Survival of rabbit and horse erythrocytes in vivo after changing the fatty acyl composition of their phosphatidylcholine

The phospholipid composition and the distribution of phospholipids over the two leaflets of the membrane have been investigated for rabbit and horse erythrocyte membranes. Phosphatidylcholine (PC) comprises 39.4% and 41.3% of the total phospholipid complement of the rabbit and horse erythrocytes, respectively. In both membranes the distribution of this phospholipid is asymmetric: 70% of the PC is present in the outer layer of the rabbit membrane and 60% in that of the horse. The major species of this phospholipid class are the (1-palmitoyl-2-oleoyl)- and the (1-palmitoyl-2-linoleoyl)PC. The disaturated species, (1,2-dipalmitoyl)PC, is present in limited amounts only. Partial replacement of the native PC from intact erythrocytes was accomplished with a purified PC specific transfer protein from bovine liver. Replacement of the native PC species with (1-palmitoyl-2-oleoyl)PC up to 40% of the total PC complement had no effect on the osmotic fragility, the shape and the in vivo survival time of both erythrocyte species. Replacement of the native PC in both rabbit and horse erythrocytes with (1,2-dipalmitoyl)PC up to 20% gave rise to an increased osmotic fragility, a shape change from discocytic to echinocytic and a significant reduction in survival time measured after reinjection of the modified cells. At 30% replacement with (1,2-dipalmitoyl)PC the resulting spheroechinocytes appeared to be cleared from the circulation within 24 h after reinjection. The conclusion can be drawn that the repair mechanisms which may exist in vivo are insufficient to cope with the drastic changes in properties of the erythrocyte membrane which are induced by replacing more than 15% of the native PC by the dipalmitoyl species.     

Minor human antibody response to a mouse and chimeric monoclonal antibody after a single i.v. infusion in ovarian carcinoma patients: a comparison of five assays

The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab)₂ (n=28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18;n=24, 3 mg). Serum samples were taken before injection and 2–3 and 6–14 weeks after administration. A double-antigen or bridging assay was developed to detect responses against both murine as well as chimeric antibodies. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) as well as three commercially available assays were used to study antibody response against the murine antibody OV-TL 3. With both the double-antigen (bridging) assay and the indirect ELISA 1 of the 28 patients (4%) injected with murine OV-TL 3 F(ab)₂ showed a HAMA reaction 6 weeks after injection, which was demonstrated to be a mixed anti-isotypic and anti-idiotypic response. None of the 24 patients injected with the chimeric MOv18 showed an anti-chimeric antibody response. The various commercially available assays demonstrated conflicting results. The double-antigen-or bridging assay is a reliable method to detect anti-murine and antichimeric antibodies. The assay can be easily adapted for use with human antibodies. The immunogenicity of OV-TL 3 F(ab)₂ and cMOv18 in patients is low, making both antibodies candidates for immunotherapy.This work was supported by a clinical research grant of the Netherlands Organization for Scientific Research (NWO 900-716-020) and by the Biocare Foundation (grant 92-05).     

Comparison of the two different phosphagen systems in the lugworm Arenicola marina

The different phosphagen systems in the lugworm Arenicola marina, the phosphotaurocyamine/taurocyamine kinase system of the body wall and the phosphocreatine/creatine kinase system of the spermatozoa, have been investigated to answer the question whether the change reflects different functional modes of these phosphagen systems. Enzyme analyses have shown that in contrast to the body wall taurocyamine kinase, creatine kinase of spermatozoa exists in at least two different forms which are compartmented in the mitochondria (creatine kinase I) and in the flagellum (creatine kinase II). Creatine kinase I is strongly attached to cell structures which require detergents and high phosphate concentrations for solubilization. The affinities of taurocyamine kinase and creatine kinase for all substrates are very similar except the extremely high K _m for creatine of both creatine kinase I and II. The level of creatine in spermatozoa is fivefold higher than taurocyamine in the body wall at similar phosphorylation potential (ATP/ADO_free) and ATP-buffer capacity (phosphagen/ATP), reflecting the higher equilibrium constants of the creatine kinase reaction compared to that of the taurocyamine kinase reaction (Ellington 1989). The high creatine concentration gives the phosphocreatine/creatine kinase system an advantage over the phosphotaurocyamine/taurocyamine kinase system for transport of energyrich phosphate at high phosphorylation potential by increasing the radial diffusion flux. The maximum diffusive flux of free ADP in spermatozoa is three orders of magnitude below the respiratory ATP production while the creatine flux would allow an unlimited energy transport over the long diffusion distance. In lugworm body wall, however, the low ATP turnover and the low diffusion distances between mitochondria and myosin-ATPases do not require a phosphagen shuttle.Abbreviations ADP _free cytoplasmic adenosine diphosphate - Ap ₅ A P₁, P₅-di(adenosine-5-) pentaphosphate - AK arginine kinase - CK creatine kinase (EC 2.7.3.2) - DTT dithiothreitol - GAPDH glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) - HOADH 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) - IEP isoelectric point - MIM mitochondria isolating medium - P _i-free cytoplasmic inorganic phosphate - (P)Arg (phospho)arginine - (P)Cr (phospho)creatine - (P)Tc (phospho)taurocyamine - SEM scanning electron microscopy - TK taurocyamine kinase - TEM transmission electron microscopy     

Vibrio cholerae phosphatases required for the utilization of nucleotides and extracellular DNA as phosphate sources

Phosphate is essential for life, being used in many core processes such as signal transduction and synthesis of nucleic acids. The waterborne agent of cholera, Vibrio cholerae, encounters phosphate limitation in both the aquatic environment and human intestinal tract. This bacterium can utilize extracellular DNA (eDNA) as a phosphate source, a phenotype dependent on secreted endo‐ and exonucleases. However, no transporter of nucleotides has been identified in V. cholerae, suggesting that in order for the organism to utilize the DNA as a phosphate source, it must first separate the phosphate and nucleoside groups before transporting phosphate into the cell. In this study, we investigated the factors required for assimilation of phosphate from eDNA. We identified PhoX, and the previously unknown proteins UshA and CpdB as the major phosphatases that allow phosphate acquisition from eDNA and nucleotides. We demonstrated separable but partially overlapping roles for the three phosphatases and showed that the activity of PhoX and CpdB is induced by phosphate limitation. Thus, this study provides mechanistic insight into how V. cholerae can acquire phosphate from extracellular DNA, which is likely to be an important phosphate source in the environment and during infection.     

Three-dimensional speckle tracking echocardiography and cardiac magnetic resonance for left ventricular chamber quantification and identification of myocardial transmural scar

Background

We compared three-dimensional speckle tracking echocardiography (3DSTE) and its strain to cardiac magnetic resonance (CMR) with delayed contrast enhancement for left ventricular (LV) chamber quantification and transmurality of myocardial scar. Furthermore, we examined the ability of 3DSTE strain to differentiate between ischaemic and non-ischaemic LV dysfunction.

Methods

In 80 consecutive patients with ischaemic and 40 patients with non-ischaemic LV dysfunction, the correlations between LV volumes and ejection fraction were measured using 3DSTE and CMR. Global and regional 3DSTE strains and total or percentage enhanced LV mass were evaluated.

Results

LV end-diastolic and end-systolic volumes and ejection fraction correlated well between 3DSTE and CMR (r: 0.83, 0.88 and 0.89, respectively). However, 3DSTE significantly underestimated volumes. Correlation for LV mass was modest (r = 0.59). All 3DSTE regional strain values except for radial strain were lower in segments with versus segments without transmural enhancement. However, strain parameters could not identify the transmurality of scar. No significant difference between ischaemic and non-ischaemic LV dysfunction was observed in either global or regional 3DSTE strain except for twist, which was lower in the non-ischaemic group (4.9 ± 3.3 vs. 6.4 ± 3.2°, p = 0.03).

Conclusion

3DSTE LV volumes are underestimated compared with CMR, while LV ejection fraction revealed excellent accuracy. Functional impairment by 3DSTE strain does not correlate well with scar localisation or extent by CMR. 3DSTE strain could not differentiate between ischaemic and non-ischaemic LV dysfunction. Future studies will need to clarify if 3DSTE strain and CMR delayed contrast enhancement can provide incremental value to the prediction of future cardiovascular events.

     

Somatic origin of inherited haemophilia A

Summary We found a partial deletion of the clotting factor VIII gene of about 2000 bp, spanning exon 5 and part of intervening sequence 4 and 5 in an isolated patient with severe haemophilia A. The mother of the patient, who appeared to be a non-carrier on the basis of coagulation assays and restriction fragment length polymorphism analysis in the family, turned out to be a mosaic for the deletion, not only in her germ cells, but also in various somatic cells. These findings suggest that the mutation is the result of an event in early embryogenesis. If mosaicism for a mutation, either gonadal or somatic, proves to be a common phenomenon in human genetics, it is imperative to reconsider genetic risks for (future) sibs of any apparently new mutant of a hereditary disease.     

Action of pancreatic phospholipase A2 on phosphatidylcholine bilayers in different physical states.

1. Saturated and unsaturated phosphatidylcholines, dispersed as liposomes in water, can be hydrolysed by phospholipase A2 from pig pancreas. A pure saturated phosphatidylcholine is hydrolysed only near its transition temperature. An unsaturated phosphatidylcholine is hydrolysed preferentially near its transition temperature, but hydrolysis can occur also above the transition temperature, albeit at a much lower rate. 2. An equimolar mixture of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, which shows cocrystallization of the paraffin chains, is hydrolyzed between 25 and 40 degrees C with a maximum at 32 degrees C, in agreement with the calorimetric scan of the phase transition. 3. An equimolar mixture of dilauroyl phosphatidylcholine and distearoyl phosphatidylcholine, which shows a monotectic behaviour, is hydrolysed at all temperatures. Hydrolysis is maximal at 0 and 40 degrees C, at which temperatures dilauroyl phosphatidylcholine and distearoyl phosphatidylcholine undergo their phase transition, respectively. 4. Both in the mixture showing cocrystallization and in the mixture in which phase separation occurs, the phosphatidylcholine species with the shorter fatty acid chains is hydrolysed at a higher rate than the longer chain fatty acid species. 5. Hydrolysis is inhibited by the presence of cholesterol in liposomes prepared of saturated phosphatidylcholine. Inhibition is complete at a cholesterol concentration of 35 mol %. Subsequent addition of filipin and amphotericin B to the mixed cholesterol-phosphatidylcholine liposomes overcomes the inhibitory effect of cholesterol.