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| 3. |
- Rydberg, Lennart, 1944, et al.
(author)
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Extracorporeal ("ex vivo") connection of pig kidneys to humans. II. The anti-pig antibody response.
- 1996
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In: Xenotransplantation. - : Wiley. - 0908-665X .- 1399-3089. ; 3:4, s. 340-53
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Journal article (peer-reviewed)abstract
- Pig kidneys were extracorporeally "ex vivo" connected to the circulation of two volunteer male dialysis patients (Breimer et al., this issue). The patients were pretreated by daily plasmapheresis for 3 consecutive days, which reduced the anti-pig lymphocytotoxic titer from 8 to 2 in the first patient and from 8 to 1 in the second patient. The anti-pig hemagglutinating titers were reduced from 32 to 4 in the first patient and from 2 to 1 in the second patient. No drugs, except heparin, were given. The perfusion lasted for 65 min in patient 1 and the experiment was terminated due to increased vascular resistance in the pig kidney. Ultrastructural investigation showed a picture similar to a hyperacute vascular rejection. Immunohistochemical studies showed a weak staining of IgM antibodies, but no IgG in the small arteries and glomeruli. The pig kidney of patient 2 was perfused for 15 min and the experiment terminated due to serious side effects of the patient. Light and electron microscopical investigation showed virtually no structural changes of the kidney tissue and immunostaining for human antibodies was negative. In both patients, serum samples collected 2-5 weeks postperfusion showed a strong anti-pig antibody titer rise (up to 512) which thereafter declined but stabilized on a higher level than before the experiment. The antibody response in the two patients was different. In patient 1, the major anti-pig antibodies directed to carbohydrate antigens were of IgG (IgG1 and IgG2 subclasses) type, while the IgM response was less prominent and virtually no IgA antibodies were produced. Despite the short duration of the perfusion in patient 2, a humoral immune response was seen that was mainly confined to the IgA immunoglobulin class (IgA1 subclass). Blood group glycospingolipid fractions, prepared from the contralateral kidney of the donor pigs, were used for immunostaining with patient serum samples. In both patients, the antibodies produced after the perfusion, mainly recognized the Galα1-3Gal epitope both as part of the "linear B" pentasaccharide but also on more complex carbohydrate structures. Patient 1 was HLA-immunized before the experiment due to a kidney allograft and had a panel reactivity of 85% before the perfusion. No change in the panel reactivity of HLA-antibodies was found after the perfusion experiments. Patient 2 had no HLA antibodies before and remained negative after the perfusion. Patient serum samples collected before and after the perfusion were tested for reactivity against human endothelial cell lines. No antibodies were generated.
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- Breimer, Michael, 1951, et al.
(author)
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Extracorporeal ("ex vivo") connection of pig kidneys to humans. I. Clinical data and studies of platelet destruction.
- 1996
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In: Xenotransplantation. - : Wiley. - 0908-665X .- 1399-3089. ; 3:4, s. 328-39
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Journal article (peer-reviewed)abstract
- The pioneering experiment by Welsh et al. (Immunological Lett 1991:29:167-170) connecting a pig kidney to the human circulation has been repeated in a modified manner. Two volunteer dialysis patients were pretreated by daily plasmapheresis on days -2,-1, and 0 to remove the naturally occurring anti-pig xenoantibodies. The anti-pig lymphocytotoxic liters were reduced from 1:8 to 1:2 in patient 1 and from 1:8 to 1:1 in patient 2. No steroids or immunosuppressive drugs were administrated before or during the experiments. A sterile pig kidney was extracorporeally ("ex vivo") connected to the patients a/v fistula using an arterial and a venous pump similar to a dialysis. The two experiments gave different results. In the first experiment the perfusion pressure was kept at 100 mmHg for the initial 25 min by reducing the pump speed until the minimum blood flow of 30 ml/min was reached. Thereafter, the pressure rose continuously and the experiment was terminated at 65 min at a perfusion pressure of 200 mmHg. The patient did not feel any discomfort during the perfusion. In the second experiment, a stable blood flow of 200 ml/min was reached at a pressure of 100 mmHg after a few minutes. The perfusion was terminated at 15 min when the patient developed chest and abdominal pain, hypotension, and electrocardiographic signs of myocardial ischemia. The patient recovered quickly. In the first experiment, small volumes of clear urine was produced until the pressure rose above 100 mmHg, which resulted in hematuria. In the second experiment clear urine (4 ml/min) was produced. (51)Chromium clearance values were after 15 min <1 ml/min for kidney 1 and 12 ml/min (8 ml/min/100 g) for kidney 2. A drastic reduction in platelet count (128 to 48 and 64 to 8 × 10(9)/1, respectively) during the passage through the kidney was found in blood samples collected simultaneously before and after the organ. No change in hemoglobin values and leucocyte counts were found. Light- and electron-microscopical analysis of the kidney tissues revealed for kidney 1 focal areas with obliteration of the glomerular and peritubular capillaries by platelets and PMN cells and severe damage of the endothelial cells comparable to a picture of a hyperacute rejection. In kidney 2, all vessels were patent but in the capillaries large amount of membrane fragments were detected by electron microscopy and a discrete damage of the endothelial cells were seen in some segments. No intact platelets were present in the vascular tree. These human experiments support the hypothesis that hyperacute rejection of pig to human xenografts is delayed in time by removal of the preformed anti-pig xenoantibodies. A new finding was a very rapid destruction of platelets occurring in the kidney of patient 2 who had very low liters of xenoantibodies. The humoral immune response is described in detail in an accompanying paper (Rydberg et al., this issue).
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| 16. |
- Fernandez-Mateos, P, et al.
(author)
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Point mutations and deletion responsible for the Bombay H null and the Reunion H weak blood groups.
- 1998
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In: Vox sanguinis. - 0042-9007. ; 75:1, s. 37-46
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Journal article (peer-reviewed)abstract
- OBJECTIVE: Definition of the molecular basis of the Reunion and the Bombay red cell and salivary H-deficient phenotypes. METHODS: Sequence and expression of FUT1 and FUT2 genes from H-deficient individuals. Family segregation analysis of the mutations responsible for the fucosyltransferase defects of H, secretor and Lewis systems. RESULTS: The Indian red cell H null Bombay phenotype depends on a new mutation of the FUT1 gene. T725-->G changing Leu242-->Arg. Their salivary nonsecretor phenotype is secondary to a complete deletion of the FUT2 gene. The red cell H weak Reunion phenotype depends on another new mutation of FUT1, C349-->T which induces a change of His117-->Tyr. Their salivary nonsecretor phenotype is due to the known Caucasian inactivating mutation G428-->A. CONCLUSION: Single prevalent FUT1 and FUT2 point mutations and a deletion are responsible for the Indian Bombay H null and the Reunion H weak phenotypes found on Reunion island. This is in contrast with other H-deficient phenotypes where sporadic nonprevalent inactivating mutations are the rule.
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- Kromer, S, et al.
(author)
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Regulation of the supply of cytosolic oxaloacetate for mitochondrial metabolism via phospho enolpyruvate carboxylase in barley leaf protoplasts .1. The effect of covalent modification on PEPC activity, pH response, and kinetic properties
- 1996
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In: Biochimica et Biophysica Acta - General Subjects. - 0304-4165 .- 1872-8006. ; 1289:3, s. 343-350
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Journal article (peer-reviewed)abstract
- The regulation of the supply of oxaloacetate (OAA) for mitochondrial metabolism via phosphoenolpyruvate carboxylase (PEPC) by covalent modification is studied in barley (Hordeum vulgare L.) leaf protoplasts in light or darkness as well as under photorespiratory or non-photorespiratory conditions. Extracts for studies on in vivo PEPC phosphorylation were prepared from barley leaf protoplasts by rapid filtration, fractionating the cell within less than 1 s. Measurements of in vitro PEPC activity were performed on samples quickly frozen in liquid nitrogen to break the cell and stop metabolism and thus preserve the in vivo activation state. The relative PEPC phosphorylation state increased upon illumination and decreased upon redarkening under photorespiratory and non-photorespiratory conditions. PEPC activity measured in the presence of malate (3 mM) under photorespiratory conditions showed the same response indicating that a light-induced increase in PEPC activity and decrease in malate sensitivity is caused by an increased phosphorylation level of the PEPC protein. PEPC activity was pH dependent. At the physiological cytosolic pH, activity was suboptimal, but most sensitive towards malate inhibition and glucose 6-phosphate stimulation. The presence of malate increased the sensitivity of PEPC activity towards pH changes. The response of PEPC activity to changing pH was not affected by changes in the activation state of the enzyme. The K-m (phosphoenolpyruvate, PEP) is about 1 mM. Upon illumination the K-m (PEP) decrease significantly. V-max was unaffected by the light treatment. The presence of physiological concentrations of glucose 6-phosphate decreased K-m (PEP) 5- to 10-fold and increased V-max by about 35%. The effect of glucose 6-phosphate was strongest (up to 7-fold) at subsaturating PEP concentrations stimulating PEPC activity to nearly maximal rates. The results show that an increase in PEPC phosphorylation state causes an increase in PEPC activity as well as in substrate affinity leading to an increased production of OAA in the light.
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| 21. |
- Kromer, S, et al.
(author)
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Regulation of the supply of oxaloacetate for mitochondrial metabolism via phospho enolpyruvate carboxylase in barley leaf protoplasts .2. Effects of metabolites on PEPC activity at different activation states of the protein
- 1996
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In: Biochimica et Biophysica Acta - General Subjects. - 0304-4165 .- 1872-8006. ; 1289:3, s. 351-361
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Journal article (peer-reviewed)abstract
- The regulation of the supply of oxaloacetate (OAA) for mitochondrial metabolism via phosphoenolpyruvate carboxylase (PEPC) by metabolites is studied in barley (Hordeum vulgare L.) leaf protoplasts in light or darkness as well as under photorespiratory or non-photorespiratory conditions. Measurements on PEPC activity were performed on samples quickly frozen in liquid nitrogen to break the cell and stop metabolism and thus preserve the in vivo activation state. Glycine, serine, pyruvate, acetyl-CoA, glycolate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and ADP had no significant effect on PEPC activity. Malate, aspartate and glutamate were strong inhibitors of PEPC activity decreasing the activity more in light versus darkness. However, at the physiological cytosolic concentration of these metabolites under the respective conditions, inhibition of PEPC activity was about the same with the exception of aspartate which inhibits more under non-photorespiratory than under photorespiratory conditions. 2-Oxoglutarate and glyoxylate decreased PEPC activity by 20 to 40% in the range of its physiological cytosolic concentration. Inhibition by physiological cytosolic concentrations of glutamine was limited. Glucose B-phosphate, fructose B-phosphate, 3-phosphoglycerate, dihydroxyacetonphosphate and P-i stimulated PEPC activity significantly in their physiological cytosolic concentration range. Physiological cytosolic concentrations of glucose 6-phosphate and fructose 6-phosphate activated PEPC activity to about the same extent under all conditions applied, while 3-phosphoglycerate and dihydroxyacetonphosphate stimulating Stronger under non-photorespiratory versus photorespiratory conditions. Moreover, dihydroxyacetonphosphate stimulated PEPC activity more in light versus darkness under non-photorespiratory conditions. P-i activation of PEPC activity decreases in light versus darkness under non-photorespiratory conditions. Stimulation of PEPC activity by citrate in its physiological concentration range is limited. Glucose 1-phosphate and AMP activated PEPC activity only at concentrations higher than their physiological levels in the cytosol. Determinations of PEPC activity in the presence of different malate/glucose 6-phosphate ratios revealed that glucose 6-phosphate totally relieved the inhibitory effect of malate. The regulatory properties of PEPC activity will be discussed in relation to its functions in C-3 plants.
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| 23. |
- Larsson, S, et al.
(author)
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Molecular cloning and biochemical characterization of carbonic anhydrase from Populus tremula x tremuloides
- 1997
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In: Plant Molecular Biology. - 0167-4412 .- 1573-5028. ; 34:4, s. 583-592
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Journal article (peer-reviewed)abstract
- A leaf cDNA library from hybrid aspen, Populus tremula x tremuloides, was constructed. From this two different cDNA clones, denoted CAla and CAlb, encoding a chloroplastic carbonic anhydrase (CA) were isolated and DNA sequenced. Analysis of the deduced amino acid sequences showed that the isolated CAs belong to the beta-CA family, and have identities around 70% to other dicotyledonous plant CAs. The two hybrid aspen cDNA clones display a high nucleotide sequence identity, only 12 nucleotides differ. Since only one gene copy of this soluble chloroplastic CA is present in the nuclear genome, we postulate that the two isolated cDNA clones are alleles. Northern blot hybridization revealed a CA transcript of ca. 1300 bases, 140 bases shorter than in pea. Western and northern blot hybridizations on crude protein extracts and on total RNA, respectively, isolated from stem and leaves, showed that hybrid aspen CA is expressed specifically in the leaf under the growth conditions used. Based on the deduced amino acid sequence, the mature hybrid aspen CA enzyme subunit has a molecular mass of 24.8 kDa. The enzyme was over-expressed in Escherichia coli, and purified by affinity chromatography. Biochemical characterization showed that the protein structure and the CO2-hydration activity are similar to the pea enzyme. Molecular characterization of a CA from a perennial plant has not previously been performed, and it demonstrates that both the structure and activity of hybrid aspen CA resembles CAs from annual plants.
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| 26. |
- Lindström, K, et al.
(author)
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Blood group glycosphingolipid expression in kidney of an individual with the rare blood group A1 Le(a-b+) p phenotype: absence of blood group structures based on the globoseries.
- 1996
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In: Glycoconjugate journal. - 0282-0080. ; 13:2, s. 307-13
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Journal article (peer-reviewed)abstract
- Total neutral glycolipid fractions were isolated from kidney and ureter tissue obtained at autopsy of an individual of the rare blood group A1 Le(a-b+) p. The amount of glycolipids isolated were 3.7 and 2.5 mg g-1 dry tissue weight for the kidney and ureter tissue, which is in the range of reference blood group P kidneys. Part of the kidney glycolipid fraction was subfractionated by HPLC. Glycolipid compounds were structurally characterized by thin-layer chromatography (chemical detection and immunostaining with monoclonal antibodies), proton NMR spectroscopy and mass spectrometry. Globotriaosyl- and globotetraosyl-ceramides, which are the major compounds in kidneys of P individuals, were absent in the p kidney, and a comparatively increased amount of monoglycosyl- and lactosylceramides was found. A shift to longer fatty acyl chains in the ceramide part of lactosylceramides was noted. Elongated globoseries compounds with five to seven sugar residues, including the blood group A type 4 chain structure, were lacking. A slight increase in neolactotetraosyl- and blood group X pentaglycosyl-ceramides was noticed. The study confirms an enzymatic block in the conversion of lactosylceramide to elongated globoseries compounds in the kidney tissue similar to that of erythrocytes of p individuals.
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| 27. |
- Magnusson, Kerstin, 1957, et al.
(author)
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Contamination and correlation with toxicity of sediment samples from the Skiagerrak and Kattegat
- 1996
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In: Journal of Sea Research. - 1385-1101. ; 35:1-3, s. 223-234
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Journal article (peer-reviewed)abstract
- The pollution state in the Skagerrak and Kattegat was investigated by determination of pollutant concentrations and toxicity of sediment samples from 11 stations in the area. A comparison was made with the sediment from a reference site near the Faroe Islands. Polycyclic aromatic hydrocarbons (PAH) and organochlorines were determined in whole sediment and heavy metals and ammonia were analysed in filtered pore water. Toxicity was bioassayed in whole sediment with Nitocra spinipes and Daphnia magna, in pore water with Mytilus edulis larvae and in solvent extracts from sediment with tests measuring etoxyresorufin-O-deethylase (EROD) activity in Oncorhyncus mykiss and rate of denitrification. Sites close to Goteborg and in an area from the Oslo fjord to the Norwegian Trench were most polluted. Sediment from the Faroe Islands was least polluted and also least toxic. Multivariate statistical analysis indicates that the different tests were sensitive to different kinds of pollutants. Effects on mussel larvae correlated strongest with the occurrence of ammonia, manganese, cadmium and PAHs, Nitocra with alpha-hexachlorocyclohexane (HCH) and p,p'-DDD, Daphnia with arsenic and gamma-HCH, fish EROD activity with benzo[ghi]perylene and unknown compounds associated with organic carbon, and denitrification with chlordanes, dieldrin and a few PAHs. The results indicate that sampling sites close to Goteborg are so polluted that harmful effects on the ecosystem probably occur.
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| 29. |
- Olling, A, et al.
(author)
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Electrospray ionization and collision-induced dissociation time-of-flight mass spectrometry of neutral glycosphingolipids.
- 1998
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In: Rapid communications in mass spectrometry : RCM. - 0951-4198. ; 12:10, s. 637-45
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Journal article (peer-reviewed)abstract
- A series of native naturally occurring neutral glycosphingolipids has been analysed by electrospray ionization tandem mass spectrometry using a hybrid magnetic sector-TOF instrument. The collision-induced dissociation products of precursor ions were detected by an orthogonal acceleration time-of-flight mass spectrometer as the second analyser. Glycosphingolipids, with mono- to hexa-saccharide chain lengths with different ceramide constituents, were studied. The result of electrospray ionization in the positive ion mode generally showed singly charged molecular ions with Na+ as adduct, [M + Na]+. The sensitivity of the electrospray ionization was greatly enhanced by addition of NaCl, LiCl (forming [M + Li]+) or KCl (yielding [M + K]+) to the sample. A comparison between the collision-induced dissociation of precursor molecular ions of monoglycosylceramides, using Na+, Li+ and K+ as adducting species, showed that the intensity of the fragment ions and the extent of the daughter ion fragmentation of the molecular ions, are dependent on the type of adduct used. The daughter ion spectra of Li+ adduct ions showed intense sequence fragment ions, both of the saccharide chain and the ceramide moiety, and were superior to those obtained using Na+ or K+. The collision-induced dissociation spectra of the [M + Li]+ ions, of glycosphingolipids containing di- to hexasaccharides, are also presented. Proposed possible fragments, resulting from the CID of the molecular ions [M + Li]+ of monoglycosylceramides, are shown.
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| 39. |
- Samuelsson, Marcus, 1967-, et al.
(author)
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Vargdebatt
- 1997
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In: Argaladei - friluftsliv en livsstil. - 1402-9235. ; :1, s. 16-
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Journal article (pop. science, debate, etc.)
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