Economic and environmental effects of the manure policy in The Netherlands: synthesis of integrated ex-post and ex-ante evaluation.

This paper summarises the results of both an ex-post evaluation of the Dutch Mineral Accounting System (MINAS) and an ex-ante evaluation of the effect of different levy-free surplus values. The MINAS system has been introduced in 1998 in order to reduce nitrate and phosphate leaching from agricultural soils. MINAS resulted in a reduction of the N surplus on dairy farms of approximately 50 kg ha(-1) to limited or no costs to the farms involved. MINAS resulted in higher costs for manure removal for intensive livestock farmers. Though emissions of N and P have decreased significantly during the last five years effects of this reduction in environmental quality cannot be observed, except for a small decrease in nitrate concentration of the upper groundwater. The ex-ante evaluation of different variants for possible future levy-free surplus levels indicated that under the lowest variant, the nitrate concentration in groundwater will exceed 50 mg per litre on 14% of the area. The environmental effect of the different variants for P were not distinguished. The lowering of the levy-free surplus for P will have a drastic effect on the intensive livestock farms. The incorporation of fertiliser P under the MINAS system would be a cheap option to reduce the P surplus.

Irradiated versus nonirradiated endothelial cells: effect on proliferation of vascular smooth muscle cells.

PURPOSE: Endovascular radiation therapy is a promising strategy for the prevention of restenosis. Radiation prevents proliferation of vascular smooth muscle cells, thereby reducing the incidence of restenosis, but may also affect the remaining endothelial cells. For this reason, a comparison was made between irradiated and nonirradiated endothelial cells and their effects on the proliferation of vascular smooth muscle cells in a coculture system was evaluated. MATERIALS AND METHODS: A coculture system was used, in which both endothelial cells and vascular smooth muscle cells were grown on opposite sides of a semipermeable membrane. After a period of growth arrest, the proliferation of vascular smooth muscle cells was measured during four subsequent days. RESULTS: The presence of endothelial cells stimulated the proliferation of vascular smooth muscle cells during the first days of analysis but had an inhibitory effect during the subsequent days (P <.5). gamma-irradiation of endothelial cells resulted in a complete blockage of the proliferation of these cells. However, irradiated endothelial cells affected the proliferation of vascular smooth muscle cells in coculture in a fashion comparable to nonirradiated endothelial cells (P >.5). CONCLUSION: The results suggest that, in endovascular radiation therapy, irradiation of endothelial cells does not change their effects on the proliferative behavior of vascular smooth muscle cells.

Human plasma phospholipid transfer protein increases the antiatherogenic potential of high density lipoproteins in transgenic mice.

Plasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoprotein particles and alters high density lipoprotein (HDL) subfraction patterns in vitro, but its physiological function is poorly understood. Transgenic mice that overexpress human PLTP were generated. Compared with wild-type mice, these mice show a 2.5- to 4.5-fold increase in PLTP activity in plasma. This results in a 30% to 40% decrease of plasma levels of HDL cholesterol. Incubation of plasma from transgenic animals at 37 degrees C reveals a 2- to 3-fold increase in the formation of pre-beta-HDL compared with plasma from wild-type mice. Although pre-beta-HDL is normally a minor subfraction of HDL, it is known to be a very efficient acceptor of peripheral cell cholesterol and a key mediator in reverse cholesterol transport. Further experiments show that plasma from transgenic animals is much more efficient in preventing the accumulation of intracellular cholesterol in macrophages than plasma from wild-type mice, despite lower total HDL concentrations. It is concluded that PLTP can act as an antiatherogenic factor preventing cellular cholesterol overload by generation of pre-beta-HDL.

Gp96/GRP94 is a putative high density lipoprotein-binding protein in liver.

We have previously shown that three high density lipoproteins (HDL)-binding proteins in liver, of 90, 110 and 180 kDa, are structurally related. In this study, these proteins are identified as gp96/GRP94. This protein is known to occur as a homodimer and has a dual subcellular localization: it is both an endoplasmic reticulum resident protein, where it is supposed to act as a chaperonin, and a plasma membrane protein, whose significance is unknown. In ultrastructural studies the plasma membrane localization of the homodimeric form was verified. The 90-kDa protein was abundantly present at the membranes of the endosomal/lysosomal vesicles as well as at the apical hepatocyte membranes, comprising the bile canaliculi. The monomeric protein is scarcely present at the basolateral membrane of the hepatocytes, but could be demonstrated in coated pits, suggesting involvement in receptor-mediated endocytosis. Labeling of the endoplasmic reticulum was virtually absent. Gp96/GRP94 was transiently expressed in COS-1 cells. However, the expressed protein was exclusively localized in the endoplasmic reticulum. Transfection with constructs in which the C-terminal KDEL sequence had been deleted, resulted in plasma membrane localized expression of protein, but only in an extremely low percentage of cells. In order to evaluate the HDL-binding capacities of this protein, stably transfected cells were generated, using several cell types. It appeared to be difficult to obtain a prolonged high level expression of gp96. In these cases, however, a marked increase of HDL-binding activity compared with the control cells could be observed.

Seeding of intravascular stents by the xenotransplantation of genetically modified endothelial cells.

A novel approach of cell seeding of stents using xenotransplanted endothelium is proposed. The advantages of this approach are that these doubly transgenic animals will provide a limitless supply of endothelial cells producing controllable levels of active compound. These foreign cells will act as Trojan horses, graciously accepted at face value by the host organism, but capable of modifying the pathophysiological response to vessel damage, typified by the process of restenosis. Once implanted, the production of the bioactive compound is under exogenous control by means of 'designer' genes coding for modified cell surface receptors, which are introduced with the transgene to provide controllable levels of compound. Interaction of an orally administered compound with the modified cell receptor will switch on the transgene, while in its absence the transgene remains dormant. We have been able to show the feasibility this type of approach has for other animal species, and it shows great potential for application to humans.

Structural relation between HDL-binding proteins in porcine liver.

We have found strong evidence for a relation between three high-density lipoprotein (HDL)-binding proteins of 90, 110, and 180 kDa in porcine liver that were detected by ligand blotting. Because HDL-binding proteins with identical molecular masses were detected in human liver, all subsequent experiments were performed with porcine liver proteins. An antiserum raised against a highly purified preparation of the 90-kDa HDL-binding protein, designated 90-PC, showed cross-immunoreactivity with the 110- and 180-kDa HDL-binding proteins. Purified protein preparations of the 90-, 110-, and 180-kDa HDL-binding proteins were obtained and analyzed by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. Under nonreducing conditions these preparations showed protein bands with the expected molecular masses. Reduction of these preparations resulted in protein bands of 90 kDa. Ligand blotting experiments with 125I-HDL showed protein bands of 90, 110, and 180 kDa under nonreducing conditions and a 90-kDa protein band in all three preparations under reducing conditions. Immunoblotting experiments with 90-PC antiserum resulted in a similar pattern. The three protein preparations were then subjected to cyanogen bromide cleavage and the resulting peptides separated on gel. Immunoblotting with the 90-PC antibody revealed a pattern of protein bands that was remarkably similar in all three protein preparations. Immunohistochemical localization studies with the 90-PC antibody showed that the HDL-binding proteins were present both at the borders of the sinusoids as well as within the hepatocellular plates.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution and characterization of HNK-1 antigens in the developing avian heart.

The heart originates from splanchnic mesoderm and to a lesser extent from neural crest cells. The HNK-1 monoclonal antibody is a marker for early migrating neural crest cells, but reacts also with structures which are not derived from the neural crest. We investigated whether heart structures are HNK-1 positive before neural crest cells colonize these target tissues. To that end, we determined the HNK-1 antigen expression in the developing avian heart on immunohistochemical sections and on Western blots. The HNK-1 immunoreactivity in the developing chick heart is compared with data from literature on the localization of neural crest cells in chick/quail chimeras. Structures with neural crest contribution, including parts of the early outflow tract and the related endocardial cushions, the primordia of the semilunar valve leaflets and the aorticopulmonary septum were HNK-1 positive. Furthermore, other structures were HNK-1 positive, such as the atrioventricular cushions, the wall of the sinus venosus at stage HH 15 through 21, parts of the endocardium at E3, parts of the myocardium at E6, and the extracellular matrix in the myocardial base of the semilunar valves at E14. HNK-1 expression was particularly observed in morphologically dynamic regions such as the developing valves, the outflow tract cushion, the developing conduction system and the autonomic nervous system of the heart. We observed that atrioventricular endocardial cushions are HNK-1 positive. We conclude that: a HNK-1 immunoreactivity does not always coincide with the presence of neural crest cells or their derivatives; (2) the outflow tract cushions and atrioventricular endocardial cushions are HNK-1 positive before neural crest cells are expected (stage HH 19) to enter the endocardial cushions of the outflow tract; (3) the observed spatio-temporal HNK-1 patterns observed in the developing heart correspond with various HNK-1 antigens. Apart from a constant pattern of HNK-1 antigens during development, stage-dependent HNK-1 antigens were also found.

Regional differences between various axial segments of the avian neural crest regarding the formation of enteric ganglia.

The vagal neural crest adjacent to somites 1-7 gives rise to the enteric ganglia along the entire digestive tract. It is generally assumed that formation of enteric ganglia in preumbilical gut is independent of the axial segment from which the neural crest originates. In post-umbilical gut, however, there is evidence that the axial segment of origin of the neural crest might be relevant to neural differentiation. In this part of the gut, we previously identified a subpopulation of HNK-1-immunoreactive cells within the enteric mesenchyme. This immunoreactivity disappeared upon formation of the enteric nervous system. We studied the interaction between various axial segments of quail neural crest and the microenvironment in a neural chicken hindgut using chorioallantoic membrane cocultures. We found that neural crest cells from various axial segments could migrate into the gut and home to the correct sites. However, whereas vagal neural crest cells differentiated into enteric neurons, neural crest cells from truncal segments mainly differentiated into melanocytes. The HNK-1-immunoreactivity within the enteric mesenchyme only disappeared when neural crest cell colonization was followed by differentiation into enteric neurons and subsequent formation of enteric ganglia. To determine whether differentiation of neural crest cells in chorioallantoic membrane cocultures was influenced by the prolonged presence of the neural tube and notochord, we developed a new coculture system, using neural crest cells cultured in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Colonization characteristics of enteric neural crest cells: embryological aspects of Hirschsprung's disease.

This study explores the development of the enteric nervous system in avian embryos. Particular emphasis was given to colonization characteristics of neural crest cells present in primitive enteric ganglia. By coculturing neuronal and aneuronal gut of quail and chicken embryos, we investigated if and when neural crest cells in primitive enteric ganglia could detach from these ganglia, migrate, and colonize adjacent chicken gut. Quail neural crest cells were identified using the quail nucleolar marker and the HNK-1 antibody. Enteric neurons were identified using three monoclonal antibodies directed against neurofilament proteins. We found that neural crest cells detached from primitive ganglia in neuronal quail gut from E6 till E9, whereas neural crest cells did not leave enteric ganglia from E10 gut. These observations show that there is a transient phase during which enteric neural crest cells can leave the gut. To determine whether neural crest cells could colonize neuronal gut we cocultured neuronal gut or the neural primordium and neuronal chicken gut (E11). We found that quail neural crest cells do not colonize neuronal E11 gut, whereas they do colonize aneuronal gut of the same age. We suggest that aneuronal gut attracts neural crest cells by diffusing factors.

Characterization of HNK-1 antigens during the formation of the avian enteric nervous system.

During vertebrate embryogenesis, interaction between neural crest cells and the enteric mesenchyme gives rise to the development of the enteric nervous system. In birds, monoclonal antibody HNK-1 is a marker for neural crest cells from the entire rostrocaudal axis. In this study, we aimed to characterize the HNK-1 carrying cells and antigen(s) during the formation of the enteric nervous system in the hindgut. Immunohistological findings showed that HNK-1-positive mesenchymal cells are present in the gut prior to neural crest cell colonization. After neural crest cell colonization this cell type cannot be visualized anymore with the HNK-1 antibody. We characterized the HNK-1 antigens that are present before and after neural crest cell colonization of the hindgut. Immunoblot analysis of plasma membranes from embryonic hindgut revealed a wide array of HNK-1-carrying glycoproteins. We found that two HNK-1 antigens are present in E4 hindgut prior to neural crest cell colonization and that the expression of these antigens disappears after neural crest colonization. These two membrane glycoproteins, G-42 and G-44, have relative molecular masses of 42,000 and 44,000, respectively, and they both have isoelectric points of 5.5 under reducing conditions. We suggest that these HNK-1 antigens and the HNK-1-positive mesenchymal cells have some role in the formation of the enteric nervous system.

Regulation of chloride transport in cultured normal and cystic fibrosis keratinocytes.

Cultured normal (N) cystic fibrosis (CF) keratinocytes were evaluated for their Cl(-)-transport properties by patch-clamp-, Ussing chamber- and isotopic efflux-measurements. Special attention was paid to a 32 pS outwardly rectifying Cl- channel which has been reported to be activated upon activation of cAMP-dependent pathways in N, but not in CF cells. This depolarization-induced Cl- channel was found with a similar incidence in N and CF apical keratinocyte membranes. However, activation of this channel in excised patches by protein kinase (PK)-A or PK-C was not successful in either N or CF keratinocytes. Forskolin was not able to activate Cl- channels in N and CF cell-attached patches. The Ca(2+)-ionophore A23187 activated in cell-attached patches a linear 17 pS Cl- channel in both N and CF cells. This channel inactivated upon excision. No relationship between the cell-attached 17 pS and the excised 32 pS channel could be demonstrated. Returning to the measurement of Cl- transport at the macroscopic level, we found that a drastic rise in intracellular cAMP induced by forskolin did in N as well as CF cells not result in a change in the short-circuit current (Isc) or the fractional efflux rates of 36Cl- and 125I-. In contrast, addition of A23187 resulted in an increase of the Isc and in the isotopic anion efflux rates in N and CF cells. We conclude that Cl(-)-transport in cultured human keratinocytes can be activated by Ca2+, but not by cAMP-dependent pathways.

High density lipoprotein-binding proteins in porcine liver. Isolation and histological localization.

The antiatherogenic properties of high density lipoproteins (HDLs) are thought to reside in their involvement in the reverse cholesterol transport pathway. Specific HDL-binding proteins could play a key role in this process. Two HDL-binding proteins of approximately 90 and 180 kd were identified in porcine liver by ligand blotting and were purified to apparent homogeneity by a combination of protein extraction, DEAE-cellulose chromatography, Con A-Sepharose chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding of 125I-HDL by these proteins could be actively competed for by unlabeled HDL but not by low density lipoprotein. Polyclonal antisera have been raised against these two proteins. Each antiserum recognized only one of the HDL-binding proteins, indicating that they are not immunologically related. Moreover, striking differences in localization were observed in immunohistochemical studies. The 90-kd protein is located within the hepatocellular plates, while the 180-kd protein is present along the lining of the sinusoids. These results suggest functional differences between the two HDL-binding proteins described.

Cultured epithelial cells from patients with cystic fibrosis have an increased expression of the 14 kDa Ca2(+)-binding protein CFA.

The Cystic Fibrosis antigen (CFA) is a 14 kDa. Ca2(+)-binding protein known to be expressed in cells of myeloid origin during normal cell differentiation. CFA serum levels are elevated in Cystic Fibrosis (CF) patients and heterozygotes. We examined the expression of CFA in different cultured epithelial cells from controls and patients with CF. The steady state level of CFA was in general higher in epithelial cells from CF patients compared to control cells and was found to increase during cell aging. The latter difference could be attributed to an increased rate of CFA synthesis rather than to an impairment of CFA degradation or secretion, as shown by pulse chase experiments.

Formation and malformation of the enteric nervous system in mice: an organ culture study.

Despite some progress in the treatment of congenital malformations of the enteric nervous system, there is no knowledge about the pathogenesis. The study of the normal formation of the enteric nervous system is hampered by the difficulty of manipulating and culturing mammalian embryos and their organs. Three methods to culture bowel explants of murine embryos, (chorioallantoic membrane grafting, organotypic tissue culture, and renal subcapsular space grafting) were compared. The three-dimensional cytoarchitecture of the bowel developed almost normally in the renal subcapsular space cultures. Using this culture system, it was found that neural crest cells colonize the murine bowel in distinct phases. The distal bowel was colonized at the 13th day of development. In a spontaneous mouse mutant model for intestinal aganglionosis, the lethal spotted mouse, the colonization of the distal 2 mm of the bowel did not occur at E13.

Chloride transport in cultured nasal epithelium of cystic fibrosis patients.

In this study, nasal polyp epithelial cells from control and cystic fibrosis (CF) patients were cultured using a method which allows multiple passages. The cells were tested in Ussing chamber experiments to study transcellular ion transport. Cultured CF nasal polyp cells did not exhibit spontaneous transcellular chloride transport in the presence of amiloride, in contrast to normal cells. Forskolin increased the short circuit current (Isc) in control but not CF cells. Forskolin and isoproterenol increased the cAMP levels in control and CF cells. Histamine, bradykinin and isoproterenol transiently increased the intracellular calcium level and caused a parallel increase of the transcellular chloride current in both normal and CF cells. The transient effects of isoproterenol were not sensitive to the beta blocker atenol and could not be mimicked by forskolin. We conclude that in cultured nasal polyp cells a difference in chloride transport activity between CF and control cells is retained following multiple passages. Our results suggest that the active state of chloride channels in nasal polyp cells does not require activation of a second messenger pathway. This apparently spontaneous activity appears to be reduced in CF cells. The calcium- but not the cAMP-dependent activation of transepithelial chloride secretion is at least partially preserved in cultured CF airway epithelium.

Immortalization of nasal polyp epithelial cells from cystic fibrosis patients.

We have developed immortalized epithelial cystic fibrosis (CF) cell lines by infecting cultured nasal polyp cells with a SV40/Adenol2 hybrid virus. The cell lines obtained are epithelial in nature as shown by cytokeratin production and morphology, although cytokeratins 4 and 13 typical of primary nasal polyp cells are produced at a much reduced rate. Ussing chamber experiments showed that the precrisis CF cell line NCF3 was able to perform trans-cellular chloride transport when activated by agents which elevate intracellular calcium. cAMP agonists had no effect on chloride flux in NCF3 as expected for CF cells. The apical chloride channels found with the patch clamp technique in NCF3 and in the postcrisis cell line NCF3A have a conductance similar to that of chloride channels found earlier in normal and CF epithelial cells. The channels show a delay in the onset of activity in off-cell patches and are not activated by increased cAMP levels in the cell. This indicates that immortalized CF epithelial cells will provide a useful model for the study of cystic fibrosis.

A model for aganglionosis in the chicken embryo.

We investigated the ability of neural crest (NC) cells to colonize hindgut, which had remained aneuronal due to bowel transection in ovo at an early stage. The fact that the bowel remained aneuronal proved that the "sacral" NC does not provide precursor cells for enteric neurons in the hindgut. HNK-1 immunostaining of aneuronal hindgut revealed cell-free, ganglionic structures at the site of the myenteric plexus and a sub-population of mesenchymal cells in the submucosa. We cocultured this particular type of aneuronal bowel with the vagal neural anlage of either quail or chick embryos on the chick chorioallantoic membrane. After 1 week coculture, it appeared that NC cell colonisation of the hindgut had taken place, although there was a difference between the quail-chick and chick-chick model. Quail NC cells had given rise to submucous plexuses and some myenteric plexuses. Chick NC cells had only colonised the submucous region. These findings indicate that the cell-free ganglionic structures hamper neural crest cell colonization in the myenteric region.

Diagnosis of innervation-related motility disorders of the gut and basic aspects of enteric nervous system development.

Motility disorders of the gut in children have become a matter of increasing concern for the pediatric surgeon. Infantile hypertrophic pyloric stenosis is the most common disease requiring surgery in early infancy. While this entity was first described as early as 1888 by Hirschsprung, its etiology and pathogenesis are still an enigma. Fortunately, its surgical treatment is simple and safe, which cannot be said of all other motility disorders of the infantile gut. Dysmotility in small bowel atresia and in gastroschisis is related to damaged smooth muscle cells caused by concomitant ischemia of the bowel wall. In contrast, the temporarily adynamic bowel of the prematurely born child, as well as Hirschsprung's disease and related disorders, is the result of anomalies of the intestinal innervation. The pathogenesis of congenital malformations of the enteric nervous system is still a mystery to surgeons and physicians alike. With his pressure studies of the colon, Swenson first recognized Hirschsprung's disease for what it was. This led to the resection of the manometrically diagnosed abnormal colon, which was found to be aganglionic. Histological investigation of the bowel wall became the decisive tool, replacing manometry, in the diagnosis of Hirschsprung's disease. Histochemical investigation of the bowel wall is not conclusive in other malformations of the enteric nervous system, since the presence or absence of enteric neurons is not the definitive factor discriminating between normally and abnormally functioning bowel. Monoclonal antibodies raised against neuron-specific markers may become important tools for differentiation within the spectrum of congenital malformations of the enteric nervous system. The immunocytochemical technique, however, does not provide sufficient information to explain the cause of innervation disorders of the gut in infancy and childhood. Primary migration disturbances or selective disappearance of enteric neurons following ischemia are highly unlikely to cause aganglionosis of the gut. With respect to the pathogenesis and etiology of Hirschsprung's disease, current research is focused on the embryonic bowel (target organ) in plexus formation. The enteric nervous system is still an enigma, although its origin is known, at least in birds. Why neural crest cells travel, along what paths, how they reach their destination, and what may go wrong during the migratory process, are questions that must be answered. There is increasing knowledge concerning the way in which neural crest cells aggregate and form plexuses in the gut. It is largely unknown why neural crest cells settle, in the bowel, at the sites of the myenteric and submucous plexus.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal antibodies in detection of choroidal melanoma.

Three monoclonal antibodies (MoAbs) prepared against cutaneous melanomas were tested against one group of 12 choroidal melanomas with indirect immunofluorescence in frozen sections. A fourth MoAb was tested in paraffin sections of a second group of 47 choroidal melanomas. One MoAb (NKI-M7) did not react with choroidal melanoma, even though it had a high sensitivity for cutaneous melanoma. A second MoAb (NKI-M6) showed a positive reaction with only 2/12 choroidal melanomas. The third MoAb (NKI/beteb) reacted with all choroidal melanomas, regardless of the cell type. MoAb NKI/C-3 was positive with 38/47 (81%) choroidal melanomas. We conclude that NKI/C-3 and NKI/beteb have a high sensitivity for both cutaneous and choroidal melanomas in frozen sections. Of these two antibodies NKI/beteb was the most specific for cutaneous naevi and melanomas.

Binding characteristics of high density lipoprotein subclasses to porcine liver, adrenal and skeletal muscle plasma membranes.

1. We compared binding characteristics of 125I-labeled high density lipoprotein (HDL) subclasses to porcine liver, adrenal and skeletal muscle plasma membranes. 2. HDL subclasses were discriminated by their buoyant densities (HDL2 and HDL3) or by their apolipoprotein (apo) content (Lp-AI (particles containing apoA-I but no apoA-II) and LpA-I/A-II (particles containing both apoA-I and apoA-II)). 3. HDL2 and HDL3 showed saturable binding to the three types of membrane preparations. 4. No differences were found in the Kds within one HDL subclass. 5. Kds and maximal binding of HDL2 were lower than these of HDL3. Unlabeled HDL2 and HDL3, but not LDL, effectively displaced 125I-HDL2 and 125I-HDL3. 6. Binding of HDL was independent of the concentration of NaCl and did not require calcium. 7. These results suggest a process mediated by a single specific receptor in porcine liver, adrenal and skeletal muscle plasma membranes. 8. We also studied binding characteristics of HDL subclasses Lp-AI and LpA-I/A-II to porcine liver membranes. LpA-I showed the highest Kd and maximal binding. 9. All types of HDL subclasses studied (i.e. HDL2, HDL3, LpA-I and LpA-I/A-II) effectively competed for binding of both Lp-AI and LpA-I/A-II, suggesting that the HDL subclasses studied bind to the same receptor by their apoA-I moiety.