Lens culinaris lectin immobilized on sepharose: binding and sugar-specific release of intact tissue culture cells.

Lens culinaris lectin (LCL) covalently linked to 2B Sepharose binds tissue culture cells to the matrix. This is prevented by hapten sugars specific for LCL. Unlike other immobilized lectins, lens culinaris lectin allows the removal of bound cells from the matrix on addition of the specific sugars in a concentration-dependent manner. Binding and release occur under physiological conditions. Released cells continue to grow.

Phosphorylation by cell surface protein kinase of bovine and human fibrinogen and fibrin.

Human and bovine fibrinogen as well as fibrin are shown to be phosphorylated by Co631 (monolayer, hamster) and RPL12 (suspension, chicken) cells by their surface protein kinase of the casein kinase II type. The phosphate label is introduced into the alpha-peptide. The kinase system phosphorylates serine residues and utilizes GTP equally well as ATP. The participation of intact cell surfaces indicates the possibility of phosphorylation of extracellular fibrinogen independently of the site of its biosynthesis.

Ecto-protein kinase activities in normal and transformed cells.

The incubation of intact uninfected and Rous sarcoma virus (RSV)-transformed chicken cells (SR-RSV-A) with micromolar amounts of [gamma-32P]ATP under physiological conditions resulted in the radioactive phosphorylation of a variety of proteins. According to the experimental protocol the detectable phosphorylation was restricted to ATP utilization at the cell surface and was catalyzed by surface located protein kinase (PK). Serine- and to a lesser extent, threonine residues were phosphorylated. With respect to this enzyme the cells under investigation showed upon incubation with phosvitin the release of surface (phosvitin) kinase into the incubation medium. Based on immunochemical analysis and PK-assays using antisera from RSV-tumor bearing rabbits (TBR-serum) the pp60v-src with its associated tyrosine kinase activity was likewise detected in appreciable amounts at the outside of RSV-transformed chicken and mammalian cells. There was no cross reactivity of TBR-serum with phosvitin kinase. Phosvitin was not phosphorylated by the immunoprecipitated pp60v-src. Whereas phosphorylation catalyzed by pp60v-src was blocked with 10 to 20 microM diadenosine 5',5''-P1P4 tetraphosphate (Ap4A) the phosvitin phosphorylation was far less sensitive towards inhibition by Ap4A, similar to the cellular pp60c-src kinase activity in uninfected cells. The functional significance of the PK activities in uninfected and RSV-transformed cells observed at their surface or in cell-free form as well as the nature of their substrates remain to be established.

Assays of cell surface protein kinase: importance of selecting cytophilic substrates.

The ability of phosvitin and histone to serve as substrates for possible protein kinase(s) at the cell surface was examined with regard to their suitability as indicators of such activity. While phosvitin did not interfere with the membrane barrier of HeLa cells, 3T3 and SV 3T3 cells, histone caused severe damage as indicated by both uptake of viability stains Trypan Blue and diamidino-phenylindol and by release of intracellular compounds such as lactate dehydrogenase, cAMP-dependent protein kinase(s), and metabolically prelabelled proteins. Where intactness of the cell membrane is prerequisite for verification of ecto-protein kinase, histone cannot be used as substrate. In contrast, we found that phosvitin is suitable for assays of cell surface located protein kinase activity.

Generation of pyrophosphate from extracellular ATP at the surface of HeLa cells.

On incubation of intact HeLa cells with micromolar concentrations of [gamma 32 P]ATP under physiological conditions, the generation of inorganic pyrophophate in the supernatant has been observed. The reaction depends on incubation time and cell number employed; the enzymatic activity is not shed into the supernatant. Isotope dilution data support that the responsible enzyme(s) is located at the cell surface.

[Drug users outside medical treatment: methodological and practical aspects]. / Consommateurs de drogues hors traitement médical: Aspects méthodologiques et pratiques.

A legal context that classifies the consumption of heroin and cocaine as an illegal act, poses a considerable methodological challenge to research on users of these substances. This is in particular the case for research on those users who are not in treatment and, therefore, cannot be recruited through treatment settings. In a research project on heroin and/or cocaine users outside treatment settings, a sample of 917 individuals was recruited through "Privileged Access Interviewers" in the whole of Switzerland. In the first part of this article, we discuss matters of reliability as well as of validity concerning this method of data collection. In the second part of the article, we discuss the use of low threshold syringe exchange schemes by the user groups represented in the sample. Only intravenous drug users frequent those services - they are however a minority in the sample (n = 238). In several regions of Switzerland syringe exchange schemes do not exist. Where they do exist, they appear to correspond to a need which they are able to cover largely. In the regions without such services, intravenous drug users get their supply of syringes more frequently from pharmacies. However, pharmacies do not compensate the absence of specific syringe exchange schemes. In regions without such schemes, injections with used syringes are more frequent. Thus, regarding Aids-Prevention, there is an urgent need to develop syringe exchange schemes in all parts of the country.

Characteristics of heroin and cocaine users unknown to treatment agencies. Results from the Swiss Hidden Population Study.

The aim of this study was to compare the characteristics of heroin or cocaine users who are not in contact with drug-treatment agencies in Switzerland to the characteristics of a group who are in treatment. A sample of 917 users of heroin and/or cocaine was recruited outside treatment settings by 31 Privileged Access Interviewers. Respondents were divided into a study group of 512 heroin and/or cocaine users not following any treatment, and a control group of 238 users who were following treatment. Respondents in the no-treatment group use drugs less frequently, are less likely to inject drugs, have a more social pattern of use and more often have the impression of controlling their drug use. They have less contact with the legal system and the police, are in a better social situation and more often perceive themselves to be in good health. In both groups, respondents whose main drug of use is heroin generally have a more problematic pattern of use than those who use mainly cocaine. There are no significant differences between the two groups regarding present HIV-risk behaviour and prevention. The data show no significant association between the duration of use of heroin or cocaine and signs for problem use. These findings support the hypothesis that drug users not in treatment and drug users in treatment are two distinct populations, in terms of profile of drug use and prevalence of social or health problems that are associated to it.

[Exchange of syringes or apprenticeship? The buses of Bienne and Geneva]. / Echange de seringues ou apprentissage? Les bus de Bienne et Genève.

In Switzerland, the health ministry (Office Fédéral de la Santé Publique) has systematically encouraged the evaluation of low threshold services. In this article, we discuss the evaluation of two of these: the buses for syringes exchange in Geneva and Bienne, the implementation of these two services, the success obtained and the contacts established. Even if the design of such an evaluation was relatively complicated, with one monitoring and two specific surveys, the principal aim of this article is not to measure the efficacy as such but to show how an learning process has occurred between the actors: government, administration, police, service's team, drug's users and neighbourhood's inhabitants. The efficacy for a long period of time and the implementation's success are largely linked to such learning processes.

Phosphorylated human lectin galectin-3: analysis of ligand binding by histochemical monitoring of normal/malignant squamous epithelia and by isothermal titration calorimetry.

The human lectin galectin-3 is a multifunctional effector with special functions in regulation of adhesion and apoptosis. Its unique trimodular organization includes the 12-residue N-terminal sequence, a substrate for protein kinase CK1-dependent phosphorylation. As a step towards elucidating its significance, we prepared phosphorylated galectin-3, labelled it and used it as a tool in histochemistry. We monitored normal and malignant squamous epithelia. Binding was suprabasal with obvious positive correlation to the degree of differentiation and negative correlation to proliferation. The staining pattern resembled that obtained with the unmodified lectin. Basal cell carcinomas were invariably negative. The epidermal positivity profile was akin to distribution of the desmosomal protein desmoglein, as also seen with keratinocytes in vitro. In all cases, binding was inhibitable by the presence of lactose, prompting further investigation of the activity of the lectin site by a sensitive biochemical method, i.e. isothermal titration calorimetry. The overall affinity and the individual enthalpic and entropic contributions were determined. No effect of phosphorylation was revealed. This strategic combination of histo- and biochemical techniques applied to an endogenous effector after its processing by a protein kinase thus enabled a detailed monitoring of the binding properties of the post-translationally modified lectin. It underscores the value of using endogenous lectins as a histochemical tool. The documented approach has merit for applications beyond lectinology.

Ecto-protein kinase substrate p120 revealed as the cell-surface-expressed nucleolar phosphoprotein Nopp140: a candidate protein for extracellular Ca2+-sensing.

A variety of cell membrane proteins become phosphorylated in their ecto-domains by cell-surface protein kinase (ecto-PK) activities, as detected in a broad spectrum of cell types. This study reports the isolation and identification of a frequent ecto-PK substrate, ecto-p120, using HeLa cells as a model. Data from MS and further biochemical and immunochemical means identified ecto-p120 as a cell-surface homologue of human nucleolar phosphoprotein p140 (hNopp140), which belongs to the family of argyrophilic (AgNOR-stainable) proteins. The superposition of (32)P-labelled ecto-nucleolar phosphoprotein p140 (ecto-Nopp140) with anti-Nopp140 immunostaining could be demonstrated in a wide range of cell lines without any exceptions, suggesting a nearly universal occurrence of cell-surface Nopp140. A previous, tentative association of ecto-p120 with the nucleoplasmic pre-mRNA-binding protein hnRNP U has thus been supplanted, since improved purification techniques have allowed unambiguous identification of this ecto-PK cell-surface substrate. Furthermore, we have shown that rapid suppression of ecto-hNopp140 phosphorylation resulted upon a rise in the free extracellular calcium, while lowering the calcium concentrations returned ecto-Nopp140 phosphorylation to the original level. It is important to note that these Ca(2+)-dependent effects on ecto-Nopp140 phosphorylation are not accompanied by alterations in the phosphorylation of other ecto-PK substrates. Our results indicate that, in addition to nucleolin, a further nucleolar protein, which was considered initially to be strictly intracellular, is identified as a cell-surface phosphoprotein.

Ecto-protein kinase release differs from cleavage by phospholipases of a glycosyl-phosphatidylinositol membrane anchor.

Ecto-protein kinases have been detected as physiological constituents of cells. One feature of ecto-phosvitin/casein kinase (ecto-PK) is its release from the surface in a soluble form when cells are incubated with exogenous substrate protein. This is interesting in view of the fact that some ecto-enzymes are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Such enzymes are known to be released from the surface through cleavage by a phospholipase activity. We therefore investigated whether bacterial phospholipase C (PI-PLC) was able to release ecto-PK from intact HeLa cells. The data show that whereas alkaline phosphatase, known to be GPI-anchored, was solubilized, the ecto-PK was neither released nor affected in its activity. Another effect of treatment of cells with phospholipases was the formation of diacylglycerol or phosphatidic acid which, however, did not occur when cells were incubated with phosvitin, the condition which induces ecto-PK release. These results coherently indicate that cellular phospholipases are not involved in the release mechanism of ecto-PK. Also, the presence of various protease inhibitors did not affect ecto-PK release. Cross-linking of cell-surface proteins by bifunctional agents of the succinimidyl-type suggest a protein-protein interaction responsible for membrane anchoring of the ecto-PK.

Autoimmune diseases: nuclear autoantigens can be found at the cell-surface.

The pathogenesis of autoimmune diseases is only partially understood. In particular, the question remains why many nuclear proteins have been identified as autoantigens. One possible mechanism for an autoimmune response to nuclear proteins involves their exposure to the immune system. In this report we discuss currently available data on the exposure of nuclear proteins by expression at the cell-surface. Although the pathways of surface expression remain unclear, the presence of nuclear proteins at the cell-surface might reflect a pathological reaction leading to an exposure of epitopes, e.g. to self-reactive B-cells. It is suggested that cell-surface expression of intracellular proteins can contribute to the generation of autoimmune diseases.

Phospho-accepting proteins in bovine sera.

Phospho-accepting proteins in bovine sera have been detected by the use of immobilized protein kinase from rat muscle and (gamma32P)-ATP in an in vitro system. A partial biochemical characterization points to the generation of typical phosphoproteins. Differences in the phosphorylation pattern between fetal serum and calf serum as demonstrated by electrophoresis in the presence of dodecylsulfate are described.

Evidence for CKI and CKII at the cell surface.

Ser/Thr-protein kinases at the cell surface (ecto-PK) use physiological concentrations of extracellular ATP for phosphorylation of endogenous cell surface proteins, as well as of soluble protein substrates in the extracellular environment (Kübler et al., 1982, 1989). One abundant ecto-PK component is believed to be a protein kinase CKII since it phosphorylates phosvitin and casein, is sensitive to heparin at low concentrations, and can use both ATP and GTP as cosubstrate. This ecto-PK activity can be detached from the surface of intact cells through interaction with exogenous substrates, a process termed "shedding" (Kübler et al., 1983). This study reports a method for the purification and identification of shedded ecto-PK. Affinity chromatography of the concentrated ecto-PK through a heparin-matrix resolved two phosvitin/casein kinase activities upon elution with a NaCl gradient, termed as peak I and peak II. Relative to the total protein load of the cells employed for ecto-PK shedding, the specific activities increased by a factor of about 10(4) times. The use of peptide substrates specific for CKI and CKII, of ATP and GTP, as well as of antibodies specific for CKII subunit, clearly identified one of the enzymes as a CKI-like entity and the other one as CKII-like. Although the spatial arrangement on the cell surface of the two related ecto-PKs is unknown, their tandem appearance together in the cell supernatant might suggest the possibility of a functional unit.

Solid-state protein kinase. A tool for post-synthetically modifying and radioactively labeling proteins in vitro.

The capacity of partially purified rat muscle protein kinase coupled to cyanogen-bromide-activated Sepharose 4B to (radio-)phosphorylate proteins in vitro was evaluated using histones from calf thymus and rat liver and certain proteins as substrates. Data are presented which point to a low substrate specificity of this enzyme. It is demonstrated that even within a short time period histones are efficiently phosphorylated without the introduction of contaminating (phospho-)proteins. Therebye phosphoserine residues are formed. The phosphorylation reaction usually performed at 30 degrees C is shown to function quite efficiently also at 4 degrees C. It proceeds even at 30 degrees C for several hours at pH values close to the physiological range without the release of proteins from the solid matrix. The phosphorus transfer can be largely increased with the use of high ATP concentrations. The stability of the substrates is sufficient to suggest a wide applicability of this solid-state protein kinase in the phosphorylation of proteins either for labeling or as a tool to modify proteins post-synthetically under gentle conditions. The solid enzyme seems to be suitable for radioactively labeling proteins of more complex biological structures, such as membrane surfaces.

Protein kinase activity and substrates at the surface of intact HeLa cells.

Evidence is presented for the location at the surface of HeLa cells of a protein kinase capable of phosphorylating surface as well as extracellular (foreign) proteins. The reaction products have been found to be proteins containing phosphoryl groups as monoesters of seryl and threonyl residues (but not of tyrosine). The enzyme is of the cyclic AMP-independent type, since neither cyclic AMP nor the heat- and acid-stable inhibitor protein (specific for cyclic AMP-dependent protein kinases) influenced its activity. Further, co-substrate ATP could in part be substituted by GTP, and the spectrum of proteins phosphorylated by the ecto-enzyme differed from that phosphorylated by cyclic AMP-dependent protein kinases. Evidence for the ecto-enzymic nature of this protein kinase includes (a) utilization of co-substrate and location of products at the surface of cells carefully controlled as being in an intact state and (b) phosphorylation of exogenous protein (phosvitin; specific serum proteins) by intact cells. Conclusive proof was gained by qualitative and quantitative comparative studies of phosphorylation in cultures with varying degrees of damaged cells either as a whole or after separation into groups of intact and damaged cells by electronic cell sorting. The results of experiments with cell sonicates excluded the possibility that either enzyme or substrates released from damaged cells were simply adsorbing to the cell surface.

Major cell surface-located protein substrates of an ecto-protein kinase are homologs of known nuclear proteins.

Cell surface polypeptides serve as substrates for a casein kinase-like ecto-protein kinase activity which is demonstrable under stringent criteria with intact cells using micromolar levels of extracellular [gamma-32P]ATP. Two major 32P-labeled proteins, designated as pp100 and pp120 after their apparent molecular masses on SDS-PAGE under reducing and nonreducing conditions, have repeatedly appeared in the phosphoprotein spectra of different cell types. We have chosen HeLa cells as a source for the biochemical characterization and isolation of pp100 and pp120. Phosphorylation of pp100 and pp120 occurs in their extracellular domains at seryl residues of amino acid side chains. Several criteria deduced from the heparin sensitivity of the ecto-protein kinase and its substrate-induced shedding into the cell supernatant indicated that surface phosphorylation is a function of the ecto-protein kinase. The radioactive phosphorylation of pp100 and pp120 which coincides with their biotinylation on 2D-blots can be reversed by mild trypsination of intact cells. Purification and enrichment of pp100 and pp120 were achieved on the basis of radioactivity detection on and isolation from 1D- and 2D-gels. Amino acid sequence analysis performed on tryptic digests of purified ecto-phosphoproteins in most cases showed significant consensus sequences between pp100 and the nuclear RNA-binding protein nucleolin while pp120 sequences proved to be related to hnRNP U, a nucleoplasmic pre-mRNA-binding protein. Immunochemical analysis using anti-nucleolin and anti-hnRNP U antibodies combined with comparative phosphorylation and characterization of the ecto-proteins with authentic nucleolin and hnRNP U further established the close relationship, suggesting surface membrane versions of the nuclear proteins.

Substrate-effected release of surface-located protein kinase from intact cells.

Protein kinase activity that is independent of cAMP has been reported to exist on the surface of intact HeLa cells. Here we report that the protein kinase activity can be released by the use of casein or phosvitin within a short period of time. The discharge of the enzyme occurs from intact cells since (i) the cells do not release intracellular material and (ii) the cultures continue to grow within any morphological alteration. As shown with phosvitin, the release of protein kinase depends on substrate concentration, incubation time, and temperature. The degree of inducible release or surface protein kinase is inversely related to cell density. Four incubations with phosvitin (1 mg/ml) are sufficient to liberate most of the enzyme, thus greatly reducing the capacity of the cells to phosphorylate cellular substrates at the surface. Within approximately 24 hr after protein kinase removal, cultures have restored their surface protein kinase. Cultured cells of different origin (rat liver, mouse cerebellum, and human lung) exhibited phosvitin-induced protein kinase release from intact cells. The possible significance of these observations with respect to extracellular protein phosphorylation is discussed.

Protein kinases in HeLa cells and in human cervix carcinoma.

Extracts of HeLa cell fractions were analyzed by DEAE- and phospho-cellulose chromatography for their range of cyclic AMP-dependent and -independent protein kinase activities phosphorylating histone and/or phosvitin; extractions were by phosphate buffered saline (soluble protein kinases) and the non-ionic detergent NP-40 (membrane-bound protein kinases). The soluble fraction contained (i) cyclic AMP-dependent histone kinases type I and II as evidenced by their behaviour on DEAE-cellulose and inhibition by specific heat- and acid-stable protein kinase inhibitor (PKI) in a dose-related manner; both types I and II as well as their purified catalytic subunit also phosphorylated protamine and - with very low efficiency - casein but not phosvitin; (ii) a histone kinase (H), insensitive to cyclic AMP and PKI, also accepting protamine as substrate but not either casein or phosvitin; (iii) a phosvitin kinase (P), insensitive to cyclic AMP and PKI, which also phosphorylates casein but not histone or protamine. These four enzyme species were also found in NP-40 extracts of 27000 x g residues which, however, contained further histone and phosvitin kinase activities as yet unspecified. NP-40 extracts of the microsomal fraction possessed, besides unspecified histone and phosvitin kinase activity, only the phosvitin kinase P and appeared to be devoid of histone kinases I, II, and H. The occurrence and ratios of the protein kinases classified suggest an ordered distribution over the diverse subcellular fractions of HeLa cells. The overall pattern of soluble and membrane-bound histone and phosvitin kinases in extracts of cervix carcinoma tissue, the in vivo correlate of HeLa cells, closely resembled that of similar extracts of HeLa cells. HeLa cells hence appear, despite their long in vitro history, to express protein kinase activities similar to those of their in vivo ancestors, recommending them as a subject for the study of (certain) human protein kinase systems.