Sarcolemma phospholipid structure investigated by immunogold electron microscopy and (31)P NMR spectroscopy with lanthanide ions.

The biological functions of plasma membranes depend greatly on the biophysical properties resulting from protein and phospholipid structure. We investigated the phospholipid structure of the normal sarcolemma membrane, which is known to be highly dysfunctional in myopathies. Combining electron microscopy and (31)P nuclear magnetic resonance (NMR) spectroscopy on isolated sarcolemma vesicles, we find that (i) the sarcolemma vesicles maintain the in-vivo cellular sidedness, (ii) the phospholipid mobility is close to that observed in model membranes (similar lateral diffusion coefficients and spin-lattice T(1) relaxation times). Using broad-band and magic angle spinning (31)P NMR spectroscopy with lanthanide ions (Pr(3+)), it is possible to quantify the distribution of phospholipids between internal and external membrane layers, showing that the trans-bilayer distribution is highly asymmetrical.

Differentiation of the P-gp and MRP1 multidrug resistance systems by mobile lipid 1H-NMR spectroscopy and phosphatidylserine externalization.

We have previously demonstrated that proton NMR spectra of fatty acid chains in erythroleukemia K562 wild-type cells and their MDR1 counterparts show variations related to the phenotype over-expressing the P-glycoprotein (P-gp). Human lung cancer cells whose multidrug resistance (MDR) counterparts over-express the multidrug resistance-associated protein MRP1 have not yet been studied by NMR. Both P-gp and MRP1 belong to the same ATP-binding cassette transporter superfamily. A comparison of NMR spectra from both these multidrug-resistance phenotypes showed that the results previously obtained on the MDR1 family are not valid for MRP1. Furthermore, flow cytofluorimetry studies with external phosphatidylserine labelling showed that P-gp and MRP1 overexpressions have strong but differentiated effects on cell lipid pools.

Inventory of wild rodents and lagomorphs as natural hosts of Fasciola hepatica on a farm located in a humid area in Loire Atlantique (France).

With the objective of studying the role of wild fauna in the epidemiology of fasciolosis disease, a definitive wild-host inventory was carried out in a french farm where infected domestic hosts (cows) cohabit with wild potential ones. Liver flukes, faecal eggs and antibodies were looked for in lagomorphs (Oryctolagus cuniculus) and rodents (Myocastor coypus, Ondatra zybethicus, Rattus norvegicus, Arvicola sapidus and micromammal species) trapped in the study area. Presence of Fasciola hepatica was detected in two species: O. cuniculus and M. coypus. Infection rates were respectively 34% (42/124) and 55% (106/193). Liver flukes were found in 78 M. coypus (n = 192) and 11 O. cuniculus (n = 35). No other species was infected by F. hepatica. The number of animals shedding fluke eggs was higher in M. coypus (49 out of 127 sampled; 38.6%) than in O. cuniculus (two out of 17 sampled; 11.7%). The results indicate that M. coypus may play a role in the maintenance and the dissemination of F. hepatica in various environments and open a discussion on the role of other natural wild hosts.

Magnetic resonance spectroscopy and fluorescence microscopy to investigate mobile lipids in sensitive, resistant and reverting K562 cells and their membranes.

The erythroleukaemic K562 cell line and its adriamycin resistant counterpart were used to study resistance, its reversion and their consequences on the levels and localisation of lipids detected in proton nuclear magnetic resonance (NMR) spectra. On whole cells, the mobile lipids giving rise to a NMR signal were significantly decreased in the resistant cells when compared to the sensitive ones; these lipids recovered partially in the reverting cells. According to the spinlattice relaxation times (T1), the lipids detected appeared to be in a similar environment in sensitive and reverting cells. In membrane-enriched fractions, mobile lipid levels were not significantly different in the sensitive and reverting cell lines but decreased in resistant ones. Moreover, lipid droplets stained with a fluorescent Nile red lipophilic probe showed the presence of highly fluorescent particles in the samples in which NMR detected high levels of mobile lipids. These results suggest the participation of cytosolic lipid droplets in NMR signals in drug sensitive and reverting cells and open the question of the relative roles of these droplets and of the membrane lipids in the lipid metabolic pathways associated with drug resistance in cancer cells.

Static and magic angle spinning (31)P NMR spectroscopy of two natural plasma membranes.

Static and magic angle spinning (31)P NMR spectroscopy was used for the first time in natural plasma membranes from erythrocytes and skeletal muscle to study phospholipid arrangement and composition. Typical static powder-like spectra were obtained showing that phospholipids were in a bilayer arrangement. Magic angle spinning narrowed spectra into two components. The first one corresponded to phosphatidylcholine and the second one to the other phospholipids with intensities in agreement with the known phospholipid composition. These findings show that NMR data previously acquired using model membranes can be transposed to studies on phospholipids in their natural environment.

Quantification of plasma lipoprotein fractions by wavelet transform time-domain data processing of the proton nuclear magnetic resonance methylene spectral region.

Quantitative analysis of lipoprotein major fractions, LDL, VLDL and HDL, is of great interest for medical purposes, for instance in liver or heart diseases, diet management or cancer. The presently available biochemical methods require time consuming ultracentrifugation. A potentially automated method is proposed, using time domain quantification by Wavelet Transform (WT-NMR) method. The aim of the present study was to evaluate, on a preliminary series of nine human plasmas, the potential interest of WT-NMR in the quantification of both NMR-visible lipids and total lipoprotein fractions. The correlation coefficients between low and intermediate density (LDL+IDL), very low density (VLDL) and high density (HDL) lipoprotein visible lipid quantifications, obtained on nine human plasmas with WT-NMR and standard biochemical methods, were 0.79, 0.84 and 0.92, respectively. For the total lipoprotein assay, i.e. including an estimation of non NMR-visible protein and free cholesterol, the correlation between WT-NMR and the biochemistry were 0.87 for LDL+IDL, 0.81 for VLDL and 0.88 for HDL.

Proton and phosphorus nuclear magnetic resonance spectroscopy of human brain tumor extracts with automatic data classification: a preliminary study.

High-resolution one-dimensional proton and phosphorus and two dimensional COSY proton Magnetic Resonance Spectroscopy were used to investigate the lipid and carbohydrate metabolism of human brain tumors. Sixteen meningioma (MG) (benign tumors) and ten glioblastoma (GB) (malignant tumors) samples from brain surgery were treated for dual extraction of lipidic and aqueous phases before NMR processing. A highly significant variation of the 1H metabolite spectral pattern was observed between benign and malignant tumors. Double extraction method combined with both 1H and 31P NMR in vitro analyses provided a large set of biochemical information which may be statistically analyzed to elucidate tumor-specific biochemical pathways and to improve interpretation of in vivo spectra.

Tumour growth modifies intravascular polyamine transport by plasma lipoproteins in the mouse.

Polyamines are polycationic compounds which are implicated in cell division and tumor growth. We have evaluated the potential role of plasma lipoproteins in the transport of major polyamines, spermine, spermidine and putrescine, and the effect of tumor growth on such transport. Plasmas of healthy male BL6/DBA2 mice and of mice bearing Lewis lung carcinoma (3LL) were fractionated by isopycnic density gradient ultracentrifugation, and polyamine content determined in lipoprotein fractions. Spermidine was the most abundant polyamine in the lipoproteins of both control and tumor-bearing mice and was principally associated with HDL (d: 1.046-1.136 g/ml); approx. 40% of total plasma polyamines was lipoprotein-associated in control mice and 60% in cancerous mice. Only minor amounts were transported by LDL (< 10% of total lipoprotein-associated polyamines), while VLDL were devoid of these substances. Marked elevations of circulating levels of LDL were found in 3LL grafted mice: in these particles however, the contents of spermidine and spermine were significantly reduced. A preferential uptake of polyamines by red blood cells could in part explain this marked reduction of LDL polyamine content, but the consequence of this reduction on the net electrical charge and biochemical function of LDL remains unknown. Elevations of plasma LDL and HDL levels in 3LL-grafted mice underlie the finding that only minor modification was detected in the putrescine content of these particles. However, it is evident that elevated total amounts of putrescine were present in the plasma of such animals. Finally, the density profile of polyamines was modified in cancerous mice in which a shift to transport in lighter apo.AI-containing HDL particles was observed for spermidine; an even more marked shift was found for spermine. In conclusion, our data demonstrate that HDL particles constitute the major plasma vehicle for polyamine transport in both control and in tumor-bearing mice.

Proton magnetic resonance spectroscopy of blood plasma in heart transplant recipients treated with cyclosporine: an early prognosis test of long-term graft tolerance.

BACKGROUND: Previous results have established the potential interest of proton magnetic resonance spectroscopy (MRS) of plasma lipoproteins in the detection of rejection processes after heart transplantation. The aim of this study was to determine whether MRS can provide a relevant long-term prognosis factor as early as 1 week after transplantation. METHODS: Eighteen patients were monitored for a mean period of 16 months after transplantation. The ratio of the sum of the MRS total line widths (TLW) for lipoprotein moieties, obtained 1 week after transplantation and cyclosporine administration, over the same sum obtained on the day of transplantation (TLW(8/0)), as well as the ratio between the corresponding intensities of methyl and methylene moieties (IR) were used to quantify the lipoprotein spectral profile. RESULTS: TLW(8/0), with a cutoff value of 0.8, seemed to have the most value in predicting rejection processes (RP) several months later. All six patients with no RP (good prognosis) and all five patients with three or more RPs (poor prognosis) during the entire 16-month follow-up period were correctly detected as early as 8 days after transplantation. The seven patients with only one or two RPs, mainly occurring during the first months after transplantation, were usually classified by MRS as having good prognosis. CONCLUSIONS: The magnetic resonance spectrum depends on both qualitative and quantitative variations in the different lipoprotein fractions, known to be carriers of cyclosporine. The magnetic resonance spectrum could thus be an early expression of the ability of these lipoproteins to modulate the cyclosporine-mediated immunosuppression.

Does a passive transport between modified plasma lipoproteins and cell membranes exist in cancer disease?

Malignant diseases increase the level of total lipids in blood and modify their distribution in lipoprotein carriers affecting lipid exchanges between serum and tissues. These exchanges take place by active and/or passive ways which coexist in most tissues. This work concerns the exploration of passive diffusion, using the red blood cell mechanism as a model. Lipid components of normal and cancerous rat erythrocytes have been investigated by Proton and Carbon high resolution Magnetic Resonance Spectroscopy (H-1 and C-13 MRS). As previously established, MRS yields the usual molar ratio cholesterol/phospholipids and moreover provides information on the length and degree of unsaturation of the phospholipid fatty acyl chains. No modification has been recorded in erythrocyte lipids between cancer and control populations. These data would suggest that erythrocytes can maintain membrane lipid homeostastis during malignancy. The numerous abnormalities noted in their membrane fuction remain to be explained.

Proton nuclear magnetic resonance spectroscopy of plasma lipoprotein: technical problems and potential interest in cancer disease.

This paper reviews several methods presently available for analysing lipoprotein NMR spectra. Two main steps can be distinguished: NMR signal processing and data analysis. Time domain (wavelet transform) and frequency domain (curve fitting) signal processing methods are compared. Statistical methods of data analysis (Ascending Hierarchical Classification, Correspondence Analysis and Principal Component Analysis) have been tested on simulated NMR data of plasma lipoprotein with different numbers of sampling points and different noise levels. These few examples clearly attest that the NMR approach to complex "mixture" (such as body fluids) analysis is emerging from its infancy. New interest in plasma lipoprotein analysis in cancer biology is finally discussed in the light of previous clinical and experimental results and of understanding of lipid metabolism in cancer.

Proton NMR spectroscopy of plasma lipoproteins: a marker of the immune function in cancer disease?

1H-NMR spectroscopy of cancer plasma statistically detects significant narrowing of the methyl and methylene line widths. This change is due to relative increase in light density lipoproteins (VLDL and LDL) compared to heavy density lipoproteins (HDL). This observation had raised great hopes for a simple and universal screening test of cancer patients. Furthermore, the same signal can be observed in the plasma of pregnant women and heart transplanted patients undergoing an immunosuppressive treatment. This signal disappears after child's birth and during graft rejection processes. These observations suggest that the test initially proposed by Fossel in 1986 reveals a specific immunological status developed by the organism in "symbiosis" with "foreign" cells, rather than a cancerous disease.

Proton NMR spectroscopy of plasma lipoproteins in the experimental Lewis lung carcinoma.

Plasma and fractionated lipoproteins from 40 Lewis Lung Carcinoma grafted mice were tested from the first day up to the fatal issue by biochemical analyses and water suppressed 1H NMR spectroscopy. We have confirmed first, that the 1H NMR spectra of plasma lipoproteins are modified by the tumoral state and could provide a useful marker of the disease as long as they are used for individual follow-up with appropriate spectral parameters. Using fractionated lipoproteins we have demonstrated secondly, that the observed spectral modifications do not result from a specific cancer lipoprotein but from quantitatively modified ratio between Very Light Density Lipoproteins and High Density Lipoproteins.

A plasma membrane protein is involved in cell contact-mediated regulation of tissue-specific genes in adult hepatocytes.

We have identified the liver-regulating protein (LRP), a cell surface protein involved in the maintenance of hepatocyte differentiation when cocultured with rat liver epithelial cells (RLEC). LRP was defined by immunoreactivity to a monoclonal antibody (mAb L8) prepared from RLEC. mAb L8 specifically detected two polypeptides of 85 and 73 kD in immunoprecipitation of both hepatocyte- and RLEC-iodinated plasma membranes. The involvement of these polypeptides, which are integral membrane proteins, in cell interaction-mediated regulation of hepatocytes was assessed by evaluating the perturbing effects of the antibody on cocultures with RLEC. Several parameters characteristic of differentiated hepatocytes were studied, such as liver-specific and house-keeping gene expression, cytoskeletal organization and deposition of extracellular matrix (ECM). An early cytoskeletal disturbance was evidenced and a marked alteration of hepatocyte functional capacity was observed in the presence of the antibody, together with a loss of ECM deposition. By contrast, cell-cell aggregation or cell adhesion to various extracellular matrix components were not affected. These findings suggest that LRP is distinct from an extracellular matrix receptor. The fact that early addition of mAb L8 during cell contact establishment was necessary to be effective may indicate that LRP is a novel plasma membrane protein that plays an early pivotal role in the coordinated metabolic changes which lead to the differentiated phenotype of mature hepatocytes.

Lysosomal glycosidase activities in rat testis during sexual maturation.

Concentrations of testosterone and its metabolite dihydrotestosterone were determined in whole rat testis during the transition from the prepuberal to the mature status. The activities of five glycosidases (beta-N-acetylhexosaminidase, alpha-L-fucosidase, beta-D-galactosidase, alpha-D-glucosidase and alpha-D-mannosidase) were also investigated in the seminiferous tubules and interstitial tissues. The same androgens and enzyme activities were measured in testis of rats treated with 17-beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5-alpha-androstan-3-on e, a potent inhibitor of the 5-alpha-reductase-mediated conversion of testosterone into dihydrotestosterone. Lysosomal glycosidic activities in seminiferous tubules were found to vary slightly with the age of the animals, and thus seemed not to depend on the hormonal status of the tested animals. In contrast, the enzyme activities were low in the immarure interstitium but increased sharply during the onset of sexual maturity. The activity of each glycosidase reached a maximum between the 45th and the 65th day, except for beta-N-acetylhexosaminidase which did not decrease significantly following the 55th day. The five lysosomal enzyme activities decreased in the interstitial compartment of rat testis treated with DMAA, suggesting a relationship between these glycosidic enzyme activities and dihydrotestosterone.

Effects of perhexiline maleate on asialo-orosomucoid receptor endocytosis and recycling in HTC cells.

Perhexiline is a lysosomotropic agent which has proved to be very valuable to certain patients suffering from angina pectoris. However long-term administration of the drug may induce hepato- and neuro-toxicity. Using HTC cells (a rat hepatoma-derived cell line) whose plasma membranes were labeled with NaB[3H]4 after oxidation by NaIO4, endocytosis and recycling of labeled asialo-orosomucoid (ASOR) receptors were investigated in the presence of 50 mumols/l perhexiline maleate. The results demonstrate that the drug induces a significant decrease of the rate of both the internalization and the recycling of ASOR receptors. The mechanisms responsible for these effects have not yet been elucidated. However, the current findings may be related to the previously observed inhibitory effect of perhexiline on cellular (Na+, K+)-ATPase and Mg++-ATPase activities. Our findings would then reflect insufficient cellular energy production, resulting from depressed ATP hydrolysis in the presence of perhexiline.

Biochemical aspects of alpha-L-fucosidase in hepatocellular carcinoma.

Biochemical characteristics of alpha-L-fucosidase (alpha-L-fucoside hydrolase, EC 3.2.1.51) were studied in tumorous and nontumorous human hepatocellular carcinoma (n = 14). Five parameters were studied: (i) specific activity, (ii) thermostability, (iii) enzyme affinity for an artificial substrate (Km), (iv) isoenzyme patterns of the glycosidase before and after neuraminidase treatment and (v) pH influence on enzyme activity. The specific activity of alpha-L-fucosidase was significantly decreased in tumoral liver when compared to nontumoral liver. The curve of pH activity constantly showed a broad optimum centered near pH 5, whereas two optima were always observed in nontumoral areas. In contrast, there was no modification of the thermostability, the substrate affinity and the isoenzyme patterns of alpha-L-fucosidase in hepatocellular carcinoma.

Pathophysiological variations in the rat liver plasma membrane serine proteinase activity.

The effects of fasting, diabetes, cholestasis, two-third hepatectomy and adrenalectomy on the rat liver plasma membrane serine proteinase activity were studied. Our results show a significant decrease of the enzyme activity during fasting (-50%), during experimental diabetes (-50%), in regenerating liver after partial hepatectomy (-70%) and after extrahepatic cholestasis (-70%). No modifications are noted when the rats are bilaterally adrenalectomized. These findings suggest that the enzyme activity may be linked to the level of circulating insulin, and may be regulated in physiological cellular proliferation so as to prevent undesirable protein degradation.

Inhibition of (Na+,K+)-ATPase and Mg++-ATPase by a lysosomotropic drug: perhexiline maleate.

Human clinical observations and in vivo studies have shown that the amphiphilic drug perhexiline maleate is responsible for lipidosis storage disorders. When the drug was incubated in vivo with rat brain homogenates, the ouabain-sensitive (Na+,K+)-ATPase and the Mg++-ATPase activities were inhibited. 50% inhibition occurred at the drug concentrations 5.10(-5) M for (Na+,K+)-ATPase and at 10(-4) M for Mg++-ATPase, respectively. Kinetic studies performed on rat brain homogenates showed a mixed type inhibition of these enzymes by perhexiline maleate. The effect of other lysosomotropic drugs (imipramine, chlorpromazine, thioridazine and tamoxifen) on (Na+,K+)-ATPase and on Mg++-ATPase activities was found to be similar to that induced by perhexiline maleate. These results indicate that the inhibitory effect of perhexiline maleate on (Na+,K+)-ATPase and Mg++-ATPase may be a common feature shared by the lysosomotropic drugs.

[Biological markers in hepatocellular carcinoma]. / Les marqueurs biologiques du carcinome hépato-cellulaire.

Primary cancer of the liver, especially common in inter-tropical Africa and South-East Asia, still remains inaccessible to a really effective therapy, except for a rapid surgical excision. Improvement of its particularly poor prognosis requires therefore early screening based on reliable biological markers. Following alpha-feto-protein, various parameters have been proposed: enzyme, ferritin, desialylated serum protein, decarboxylated prothrombin... However, alpha-feto-protein remains, in practice, the reference diagnostic test, in spite of a moderate specificity below 500 ng/ml and the fact that it is frequently missing in early cancers. Its diagnostic score may be improved either by the use of monoclonal antibodies, or by determining the ratio of fucosylated form, or by concomitant use of other markers: alpha-L-fucosidase, decarboxy-prothrombin.