Decrease of human serum fucosyltransferase as an indicator of successful tumor therapy.

Surgical removal of colon carcinomas leads to a decrease in the rate of incorporation of [14C]fucose into its endogenous acceptor in human serum; normal incorporation rates are attained within 14 days. A similar time course has been determined for alpha2- and alpha3-fucosyltransferase when either desialo- or desialodegalactofetuin are employed as exogenous acceptors. A correlation has also been seen between transferase activity and the therapeutic response of patients with breast cancer. These results indicate that the determination of fucosyltransferase activity can facilitate the diagnosis of neoplasia, and the success of surgery, chemotherapy, or radiation.

Selective utilization of endogenous unsaturated phosphatidylcholines and diacylglycerols by cholinephosphotransferase of mouse lung microsomes.

In the presence of CMP, cholinephosphotransferase of mouse lung microsomes catalyzes the conversion of endogenous phosphatidylcholines into 1,2-diacyl-sn-glycerols and CDPcholine. 2. In this conversion cholinephosphotransferase shows a distinct preference for those molecular species of phosphatidylcholine which contain an unsaturated fatty acid. The enzyme hardly utilizes endogenous depalmitoylglycerophosphocholine as a substrate. 3. Membrane-bound 1,2-diacyl-sn-glycerols were also prepared by treatment of mouse lung microsomes with a pure phospholipase C from Bacillus cereus. These 1,2-diacyl-sn-glycerols were subsequently utilized as substrate by cholinephosphotransferase in the formation of phosphatidylcholine. In the latter reaction, cholinephosphotransferase exhibited a pronounced preference for unsaturated 1,2-diacyl-sn-glycerols and hardly utilized the endogenous 1,2-depalmitoyl-sn-glycerol. 4. The low affinity of cholinephosphotransferase for either dipalmitoylglycerophosphocholine or 1,2-dip

Studies on the aetiology of coeliac disease: no evidence for lectin-like components in wheat gluten.

In an approach to examine the lectin-hypothesis in the pathogenesis of coeliac disease, the presence of lectin-like components in three wheat gluten preparations known to induce coeliac disease, gliadin, Frazer fraction III and an acetic acid/ethanol extract of gluten, was investigated. Lectin-like components in these wheat gluten preparations were traced in binding studies employing a variety of model glycoproteins glycosylated with the different types of N-linked oligosaccharides, i.e., those of the high mannose-, complex- and hybrid-type. Binding affinity of wheat proteins to these glycoproteins was analyzed by affinity dotting and blotting techniques and was compared to that of the well characterized lectins Galanthus nivalis agglutinin, Concanavalin A and wheat germ agglutinin. Though the three wheat gluten preparations exhibited binding reactivity for distinct model glycoproteins, no correlation was found between the type of N-glycosylation of the model glycoproteins and their binding capability to the different wheat gluten preparations. Moreover, binding of the three gluten preparations to the model glycoproteins could not be inhibited by competitive saccharides (methyl-alpha-D-mannopyranoside, N-acetyl-D-glucosamine, mannan). Enzymatic deglycosylation of the ligand glycoproteins with endo-beta-N-acetylglucosaminidase H (Endo H, EC 3.2.1.96) or peptide N-glycosidase F (PNGase F, EC 3.5.1.52) abolished their binding reactivity for the plant lectins, but did not affect binding of the wheat gluten preparations. These results give no evidence for the presence of lectin-like components in wheat gluten preparations and do question the 'lectin hypothesis' of coeliac disease.

A human-SCID mouse model for allergic immune response bacterial superantigen enhances skin inflammation and suppresses IgE production.

Chronic skin colonization with Staphylococcus aureus is a well-known feature in atopic dermatitis. The aim of this study was to develop a human-SCID mouse model to analyze the possible role of bacterial superantigens in human allergic immune responses under in vivo conditions. SCID mice were reconstituted with peripheral blood mononuclear cells (between 2 and 9 x 10(7) cells per mouse) from atopic dermatitis patients sensitized to house dust mite allergen (Der p). Total and Der p specific antibody production required the following conditions: (i) injection of Der p; (ii) presence of CD14+ antigen-presenting cells; and (iii) IL-4 as shown by the inhibitory effect of human soluble IL-4 receptor on immunoglobulin E production. This model was used to study the immunomodulatory effects of the superantigen staphylococcal enterotoxin B in comparison with Der p. In intraperitoneally reconstituted human-SCID mice, topical treatment was ineffective in inducing skin inflammation. Therefore, additionally to intraperitoneal transfer, peripheral blood mononuclear cells from atopic donors were also injected intradermally. Such reconstituted SCID mice were then exposed via the skin to either Der p, staphylococcal enterotoxin B, or a combination of both. Maximal effects on epidermal inflammation and dermal T cell infiltration were obtained with staphylococcal enterotoxin B and Der p. Staphylococcal enterotoxin B alone was less effective and Der p only stimulated dermal T cell infiltration. These findings support the hypothesis that bacterial superantigens can act as trigger factors in allergic skin inflammation.

Heterogeneous turnover of terminal and core sugars within the carbohydrate chain of dipeptidylaminopeptidase IV isolated from rat liver plasma membrane.

Dipeptidylaminopeptidase IV, a plasma membrane-bound glycoprotein, is characterized by an intramolecular heterogeneous turnover of the protein backbone and carbohydrate chain. The faster turnover of the latter is restricted only to the outer sugars. The inner core sugars D-mannose and N-acetyl-D-glucosamine turn over at the same rate as the protein backbone.

Bioavailability of zinc from zinc-histidine complexes. I. Comparison with zinc sulfate in healthy men.

Zinc supplementation is beneficial in some clinical conditions. Histidine has been shown to improve zinc absorption in animals. To test its influence on zinc absorption in humans, we studied the bioavailability of zinc from zinc-histidine complexes as compared to zinc sulfate in 10 healthy volunteers. Ingestion of zinc complexed with histidine at a ratio of 1:2 or 1:12 increased serum-zinc concentration 25% more than ingestion of zinc sulfate. Calculated uptake was 30-40% increased with zinc histidine over zinc sulfate. Urinary excretion was not different with any preparation. Application of 15 mg zinc as zinc histidine 1:2 gave an identical serum-zinc response as 45 mg zinc taken as zinc sulfate. Zinc histidine complexes are better absorbed than zinc sulfate in humans.

Bioavailability of zinc from zinc-histidine complexes. II. Studies on patients with liver cirrhosis and the influence of the time of application.

Supplementation may benefit patients with liver cirrhosis. Zinc uptake from zinc-histidine complex 1:2 was assessed in eight patients with liver cirrhosis. Influence of the time of application was also studied. Compared with healthy controls, patients showed a 40% lower uptake of zinc after 20 mg zinc from zinc histidine. Bioavailability was identical when zinc was taken 6 h or 1 h before a meal but an order of magnitude greater than with or 1 h after a meal. No significant increase of serum zinc was found when zinc was given with a meal or 1 h after. Lower doses of zinc-histidine complexes than of zinc sulfate may be used to supplement patients with liver cirrhosis. Time of application is of great importance if this substitution is to be successful.

Fetal fibronectin as a marker for an imminent (preterm) delivery. A new technique using the glycoprotein lectin immunosorbent assay.

We have developed a new 'glycoprotein lectin immunosorbent assay' (GLIA) which permits the obstetrician to identify accurately pregnant women at risk for preterm delivery. This GLIA uses two lectins for the quantitative detection of glycosylation variants of fibronectins, namely, Maackia amurensis lectin (MAA) for the detection of fetal fibronectin (fFN), and Sambucus nigra lectin (Elderberry bark lectin; SNA). Fibronectin was quantitated in cervicovaginal secretions, amniotic fluid, and plasma of pregnant women. Detection of fFN in cervicovaginal secretions was considered to indicate a high risk of imminent delivery. The results were as follows: (1) The GLIA could differentiate between pregnant women after the onset of labour and/or with rupture of membranes and women without any signs of an imminent delivery (sensitivity 94%, specificity 96%, p < 0.001). (2) Differentiation was possible between asymptomatic pregnant women delivering within 10 days of sampling or after more than 10 days (sensitivity 93%, specificity 99%; p < 0.001). (3) If fFN was present in the cervicovaginal secretions, delivery occurred within 10 days of sampling irrespective of preterm delivery or delivery at term (p < 0.001). Thus, this GLIA is a useful assay for identifying those asymptomatic pregnant women who will deliver within 10 days of sampling.

The release of cytokines, adhesion molecules, and extracellular matrix parameters during and after reperfusion in human liver transplantation.

Poor initial graft function may increase postoperative morbidity including the risk of early allograft rejection. Various mediators, including immunostimulatory cytokines, may be released during reperfusion in relation to the extent of preservation and reperfusion injury. For this purpose, 81 patients with 85 liver transplants were monitored for cytokines, adhesion molecules, extracellular matrix (ECM) parameters, and neopterin at predefined time-points during and after transplantation. To estimate the origin of cytokine release, blood was obtained central and hepatic venously for the first 48 hr after reperfusion and subsequently from a peripheral vein. One-year patient survival was 88.9%; no relation to initial graft function was observed. Poor initial graft function failed to increase the risk for subsequent infectious complications but was associated with an increased risk of early allograft rejection. The incidence of steroid-resistant rejection was significantly increased in patients with poor initial graft function (35.7% versus 12.7% in patients with good and moderate initial graft function; P < or = 0.05). Various cytokines, adhesion molecules, and ECM parameters including sTNF-RII, sIL-2R, IL-8, IL-10, sVCAM-1, E-selectin, hyaluronic acid, sialic acid, and laminin correlated significantly with the extent of preservation and reperfusion injury. Although none of these parameters was more appropriate in determining the extent of preservation and reperfusion injury than currently established parameters (AST, ALT, and color and amount of bile production), the combined increase in these parameters may not only promote tissue repair but may also perpetuate liver allograft injury and thereby cause significant morbidity. Besides cytokines and adhesion molecules, the ECM may play a pivotal role in determining repair or ongoing tissue injury. Ongoing changes at the microvasculature and basement membrane may result in an increase of local and circulating cytokines and adhesion molecules, which increase the risk of subsequent early allograft rejection. Furthermore, the increase in sTNF-RII, E-selectin, and laminin during reperfusion was predictive of subsequent development of acute allograft rejection. These observations may be of value for further strategies to decrease reperfusion injury and prevent early allograft rejection.

A comitogenic serum glycoprotein in T lymphocyte activation.

The production of Interleukin-2 is induced in helper cells, probably of T-cell origin, after stimulation by Interleukin-1. PHA is known to induce production of Interleukin-1 and Interleukin-2 in human PBL. We observed that PHA-induced mitogenesis in PBL requires the presence of a 90-KD-serum glycoprotein, which we were able to distinguish from other known serum proteins of similar molecular weight. Its biological activity can be destroyed by removal of its sialic acid residues. Evidence presented in this paper indicates that the function of this protein is related to the induction of synthesis and/or release of Interleukin-2. The presence of PHA does not seem to be essential at the level of Interleukin-2 production; the glycoprotein is able to induce Interleukin-2 in cooperation with a soluble mediator, which is produced by adherent cells. This mediator causes T-cell mitogenesis in PBL, provided serum or the 90-KD glycoprotein is present in the culture. It is not able to support the growth of a CTL line. We suggest the name PHILIP (Plasmatic Human Interleukin-2-Inducing Protein) for the 90-KD molecule.

In an epidemiological sample the apolipoprotein E4 allele is associated to dementia and loss of memory function only in the very old.

The apolipoprotein E4 allele has been reported to be associated with late onset Alzheimer's disease. Here we report the relation of several neuropsychological test parameters and the diagnosis of dementia to the apolipoprotein E polymorphism in an epidemiological sample of 477 subjects aged 70-103 years. The apolipoprotein E4 allele was found to be associated with reduced performance in several sensitive neuropsychological memory tests and with diagnosis of dementia only in the oldest subjects (> 84 years). The association with dementia in this population based sample was much weaker than previously described and became only significant in a logistic regression analysis when age was included in the model.

[Concanavalin A- Sepharose affinity chromatography for routine microanalysis of gamma-glutamyltransferase (author's transl)]. / Concanavalin A-Sepharose-Affinitätschromatographie im Mikromassstab fur Serienanalysen am Beispiel der Gamma-Glutamyltransferase.

The separation of two molecular forms of liver gamma-glutamyltransferase is achieved by Con A-Sepharose chromatography, an adult type with high affinity to Con A and a fetal type without binding capacity to this lectin. Now we present a new method using micro-columns for affinity chromatography (gel volume 2 ml). By this rapid and inexpensive procedure it is possible to study this enzyme separation for its use in the diagnosis of liver diseases.

HFE gene mutation and transferrin saturation in very low birthweight infants.

AIM: To determine if there is an association between high transferrin saturation and the C282Y HFE gene mutation in very low birthweight (VLBW) infants. METHODS: One hundred and forty three VLBW infants receiving recombinant erythropoietin and 3 to 9 mg/kg/day of enteral iron were studied. Genomic DNA was extracted from filter paper cards. The C282Y mutation was determined by restriction fragment length polymorphism analysis. RESULTS: Six infants were heterozygous for the mutation; none was homozygous. Ten infants had a transferrin saturation above 80% at least once. No infant was positive for both transferrin saturation above 80% and the mutation. CONCLUSIONS: The data strongly suggest that there is no association between high transferrin saturation and the HFE gene mutation in VLBW infants during the first weeks of life.

Use of physiological substrates for zymograms of disaccharidases after separation in immobilized pH gradients.

A new technique is described for in situ visualization of the activity of intestinal disaccharidases after isoelectric focusing in immobilized pH gradients using their physiological substrates. The reaction principle is based on the oxidation of D-glucose, liberated by the disaccharidases, into D-gluconolactone and the production of NADH by glucose dehydrogenase. At the sites of enzymatic activity, tetrazolium salts present in the reaction mixture are reduced to relatively water-insoluble formazans by NADH. The rate of formazan production is increased by the presence of phenazine methosulfate. An additional modification of the technique involves the use of polyvinyl alcohol in the substrate solution. Due to the increase in the viscosity of the substrate solution, leakage of the enzyme from the IPG gels is minimized. Incubation times can thus be prolonged without loss of resolution and band-blurring.

Two-dimensional maps in very acidic immobilized pH gradients.

Up to the present time it has been impossible to perform two-dimensional (2-D) separations in very acidic immobilized pH gradients (IPG), due to the lack of suitable buffering acrylamido derivatives to be incorporated into the polyacrylamide matrix. The advent of the pK 3.1 buffer (2-acrylamido glycolic acid; Righetti et al., J. Biochem. Biophys. Methods 16, 1988, 185-192) allowed the formulation of such acidic gradients. We report here separations in IPG pH 2.8-5.0 intervals of polypeptide chains from total lysates of rat intestinal and liver cells and 30S and 50S ribosomal proteins from Halobacterium marismortui. Conditions are given for highly reproducible first and second dimension gels and for a proper silver staining of 2-D maps with practically no background deposition.

Two-dimensional maps in the most extended (pH 2.5-11) immobilized pH gradient interval.

In conventional isoelectric focusing in soluble, amphoteric buffers, it has been quite difficult to produce two-dimensional (2-D) separations in pH intervals greater than pH 4-8. In general more alkaline proteins were analyzed by non-equilibrium IEF in the first dimension. Even with the advent of immobilized pH gradients (IPG), separations could be extended to pH gradients not wider than pH 3-10, due to a lack of suitable buffers. Since more acidic and more alkaline acrylamido buffers have recently been synthesized, we have been able to optimize what is believed to be the widest possible immobilized pH gradient, a pH 2.5-11 span. We report here for the first time 2-D separations of total tissue lysates in such extended pH 2.5-11 gradients. It appears that, with the IPG technique, close to 100% of all possible cell products can be displayed in a single 2-D map.

Increased expression of annexin I and thioredoxin detected by two-dimensional gel electrophoresis of drug resistant human stomach cancer cells.

The therapy of advanced cancer using chemotherapy alone or in combination with radiation or hyperthermia yields an overall response rate of about 20-50%. This success is often marred by the development of resistance to cytostatic drugs. Our aim was to study the global analysis of protein expression in the development of chemoresistance in vitro. We therefore used a cell culture model derived from the gastric carcinoma cell line EPG 85-257P. A classical multidrug-resistant subline EPG85-257RDB selected to daunorubicin and an atypical multidrug-resistant cell variant EPG85-257RNOV selected to mitoxantrone, were analysed using two-dimensional electrophoresis in immobilized pH-gradients (pH 4.0-8.0) in the first dimension and linear polyacrylamide gels (12%) in the second dimension. After staining with coomassie brilliant blue, image analysis was performed using the PDQuest system. Spots of interest were isolated using preparative two-dimensional electrophoresis and subjected to microsequencing. A total of 241 spots from the EPG85-257RDB-standard and 289 spots from the EPG85-257RNOV-standard could be matched to the EPG85-257P-standard. Microsequencing after enzymatic hydrolysis in gel, mass spectrometric data and sequencing of the peptides after their fractionation using microbore HPLC identified that two proteins annexin I and thioredoxin were overexpressed in chemoresistant cell lines. Annexin I was present in both the classical and the atypical multidrug-resistant cells. Thioredoxin was found to be overexpressed only in the atypical multidrug-resistant cell line.

[Communication and integration of medical documents for the teleconference use. An outcome of the BERMED project]. / Kommunikation und Integration medizinischer Dokumente für Telekonferenz-Anwendungen: Ein Ergebnis aus dem Projekt BERMED.

The collaboration between physicians is supported in the BERMED project by implementing the remote access to distributed patient data and the realisation of computer-based medical conferencing. This requires the integration of the multimedia data in form of a meta-patient record for our medical application systems. These applications are supported by a distributed information management promoting access to different information systems, imaging modalities and digital archival storage systems. The features of image processing are concerned with quantification, segmentation and 3-D-visualisation to obtain additional information. This paper gives an overview of the actual state of the teleconferencing system in radiology.

The influence of portosystemic shunting on zinc and vitamin A metabolism in liver cirrhosis.

To elucidate the possible role of portosystemic shunting on zinc and vitamin A deficiency which has been described in patients with cirrhosis of the liver, a study on 37 hospitalized patients with liver cirrhosis was performed. Patients with surgical portosystemic shunt were found to have a significantly lower levels of zinc, vitamin A and retinol-binding protein (RBP) than controls and patients with cirrhosis without shunt. Patients with portal hypertension--considered to have spontaneous shunting--also has lower levels than those without this symptom. A significant correlation between zinc and vitamin A and RBP levels, respectively, was found. Also an increased renal zinc output was demonstrated. An influence of portosystemic shunting on zinc deficiency and subsequent vitamin A deficiency by decreased RBP release is concluded. The importance of these metabolic disorders for clinical symptoms is discussed.

Trace elements and carrier proteins in the aged.

Serum level of trace elements can be used as a marker for diagnosis and management of diseases. We evaluated the effects of age, sex and diurnal rhythm on serum concentration of Cu, Fe, Zn and their carrier proteins. The subjects (N=336) were participants of the randomized multidisciplinary Berlin Aging Study (BASE), stratified for age (70-103 yrs) and sex. There is no diurnal variation for the carrier proteins coeruloplasmin, transferrin and albumin. The age-related decline of these proteins is not significant. Not only serum Fe but also Zn levels undergo a progressive decrease during the day. In the aged the serum concentration of both trace elements decreased, but the most important changes are diurnal variations. To avoid mistakes in the interpretation of clinical findings, so called time-quantified reference values are recommended.