Physiological Precursor Research Articles

Characterization of Ligand Binding to the Bifunctional Key Enzyme in the Sialic Acid Biosynthesis by NMR: II. INVESTIGATION OF THE ManNAc KINASE FUNCTIONALITY

N-Acetylmannosamine (ManNAc) is the physiological precursors to all sialic acids that occur in nature. As variations in the sialic acid decoration of cell surfaces can profoundly affect cell-cell, pathogen-cell, or drug-cell interactions, the enzymes that convert ManNAc into sialic acid are attractive targets for the development of drugs that specifically interrupt sialic acid biosynthesis or lead to modified sialic acids on the surface of cells. The first step in the enzymatic conversion of ManNAc into sialic acid is phosphorylation, yielding N-acetylmannosamine-6-phosphate. The enzyme that catalyzes this conversion is the N-acetylmannosamine kinase (ManNAc kinase) as part of the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Here, we employed saturation transfer difference (STD) NMR experiments to study the binding of ManNAc and related ligands to the ManNAc kinase. It is shown that the configuration of C1 and C4 of ManNAc is crucial for binding to the enzyme, whereas the C2 position not only accepts variations in the attached N-acyl side chain but also tolerates inversion of configuration. Our experiments also show that ManNAc kinase maintains its functionality, even in the absence of Mg(2+). From the analysis of the STD NMR-derived binding epitopes, it is concluded that the binding mode of the N-acylmannosamines critically depends on the N-acyl side chain. In conjunction with the relative binding affinities of the ligands obtained from STD NMR titrations, it is possible to derive a structure-binding affinity relationship. This provides a cornerstone for the rational design of drugs for novel therapeutic applications by altering the sialic acid decorations of cell walls.

Mass spectroscopic analysis of phosphatidylinositol synthesis using 6-deuteriated-myo-inositol: comparison of the molecular specificities and acyl remodelling mechanisms in mouse tissues and cultured cells

Mammalian cell PtdIns (phosphatidylinositol) in vivo is enriched in the sn-1-stearoyl 2-arachidonoyl species, the physiological precursor of phosphatidylinositol 4,5-bisphosphate. Mechanisms regulating this specificity are unclear but are typically lost for cells in culture. We used ESI-MS (tandem electrospray ionization-mass spectrometry) to determine the molecular species of PtdIns synthesized by mouse tissues in vivo compared with cultured cells in vitro. After incorporation of deuteriated myo-d(6)-inositol over 3 h, endogenous and newly synthesized PtdIns and lysoPtdIns species were quantified from precursor scans of m/z 241(-) and m/z 247(-) respectively. PtdIns was synthesized as a wide range of species irrespective of the final membrane composition. Analyses of isotope enrichments argued against acyl remodelling as the major regulatory mechanism: composition of the lysoPtdIns pool under all conditions reflected that of either endogenous or newly synthesized PtdIns and was always at equilibrium. The kinetics of PtdIns synthesis, together with the prolonged time scale required for achieving final equilibrium compositions suggest that selective transport between membranes and/or hydrolysis of selected molecular species are the most probable mechanisms regulating compositions of PtdIns and, ultimately, phosphatidylinositol 4,5-bisphosphate.

L-galactono-gamma-lactone dehydrogenase aand vitamin C content in fresh-cut potatoes stored under controlled atmospheres.

L-galactono-gamma-lactone dehydrogenase (GLDH) activity and vitamin C content as ascorbic acid (AA) plus dehydroascorbic acid (DHA) were evaluated in five potato tuber cultivars (Agata, Altesse, Franceline, Manon, and Monalisa). The effect of fresh-cutting and subsequent refrigerated storage of Manon potato under different atmospheres (air, 20% CO(2) + air, 100% N(2), and vacuum packaging) on GLDH activity and vitamin C content was also determined. GLDH from the five potato tuber cultivars showed typical inhibition kinetics by high substrate concentration in the synthesis of AA from its physiological precursor L-galactonic acid-gamma-lactone (GL). GLDH activity was not correlated with the corresponding vitamin C content in any potato tuber cultivar. GLDH from all the cultivars presented a major isoform with isoelectric point (IEP) 5, which changed to IEP = 4.3 after minimal processing. In addition, the GLDH-catalyzed synthesis of AA by the new isoform showed typical Michaelis kinetics, in which the enzyme became more efficient to catalyze the reaction. Whether the change in the isoform pattern was due to either post-translational modifications or de novo synthesis of a new isoenzyme remains unanswered. Fresh-cutting increased GLDH activity from 4.7-fold (vacuum packaging) to 11-fold (air) after 6 days. In addition, 100% of vitamin C content was retained in air and decreased in the rest of atmospheres after this storage period, following the sequence vacuum packaging (89%) > 100% N(2) (78%) > 20% CO(2) + air (63%). This tendency was correlated with the corresponding GLDH activity detected in each storage atmosphere, except in the case of 20% CO(2) + air. Vacuum packaging proved to be the best storage condition, because fresh-cut potatoes did not turn brown and retained 89% of initial vitamin C content.

Nardilysin cleaves peptides at monobasic sites.

Nardilysin (N-arginine dibasic convertase, EC 3.4.24.61) was first identified on the basis of its ability to cleave peptides containing an arginine dibasic pair, i.e., Arg-Arg or Arg-Lys. However, it was observed that an aromatic residue adjacent to the dibasic pair (i.e., Phe-Arg-Lys) could alter the cleavage site. In this study we determined whether nardilysin can cleave peptides at a single basic residue. Nardilysin cleaves beta-endorphin at the monobasic site, Phe(17)-Lys(18), with a k(cat)/K(m) of 2 x 10(8) M(-)(1) min(-)(1). This can be compared to a k(cat)/K(m) of 8.5 x 10(8) M(-)(1) min(-)(1) for cleavage between a dibasic pair in dynorphin B-13. Nardilysin also cleaves calcitonin at His-Arg and somatostatin-14 at Cys-Lys. We examined the hydrolysis of fluorogenic peptides based on the beta-endorphin 12-24 sequence, Abz-T-P-L-V-T-L-X(1)-X(2)-N-A-I-I-K-Q-EDDnp. Nardilysin hydrolyzes the peptides when X(1)-X(2) = F-K, F-R, W-K, M-K, Y-K, and L-K. The kinetics of cleavage at F-K and F-R are similar; however, K-F is not hydrolyzed. Nardilysin cleaves at two monobasic sites M-K and F-R of the kallidin model peptide Abz-MISLMKRPPGFSPFRSSRI-NH(2), releasing desArg(10) kallidin (KRPPGFSPF). However, nardilysin does not release desArg(10) kallidin from the physiological precursor low molecular weight kininogen. These studies extend the range of potential substrates for nardilysin and further substantiate that nardilysin is a true peptidase.

Involvement of 5-hydroxytryptophan in melanogenesis.

Tyrosine is generally considered to be the physiological precursor of melanins and tyrosinase the enzyme responsible. However, recent studies have shown that also peroxidases are involved in the biosynthesis of melanins. These enzymes use hydrogen peroxide to oxidise various phenol substrates. In this paper, we used a substrate other than tyrosine, i.e. 5-hydroxytryptophan, to verify if its peroxidase/H2O2-mediated oxidation gave rise to the formation of melanin. We also subjected 5-hydroxytryptophan to the action of tyrosinase, for comparison purposes. We observed that both enzymes converted this substrate to melanin and that peroxidase, in the presence of hydrogen peroxide, was much more effective than tyrosinase in catalysing the oxidative polymerization of 5-hydroxytryptophan, with the formation of insoluble black melanin-like pigments. Samples deriving from the reaction-substrate enzyme were ultrafiltered at different times through an Amicon ultrafiltration cell equipped with an Amicon Diaflo XM-50 membrane, in order to remove the enzyme, and immediately lyophilised. The resulting samples were analysed by matrix assisted laser desorption/ionisation (MALDI) mass spectrometry, which clearly identified several oligomer species in the reaction mixture. This work was undertaken to investigate the possible precursors of neuromelanin and the enzyme responsible for melanogenesis in brain, since although the central nervous system does not contain tyrosinase, it is rich in peroxidase and hydrogen peroxide.

The effect of walking on regional blood flow and acetylcholine in the hippocampus in conscious rats

Recent studies in our laboratory have demonstrated that stimulation of the septal complex (i.e., the medial septal nucleus and the nucleus of the diagonal band) increases extracellular acetylcholine (ACh) release and, consequently, results in an increase in regional cerebral blood flow in the hippocampus (Hpc CBF) via activation of the nicotinic ACh receptors (nAChRs) [Neurosci. Lett. 107 (1989) 135; Neurosci. Lett. 112 (1990a) 263]. The present study aimed to examine the effects of walking on Hpc CBF, measured by laser Doppler flowmetry, in conscious rats. Walking at a moderate speed (4 cm/s) on a treadmill for 30 s produced increases in Hpc CBF and mean arterial pressure (MAP), reaching 107±1% and 105±1% of the prewalking control values, respectively. Walking for 3 min produced an increase in ACh release in the extracellular space of the hippocampus. The increase in Hpc CBF during walking was attenuated by mecamylamine (abbreviated as MEC here; 2 mg/kg, i.v.), a nAChR antagonist permeable to the blood–brain barrier (BBB), but not by hexamethonium (denoted as C6 here; 20 mg/kg, i.v.), a nAChR antagonist impermeable to the BBB, while the walking-induced increase in MAP was abolished by either agent. The response of Hpc CBF and MAP were not altered by atropine (abbreviated as ATR here; 0.5 mg/kg, i.v.), a muscarinic AChR antagonist permeable to the BBB. The increase in Hpc CBF during walking was attenuated by N ω -nitro- l-arginine methyl ester ( l-NAME, 3 and 30 mg/kg, i.v.), a nitric oxide synthase (NOS) inhibitor, and the reduced responses were reversed following the intravenous (i.v.) administration of a physiological precursor of NO, l-arginine (600 mg/kg). The results suggest that the increase in Hpc CBF during walking is independent of MAP and attributable at least to activation of the nAChRs by the cholinergic vasodilator nerves projecting to the hippocampus and to production of NO in the hippocampus.

Elicitation of sleep-onset REM periods in normal individuals using the sleep interruption technique (SIT)

Use of the sleep interruption technique (SIT) to elicit sleep onset REM periods (SOREMPs) in normal individuals is introduced along with its theoretical bases, empirical findings, and potential applications. Capitalizing upon the circadian and ultradian nature of REM sleep, the SIT has been developed to examine various psychophysiological characteristics related to REM sleep. The SIT allows us to: (1) obtain SOREMPs at the discretion of the researcher; (2) avoid the contaminating effects of preceding non-REM (NREM)-REM stage ordering on subsequent target sleep episodes; and (3) obtain many REM episodes in a short time by repeating the sleep interruptions. The SIT has been applied in several studies, such as examination of physiological precursors to REM periods, correlates of dream mechanisms, and induction of sleep paralysis in normal individuals. Guidelines for eliciting SOREMP using the SIT, including the parameters to be manipulated, are provided, e.g. NREM duration before sleep interruption, time of night of awakenings, duration of sleep interruption and tasks employed. Directions for further research such as determining optimal type of task to promote SOREMP occurrences, generalization of SOREMP as usual REM periods, and forms of SOREMP occurrences under different conditions in normal individuals and clinical patients are discussed. Finally, possible future uses of the SIT, including combining this technique with new technologies, are also suggested.

Biochemical engineering of the acyl side chain of sialic acids stimulates integrin-dependent adhesion of HL60 cells to fibronectin.

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis of several diseases. The physiological precursor of all sialic acids is N-acetyl- D-mannosamine. The N-acyl side chain of sialic acid can be modified by exposure of cells to synthetic N-acyl-modified D-mannosamines. In a new experimental approach cells were cultivated in the presence of N-propanoyl- D-mannosamine. This unnatural precursor of sialic acid is taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acid in vitro and in vivo. Here we report on the biological consequences of the incorporation of the unnatural N-propanoylneuraminic acid into glycoconjugates of HL60 cells. Biochemical engineering of the acyl side chain of neuraminic acids activates beta(1)-integrins (VLA4 or VLA5), resulting in an increased adhesion of HL60 cells to fibronectin.

Induction of Atypical Ductal Hyperplasia in Mouse Mammary Gland Organ Culture

Induction of Atypical Ductal Hyperplasia in Mouse Mammary Gland Organ Culture

Cytokinin and its precursors for improving growth and yield of rice

Phytohormones are well known for their regulatory role in plant growth and development and cytokinins represent one of the five well-established classes of phytohormones. A field experiment was conducted to evaluate the effect of a synthetic cytokinin (kinetin) and its physiological precursors (adenine+isopentyl alcohol) on growth and yield of rice. Five levels (10 −2–10 −6 M) of each, kinetin and adenine (ADE) plus isopentyl alcohol (IA) were used, in addition to an untreated control. Treatments were applied by dipping roots of rice seedlings in solutions of cytokinin or its precursors for an hour just before transplanting. The precursors were more effective in comparison with pure cytokinin. The plant height was increased by 6.5% over control when treated with 10 −5 M ADE+IA. The application of ADE+IA at the rate of 10 −4 M significantly increased the number of tillers (34.7%), number of panicles (38.5%) and paddy yield (21.6%) compared to control. Moreover, nutrient (NPK) contents were also significantly improved by 83.1, 36.4 and 26.0% in straw and 53.2, 27.7 and 10.8% in grain, respectively, as compared to control. The findings support the hypothesis that exogenous supply of cytokinin or its precursors in the root zone could improve the growth and yield of treated plant. However, it is difficult to conclude in this study that the precursors were metabolized into cytokinins outside the roots by rhizosphere/rhizoplane microorganisms before being absorbed by the plant roots or converted into cytokinins within the plant tissues. The greater effectiveness of precursors compared to kinetin could be attributed to a gradual conversion of ADE plus IA into cytokinins resulting in a continuous supply over a period of time.

Biochemical engineering of the N-acyl side chain of sialic acid: biological implications.

N-Acetylneuraminic acid is the most prominent sialic acid in eukaryotes. The structural diversity of sialic acid is exploited by viruses, bacteria, and toxins and by the sialoglycoproteins and sialoglycolipids involved in cell-cell recognition in their highly specific recognition and binding to cellular receptors. The physiological precursor of all sialic acids is N-acetyl D-mannosamine (ManNAc). By recent findings it could be shown that synthetic N-acyl-modified D-mannosamines can be taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acids in vitro and in vivo. Successfully employed D-mannosamines with modified N-acyl side chains include N-propanoyl- (ManNProp), N-butanoyl- (ManNBut)-, N-pentanoyl- (ManNPent), N-hexanoyl- (ManNHex), N-crotonoyl- (ManNCrot), N-levulinoyl- (ManNLev), N-glycolyl- (ManNGc), and N-azidoacetyl D-mannosamine (ManNAc-azido). All of these compounds are metabolized by the promiscuous sialic acid biosynthetic pathway and are incorporated into cell surface sialoglycoconjugates replacing in a cell type-specific manner 10-85% of normal sialic acids. Application of these compounds to different biological systems has revealed important and unexpected functions of the N-acyl side chain of sialic acids, including its crucial role for the interaction of different viruses with their sialylated host cell receptors. Also, treatment with ManNProp, which contains only one additional methylene group compared to the physiological precursor ManNAc, induced proliferation of astrocytes, microglia, and peripheral T-lymphocytes. Unique, chemically reactive ketone and azido groups can be introduced biosynthetically into cell surface sialoglycans using N-acyl-modified sialic acid precursors, a process offering a variety of applications including the generation of artificial cellular receptors for viral gene delivery. This group of novel sialic acid precursors enabled studies on sialic acid modifications on the surface of living cells and has improved our understanding of carbohydrate receptors in their native environment. The biochemical engineering of the side chain of sialic acid offers new tools to study its biological relevance and to exploit it as a tag for therapeutic and diagnostic applications.

Ruthenium(III) readily abstracts NO from L-arginine, the physiological precursor to NO, in the presence of H2O2. A remarkably simple model system for NO synthases.

Reaction of [Ru(Hedta)Cl]- with H2O2 in the presence of arginine, produces NO, in the form of an Ru(II)-(NO+) complex and citrulline which is a remarkably simple model system for the physiological NO synthase reaction.

Degradation mechanism and stability of 5‐aminolevulinic acid

The physiological substance and precursor of the heme synthesis 5-aminolevulinic acid (ALA) is a promising prodrug for photodiagnosis and photodynamic therapy of epithelial tumors, particularly in urological and gynecological tissues. For the clinical use of this substance, a chemically stable and sterile drug formulation is required. In the present study, degradation mechanism of ALA in aqueous solution and possibilities to improve its stability were examined. A capillary electrophoretic method was developed that was suitable for the quantification of ALA and of two degradation products. The intermediate degradation product was 2, 5-dicarboxyethyl-3,6-dihydropyrazine, which was further oxidized to 2,5-dicarboxyethylpyrazine. The structures of the degradation products were proven by (1)H and (13)C nuclear magnetic resonance spectroscopy. ALA degradation was very efficiently inhibited by adjusting the pH of the aqueous solution to a value <5 and by purging with nitrogen. Additives such as antioxidants did not improve the ALA stability. These results demonstrated that low pH ALA aqueous solution may be one possible dosage form to be considered for market introduction.

Degradation mechanism and stability of 5‐aminolevulinic acid

The physiological substance and precursor of the heme synthesis 5‐aminolevulinic acid (ALA) is a promising prodrug for photodiagnosis and photodynamic therapy of epithelial tumors, particularly in urological and gynecological tissues. For the clinical use of this substance, a chemically stable and sterile drug formulation is required. In the present study, degradation mechanism of ALA in aqueous solution and possibilities to improve its stability were examined. A capillary electrophoretic method was developed that was suitable for the quantification of ALA and of two degradation products. The intermediate degradation product was 2,5‐dicarboxyethyl‐3,6‐dihydropyrazine, which was further oxidized to 2,5‐dicarboxyethylpyrazine. The structures of the degradation products were proven by 1H and 13C nuclear magnetic resonance spectroscopy. ALA degradation was very efficiently inhibited by adjusting the pH of the aqueous solution to a value <5 and by purging with nitrogen. Additives such as antioxidants did not improve the ALA stability. These results demonstrated that low pH ALA aqueous solution may be one possible dosage form to be considered for market introduction. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 1335–1341, 2000

Increased endothelium--monocyte interactions in salt-sensitive hypertension: effect of L-arginine.

Although adhesion of monocytes to endothelial cells is considered as one of the initial factors leading in the long term to the development of atherosclerosis, the effects of hypertension on monocyte-endothelial cell interactions are still largely unknown. Thus we evaluated whether hypertension affects adhesion of monocytes on rat carotid endothelium, and whether this adhesion may be modified by long-term treatment with L-arginine, the physiologic precursor of nitric oxide (NO). Hypertension was induced in Dahl rats using a sodium-rich diet (8%), in the absence or the presence of L-arginine (1.25 g/L in drinking water). After 1 month, the carotid arteries were isolated, opened longitudinally, and incubated in the presence of 2 x 10(6) monocytes previously rendered fluorescent by incubation with tetramethyl rhodamine isothiocyanate (TRITC), and adherent cells were counted under fluorescence microscopy. In parallel, the production of NO was evaluated in vitro in isolated aorta and isolated hearts. Hypertension markedly increased adhesion of monocytes on carotid endothelium, and this was reduced by L-arginine. Hypertension also reduced an index of NO release at the level of the aorta and the coronary circulation. This impaired release of NO was partially prevented by L-arginine. Thus hypertension was associated with an increased adhesion of monocytes, which is probably due at least in part to a decreased production of NO. The increased adhesion was partly reduced by L-arginine, possibly secondary to an increased production of NO. Such an increased adhesion of monocytes may contribute the increased cardiovascular risk in hypertension.

Effects of L-arginine on Vascular Smooth Muscle Cell Proliferation and Apoptosis after Balloon Injury

Nitric oxide (NO) inhibits neointimal formation in experimental models of restenosis, but the mechanisms have not been fully elucidated. This study examined whether the beneficial effect of L-arginine, the physiological NO precursor, was associated with alteration of the apoptotic and proliferative activities of vascular smooth muscle cells (VSMCs) in the vessel wall after arterial injury. Balloon injury was performed in the rat carotid-artery injury model. Rats were treated with L-arginine (2.25% in the drinking water) or normal drinking water, and sacrificed at 1, 2 and 14 days postinjury. Apoptosis was assessed by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling (TUNEL), and proliferation by proliferating cell nuclear antigen (PCNA) immunohistochemistry. Treatment with L-arginine increased the luminal area at 14 days postinjury (0.26 +/- 0.03 mm2 vs 0.14 +/- 0.04 mm2; p < 0.05), and this effect was attributable to a reduction in neointimal formation (0.11 +/- 0.03 mm2 vs 0.23 +/- 0.04 mm2; p < 0.05), while L-arginine did not affect vascular remodeling, as indicated by the total vessel area. The decreased neointimal area at 14 days after balloon injury contained a reduced percentage of TUNEL positive (0.1 +/- 0.1% vs 2.0 +/- 0.6%; p < 0.05), and PCNA positive (13.0 +/- 2.6% vs 27.2 +/- 5.9%; p < 0.05) nuclei, respectively. L-arginine did not influence the apoptotic or proliferative activities of VSMCs at earlier time points postinjury. The favourable effect of L-arginine in the rat model of arterial injury is associated with inhibition of VSMC proliferative activity in the vessel wall and is not explained by increased VSMC apoptosis.

Neuroendocrine, immunologic, and microvascular systems interactions in rheumatoid arthritis: physiopathogenetic and therapeutic perspectives

Objective: To review the “core” systems interactions in rheumatoid arthritis (RA): neuroendocrine, immunologic, and microvascular, and to interpret an integrated physiopathogenesis of the disease, beginning at a preclinical phase of risk factors to the later stages of manifest clinical inflammation. Methods: Publications on stress reactions, serum hormonal levels, biological mediators of inflammation and vascular alterations in RA during its preclinical phase, course of active disease, including pregnancy, and hormonal therapy of active disease were retrieved. In addition, experimental reports on biological models of the disease were considered. Levels of adrenal and gonadal steroids (ie, glucocorticosteroids [GCS], dehydroepiandrosterone [DHEA], its sulfate [DHEAS], estradiol [EZ], and testosterone [T]), as well as prolactin (PRL) and other hormones, biological mediators, vascular endothelial system (VES) interactions with hormones, and immunologic mediators of inflammation in RA, were reviewed and interpreted. Results: Women with premenopausal onset of RA not previously treated with GCS had lower basal serum levels of adrenal androgens, that is, DHEA or DHEAS, both before and after onset of clinical disease, compared with controls. Risk factors, including hormonal, immunologic, and hereditary indicators, were found to be uniformly present many years before clinical onset in such younger women, as compared with a frequency of circa 15% in matched controls. Also, a history of heavy cigarette smoking significantly predicted the onset of RA in perimenopousal women, and in men, suggesting that vascular endothelial alterations predispose to the disease. In the same prospective study, 1 or more of 4 risk factors were present an average of 12 years before clinical onset of disease in 83% of male RA cases versus 26% in matched controls (ie, sensitivity of 83% and specificity of 74%). Early RA patients may have lower serum cortisol levels than normal controls, and less than expected for the degree of ongoing inflammation, as wel as having upregulated PRL levels. Conclusions: Among persons genetically prone to RA, the “core” systems are hypothesized to become “remodeled” during a long preclinical phase as a result of chronic imbalances in their interactive homeostasis. This hypothesis needs to be critically assessed in further studies of such physiological precursors of disease as well as stressors in the development and course of RA. Optimal hormonal management of biological mediators of RA is also a priority challenge for disease control in the future. Relevance: Evidence indicates that men and women who are susceptible topremenopausal onset of RA can each be identified long before their clinical onsets of disease, and that productive research in primary preventions is an achievable objective. Disease prevention objectives are central in the public health strategy of the National Arthritis Action Plan and of the US Public Health Service “Healthy People 2000” (and 2010 proposed). Success in such prevention goals can be expected to significantly reduce the enormous burden of this common disease, which affects all segments of the population.

THE USE OF L-ARGININE DURING LIVER PROCUREMENT FROM NON HEART BEATING DONOR PIGS. HISTOLOGICAL EVALUATION.

207 We have previously seen using normothermic recirculation (NR) with cardiopulmonary bypass (CPB) in non heart beating donors (NHBD) that liver transplantation (Ltx) was feasible. However after 40 minutes of cardiac arrest (CA) although the preservation of the hepatocyte function was possible, the specific sensibility of the biliary tract to ischemic injury burdened long term survival. L-arginine a physiological nitric oxide precursor has shown to be effective in reducing the ischemia-reperfusion injury in some experimental perfused liver models. AIM. To evaluate the effect of L-arginine on survival and histological changes in liver transplantation from NHBD pigs after 40 minutes of CA. MM. 25 out-bread weanling pigs (25-30 kg) were transplanted with an allograft from NHBD in which CA was produced by injection of KCl. After 40 minutes of WIT, extracorporeal circulation and tissue oxigenation at 37°C was used during 30 minutes of NR. Afterwards the pig was gradually cooled until a temperature of 15°C. Thereafter liver procurement was completed with UW perfusion and liver transplantation was performed after 6 hours of cold ischemia in the standard manner. Two groups were designed: LA group (n=10) treated with L-Arginine (400 mg/kg) during NR; and a control group (n=15). Liver biopsies were taken in the donor at the beginning, at the end of NR, and in the recipient before and one hour after reperfusion. On postoperative day 5 (5D) the animals were sacrifized and a liver segment was taken. All specimens were evaluated by light microscopy. At 5D the necrotic area percentage was calculated by morphometric analysis. RESULTS. During liver procurement and Ltx, sinusoidal inflammation and dilatation, congestion and hepatocellular vacuolization were more severe in the control group. Biopsies taken at 5D showed: (Table)TableCONCLUSIONS. The use of L-arginine during liver procurement from NHBD by using NR increases survival and significantly ameliorates the histological damage produced after warm ischemia preserving the biliary tract.

Biochemical Engineering of Neural Cell Surfaces by the SyntheticN-Propanoyl-substituted Neuraminic Acid Precursor

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration, and pathogenesis of diseases. During times of intense plasticity within the nervous system, such as development and regeneration, sialylation of neural cells is distinct from the time of its maintenance. In this study, a synthetic precursor of neuraminic acid, N-propanoylmannosamine (N-propanoyl neuraminic acid precursor (P-NAP)), is applied to the culture medium of oligodendrocyte progenitor cells, microglia, astrocytes, and neurons from neonatal rat brains to alter sialylation of glycoconjugates within these cells. P-NAP is metabolized and incorporated as N-propanoyl neuraminic acid into glycoproteins of the cell membrane. P-NAP stimulates the proliferation of astrocytes and microglia but not of oligodendrocyte progenitor in vitro. However, P-NAP increases the number of oligodendrocyte progenitor cells expressing the early oligodendroglial surface marker A2B5 epitope. In the presence of P-NAP, cerebellar neurons (but not astrocytes) in microexplant cultures start to express the oligodendroglial progenitor marker A2B5 epitope, which is normally undetectable on these cells. The controls, which were performed in the absence of any additive or in the presence of the physiological precursor of neuraminic acid, N-acetylmannosamine, did not show any increase in A2B5 expression.

Hypocholesterolemic properties of nitric oxide. In vivo and in vitro studies using nitric oxide donors

Previous results suggested that changes in the activity of nitric oxide (NO) can influence metabolism of apo B-containing lipoproteins. Therefore, we studied effects of exogenous NO donors and physiological NO precursors on metabolism of these lipoproteins. In rabbits, addition of 0.03% sodium nitroprusside (NaNP) to a semipurified, cholesterol-free, casein diet counteracted the elevation of LDL cholesterol induced by this diet but did not alter liver lipids after 4 weeks of feeding. In HepG2 cells, addition of nontoxic concentrations of another NO donor, S-nitroso- N-acetylpenicillamine (SNAP) to culture medium caused a dose-dependent reduction of medium apo B after 24 h. At the concentration 0.5 mM, SNAP significantly decreased medium apo B by 50% without altering total synthesis and secretion of proteins and without altering rates of cellular sterol synthesis. In cells incubated with l-arginine, reduction of medium apo B was not associated with increased NO production whereas in those exposed to N–OH–Arg medium apo B levels were not altered. We concluded that synthetic NO donors can reduce hypercholesterolemia by affecting apo B metabolism directly in the liver, via the sterol-independent mechanism.