Monoclonal antibodies against salt-resistant rat liver lipase. Cross-reactivity with lipases from rat adrenals and ovaries.

To obtain monoclonal antibodies against rat salt-resistant liver lipase, mice were immunized with enzyme purified from heparin-containing rat liver perfusates. Hybridomas were screened for antibody production by means of an enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay. Five hybridoma cell lines secreting antibodies against rat liver lipase indicated as A, B, C, D and E, have been obtained. All antibodies possess gamma one (gamma 1) heavy chains and kappa (kappa) light chains. The antibodies precipitate salt-resistant lipase from rat post-heparin plasma, are positive in ELISA, inhibit liver lipase activity and bind monospecifically with the enzyme as shown by immunoblotting. The monoclonal antibodies showed no significant reactivity with human liver lipase. The salt-resistant lipases of rat adrenals and ovaries are also precipitated by the monoclonal antibodies directed against the liver enzyme. Therefore, the heparin-releasable lipases of the liver, adrenals and ovaries possess identical epitopes.

Amino acid transport in plasma membrane vesicles from isolated rat liver parenchymal cells.

Plasma membrane vesicles were prepared from isolated rat liver parenchymal cells. The transport of several amino acids was studied and found to be identical to that in membrane vesicles from whole liver tissue.

Orientation of rat-liver plasma membrane vesicles. A biochemical and ultrastructural study.

Using both biochemical and morphological methods, the membrane orientation of plasma membrane vesicles from rat liver which are capable of catalysing the active transport of amino acids was investigated. In intact vesicles, the plasma membrane enzyme (Na+ + K+)-ATPase displays only a minor portion of its total activity which is greatly increased upon vesicle disruption. The same intact vesicles show an almost maximal binding of ouabain, which binds only to the extracellular side of the plasma membrane. A freeze-fracture analysis of the vesicles shows that a distinct population of relatively large vesicles have predominantly the in vivo membrane orientation. These large vesicles are labelled with numerous filipin-sterol complexes following exposure to the cholesterol probe, filipin, and are therefore assumed to be plasma membrane vesicles. A population of smaller vesicles with mainly an inside-out orientation were not labelled with filipin and are probably microsomes. The data obtained with both biochemical and ultrastructural techniques indicate that the plasma membrane vesicles isolated from rat liver for transport studies are mostly (at least 70%) orientated as in vivo, i.e. inside-in.

Heterogeneous distribution of the sodium-dependent alanine transport activity in the rat hepatocyte plasma membrane.

The localization of the sodium-dependent alanine uptake activity in rat liver cells was studied. Fractions representative of the canalicular, the contiguous (lateral) and the blood-sinusoidal surface of the hepatocyte were isolated by means of centrifugal fractionation and density gradient centrifugation. The distribution of various marker-enzyme activities in conjunction with the occurrence of alanine transport activity was studied both in fractions obtained after zonal density gradient centrifugation, and in the subcellular fractions mentioned above. It is concluded that the sodium-dependent alanine transport activity is primarily located in the blood-sinusoidal plasma membrane of the hepatocyte.

[Efficacy of agreements within the Enchede Stroke Service to refer patients with a stroke from the stroke unit in the hospital to a nursing home for short-term rehabilitation]. / Effectiviteit van afspraken binnen de Enschedese stroke-service om patiënten met een beroerte adequaat te verwijzen van de stroke-unit in het ziekenhuis naar een verpleeghuis voor kortdurende reactivering.

OBJECTIVE: To assess the efficacy of agreements within the Enschede Stroke Service to refer patients with a stroke from the stroke unit in the hospital to a nursing home for short-term rehabilitation. DESIGN: Prospective, partly retrospective. METHOD: All patients who were referred from the stroke unit at Medisch Spectrum Twente to the CVA Rehabilitation Unit (CRU) in the period 1 July 1999-31 July 2003 were included. Referral took place via an active multidisciplinary approach and specific referral agreements. The primary outcome was the number of patients that could be discharged home after rehabilitation. In addition, we assessed the influence on final discharge destination of age, the Barthel and Rankin scores at the time of admission to the CRU and the medical complications during the period of rehabilitation. RESULTS: 232 patients were included (133 women and 99 men, mean age 76.4 years). Within 3 months, 63% of the patients were discharged home. After 6 months, 82% had returned home. 8% of the patients died within 6 months and 9% had to stay in a nursing home permanently. Of the patient aged 80 years or older, 75% could return home within 6 months. Patients with poor Barthel and Rankin scores and medical complications had a smaller chance of being discharged home. CONCLUSION: Effective referral of patients from the stroke unit to a nursing home for short-term rehabilitation is possible. With adequate patient selection, the use of good referral agreements and multidisciplinary consultations, most patients could finally return home.

Immunoelectron microscopical localization of lysosomal beta-galactosidase and its precursor forms in normal and mutant human fibroblasts.

Immunoelectron microscopy was performed to study the biosynthesis of lysosomal beta-galactosidase (beta-gal) in normal and mutant human fibroblasts. Using polyclonal and monoclonal antibodies we show in normal cells precursor forms of beta-gal in the rough endoplasmic reticulum (RER) and in the Golgi apparatus throughout the stack of cisternae. In the lysosomes virtually all beta-gal exists as a high molecular weight multimer of mature enzyme. In the autosomal recessive disease GM1-gangliosidosis caused by a beta-gal deficiency and in galactosialidosis, associated with a combined deficiency of lysosomal neuraminidase and beta-gal, precursor forms of the latter enzyme are found in RER, Golgi and some labeling is present at the cell surface. The lysosomes remain unlabeled, indicative for the absence of enzyme molecules in this organelle. In galactosialidosis fibroblasts also no mature beta-gal is found in the lysosomes but in these cells the presence of the monomeric form can be increased by leupeptin (inhibition of proteolysis) whereas addition of a partly purified 32 kDa "protective protein" results in the restoration of high molecular weight beta-gal multimers in the lysosomes.

The subcellular localization of soluble and membrane-bound lysosomal enzymes in I-cell fibroblasts: a comparative immunocytochemical study.

Using electron microscopic immunocytochemistry with gold probes, we have studied the localization of acid alpha-glucosidase, N-acetyl-beta-hexosaminidase and beta-glucocerebrosidase in cultured skin fibroblasts from control subjects and patients with mucolipidosis II (I-cell disease). In control fibroblasts, a random distribution of acid alpha-glucosidase and N-acetyl-beta-hexosaminidase within the lysosomes was observed, whereas beta-glucocerebrosidase was found to be localized on or near the lysosomal membrane. The observations confirm the soluble character of acid alpha-glucosidase and N-acetyl-beta-hexosaminidase and the membrane-bound character of beta-glucocerebrosidase. In I-cell fibroblasts an abnormal localization of the two soluble enzymes was found. Labeling in lysosomes was very weak, but instead, small 'presumptive' vesicles containing both enzymes were detected throughout the cytoplasm and close to the plasma membrane. These vesicles could be involved in the secretion of the two enzymes. In contrast, a normal membrane-bound lysosomal localization was observed for beta-glucocerebrosidase. It is concluded that the intracellular transport of beta-glucocerebrosidase to the lysosomes can occur even when the mannose-6-phosphate recognition system is defective. This explains the normal activity of beta-glucocerebrosidase in I-cells in contrast to the deficiency of most other lysosomal enzymes.

The amino acid sequence of hamster pancreatic ribonuclease.

The amino acid sequence of golden hamster pancreatic ribonuclease was determined by analysis of tryptic, chymotryptic, thermolytic, and CNBr peptides and by automatic sequence analysis of the intact protein. Like all RNases with an Asn-Met-Thr sequence at positions 34-36, hamster RNase is glycosylated at position 34 with a complex-type carbohydrated chain. Val-17, Ala-18, His-55, His-76 and Ala-90 have never been observed in other pancreatic RNases. Ala-90 replaces Ser-90, which had been invariant in all mammalian RNases studied so far. The amino acid sequence of hamster RNase differs at 15 positions from that of another Cricetidae rodent, the muskrat. The similarity between both ribonucleases was used to confirm a few less certain parts of the muskrat RNase sequence. The replacement rate of the RNases of the Cricetidae appeared to be higher than the average rate in the mammals, but much lower than the rate in another myomorph family, the Muridae (mouse and rat). Possibly, in many respects, the Cricetidae underwent less evolutionary change in recent times than the evolutionarily highly successful Muridae.

Ultrastructural localization of glucocerebrosidase in cultured Gaucher's disease fibroblasts by immunocytochemistry.

The subcellular localization of glucocerebrosidase was studied in cultured skin fibroblasts from eight patients with Gaucher's disease. The enzyme, in situ, was visualized under the electron microscope by incubating ultrathin frozen sections of fibroblasts with antibodies against glucocerebrosidase, followed by a second incubation with goat anti- (rabbit IgG) coupled to colloidal gold. In control cells, most of the gold label was found in lysosomes, associated with the membrane. Labelling of the rough endoplasmic reticulum (RER) and Golgi complex was also observed. In fibroblasts from three Gaucher's disease patients without neurological symptoms (type 1 disease), a near normal amount of cross-reactive material (CRM) was detected in lysosomes, but in a fourth such patient, the lysosomal CRM was reduced. Little lysosomal glucocerebrosidase was detected in cells from patients with the acute neuronopathic form (type 2) or the subacute neuronopathic form (type 3) of Gaucher's disease. CRM in lysosomes correlates with amount of mature, 59 kDA glucocerebrosidase which is undetectable in type 2 and type 3 Gaucher's disease cell lines. These findings demonstrate that different mutations in the gene for glucocerebrosidase result in mutant proteins that have different intracellular localizations. They also suggest that there is a relationship between the amount of cross-reactive material in the lysosomes and the phenotypic expression of the disease.

Characteristics of maltase activity in amniotic fluid.

The nature and origin of maltase activity present in amniotic fluid, and used as a marker enzyme in the prenatal monitoring of cystic fibrosis, has been studied. Using monoclonal antibodies against human intestinal disaccharidases and via heat inactivation experiments it is shown that the maltase activity found in amniotic fluids from pregnancies of 16-24 wk of gestational age originates completely from sucrase-isomaltase; no maltase-glucoamylase could be detected. With various monospecific antibodies the possible contribution of non-intestinal brush border enzymes to the total maltase pool could be excluded: neither renal nor lysosomal maltase appeared to be present.

Human hepatoma (Hep G2) cultures contain salt-resistant triglyceridase ("liver lipase").

The culture fluid of Hep G2 human hepatoma cells contains triglyceridase activity resistant to high-salt concentrations. The lipase binds to Sepharose-heparin columns from which it can be eluted by 0.8 to 0.9 M NaCl. The nature of this lipase was studied using antibodies raised against "liver" lipases from human and rat origin. The anti-rat liver lipase inhibits both the postheparin human and rat plasma enzyme while the anti-human liver lipase has no effect on the rat enzyme. The lipase of the Hep G2 cultures showed affinity to the antibodies raised against rat as well as human "liver" lipase as shown by inhibition experiments. These results show that Hep G2 cells secrete "liver" lipase and that there seems to exist a structural homology between the lipases from rat and human origin.

Amino acid-dependent sodium transport in plasma membrane vesicles from rat liver.

The L-alanine-dependent transport of sodium ions across the plasma membrane of rat-liver parenchymal cells was studied using isolated plasma membrane vesicles. Sodium uptake is stimulated specifically by the L-isomer of alanine and other amino acids, whose transport is sodium-dependent in rat-liver plasma membrane vesicles. The L-alanine-dependent sodium flux across the membrane is inhibited by an excess of Li+ ions, but not by K+ or choline ions. Sodium transport is sensitive to -SH reagents and ionophores, and is an electrogenic process: a membrane potential (negative inside) can enhance L-alanine-dependent sodium accumulation. The data presented provide further evidence for a sodium-alanine cotransport mechanism.

Mechanism of the bactericidal action of myeloperoxidase: increased permeability of the Escherichia coli cell envelope.

The killing of Escherichia coli by isolated human myeloperoxidase plus hydrogen peroxide plus chloride ions was shown to proceed via an increased permeability of the bacterial cell wall. A correlation between the extent of the increased permeability and the number of surviving colony-forming units was found (P less than 0.0005). The same phenomenon was observed with isolated human neutrophils. The permeability increase was shown to be limited, because low-molecular-weight substrate became freely permeant, but the bacteria retained their permeability barrier for protein. Furthermore, disruption of the permeability barrier was followed by destruction of cytoplasmic protein. The active antibacterial agent was probably hypochlorous acid, the direct product of the system, rather than singlet oxygen, the nonenzymic product of hypochlorous acid and hydrogen peroxide. This is concluded from the fact that the myeloperoxidase system could be mimicked by hypochlorous acid, whereas conditions that favor formation of singlet oxygen did not enhance this effect. The relevance of the system for killing of bacteria at neutral pH is discussed.

Active transport by membrane vesicles from anaerobically grown Escherichia coli energized by electron transfer to ferricyanide and chlorate.

Active transport of amino acids by membrane vesicles from Escherichia coli, grown anaerobically on glucose in the presence of nitrate, can be energized under anaerobic conditions by electron transfer in the nitrate respiration system with formate as electron donor and nitrate as acceptor. A high rate of amino acid transport is also obtained under anaerobic conditions by electron transfer from formate to the nitrate analogue chlorate or to the membrane-impermeable electron acceptor ferricyanide. Electron transfer from formate to nitrate results in the generation of an electrical potential as is indicated by the uptake of the lipophilic cation triphenylmethylphosphonium. Ferricyanide accpets electrons from at least two sites of the nitrate respiration system. One of these sites appears to be nitrate reductase, because cytochrome b, reduced by formate, is completely reoxidized by ferricyanide and glutamate transport energized by formate plus ferricyanide and formate plus nitrate are affected by the same electron transfer inhibitors. A second site of electron transfer to ferricyanide appears to be located prior to nitrate reductase in the nitrate respiration system, since formate is oxidized at a higher rate in the presence of ferricyanide than with nitrate while formate/ferricyanide energizes transport of amino acids at a lower rate than formate/nitrate. Moreover, electron transfer inhibitors block electron transfer from formate to nitrate to a significantly higher extent than from formate to ferricyanide. The effects of irradiation of the membrane vesicles with near ultra-violet light suggest that quinones play an essential role in the electron transfer from formate to nitrate or ferricyanide. Irradiation blocks completely formate-dependent nitrate and ferricyanide reduction and active transport driven by formate/nitrate and formate/ferricyanide, but has hardly any effect on the activity of formate dehydrogenase and on ascorbate/phenazine methosulphate/oxygen-driven transport. Similar effects of ferricyanide have been observed in membrane vesicles from E. coli, grown anaerobically in the presence of fumarate. In these membrane vesicles a high rate of lactose and triphenylmethylphosphonium uptake under anaerobic conditions is obtained by electron transfer from glycerol 1-phosphate to fumarate and also to ferricyanide and evidence has been presented for the involvement of cytochromes in these electron transfers.

Synthesis and intracellular localization of chick acid alpha-glucosidase in chick erythrocyte-human fibroblast heterokaryons. A model system for the study of lysosomal enzyme synthesis.

The synthesis and localization of chick acid alpha-glucosidase has been studied in chick erythrocyte-human fibroblast heterokaryons. Monospecific antibodies raised against purified chick liver acid alpha-glucosidase were used. It was found that the acid alpha-glucosidase in the heterokaryons is of chick origin, and is localized in the same lysosomes as the human lysosomal enzymes. It is concluded that chick erythrocyte-human fibroblast heterokaryons provide a useful model system for the study of lysosomal enzyme synthesis and routing.

Kinetics and mechanism of the bactericidal action of human neutrophils against Escherichia coli.

A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the kinetics of phagocytosis, perforation of the bacterial cell envelope, and inactivation of bacterial proteins by human neutrophils. This particular E. coli mutant strain has no lactose permease, but constitutively forms the cytoplasmic enzyme beta-galactosidase. This implies that the artificial substrate ortho-nitrophenyl-beta-D-galactopyranoside cannot reach the beta-galactosidase unless the bacterial cell envelope has been perforated. Thus, the integrity of the E. coli envelope can be measured simply by the activity of beta-galactosidase with this substrate. Indeed, ingestion of E. coli ML-35 by human neutrophils was followed by perforation of the bacteria (increase in beta-galactosidase activity). Subsequently, the beta-galactosidase activity decreased due to inactivation of the enzyme. With a simple mathematical model and a curve-fitting computer program, we have determined the first-order rate constants for phagocytosis, perforation, and beta-galactosidase inactivation. With 32 normal donors, we found an interdonor variation in these rate constants of 20% to 30% (SD) and an assay variance of 5%. The perforation process closely correlated with the loss of colony-forming capacity of the bacteria. This new assay measures phagocytosis and killing in a fast, simple, and accurate way; it is not hindered by extracellular bacteria. Moreover, this method also measures the postkilling event of inactivation of a bacterial protein, which permits a better detection of neutrophils deficient in this function. The assay can also be used for screening neutrophil functions without the use of a computer program. A simple calculation suffices to detect neutrophil abnormalities. Neutrophils from patients with chronic granulomatous disease (CGD) showed an impaired rate of perforation and thus also of inactivation. Neutrophils from myeloperoxidase-deficient patients or from a patient with the Chediak-Higashi syndrome only showed a retarded inactivation of beta-galactosidase, but normal ingestion and perforation. The role of myeloperoxidase in the killing process is discussed. Although myeloperoxidase does not seem to be a prerequisite for perforation, it probably plays a role in bacterial destruction by normal cells, because the inactivation of bacterial proteins seems strictly myeloperoxidase dependent.