advantageous to decrease oxidative stress.
Keywords: Iron overload; Cardiopulmonary bypass; Antioxidant; Chelation; Transferrin; Neonate;