[Management of anal canal carcinoma]. / Prise en charge du carcinome du canal anal.

Although anal canal squamous cell carcinoma is rare, the general practitioner should consider this diagnosis in a patient with persistent lower abdominal symptoms. While classically observed in older women, an increased incidence is also seen in HIV-positive patients or patients with a history of human papillomavirus infection. Initial diagnosis and local work-up require assessment by a proctologist. Standard curative treatment combines radiotherapy with 5-FU- and MMC-based chemotherapy. Salvage surgery should be discussed in case of local relapse. The general practitioner, the proctologist and the radiation oncologist, all participate in post-treatment surveillance.

[Ovarian cancer screening: recommendations for clinical pratice]. / Dépistage du cancer de l'ovaire: recommandations pour la pratique clinique.

Ovarian cancer accounts for a minority of female cancers but remains the leading cause of death from gynaecologic cancers and the fifth leading cause of all cancer-related deaths among women. More than two thirds of cases of ovarian cancer are diagnosed once the disease becomes symptomatic, i.e. at an advanced stage. Though early detection constitutes a legitimate aim, no screening approach has yet been shown to reduce ovarian cancer mortality. In particular, ovarian imagery with endovaginal ultrasonography and serum tumor markers (CA-125) dosage performed in asymptomatic individuals have not proven their efficacy. Screening of asymptomatic women is not therefore recommended because of the limited prevalence of ovarian cancer in the general population.

[Surveillance following curative therapy for breast cancer]. / Surveillance après traitement curatif du cancer du sein.

Surveillance following curative therapy for localised breast cancer has two major aims: 1) diagnosis of recurrence or a second tumor: regular interviews and physical examinations with a frequency adapted to each case, and yearly mammography (+/- ultrasonography), are the only recommended procedure. Additional screening is not indicated in asymptomatic patient. 2) Screening for complications and side effects due to treatment: tamoxifen treatment should be stopped one week prior to any surgery or long trip to avoid thromboembolic events, and any metrorrhagia should be investigated due to the risk for endometrial cancer. Osteoporosis and arthralgia are frequently observed with aromatase inhibitors.

New procedures for glycophorin A purification with high yield and high purity.

Glycophorin A (GPA) is the major glycoprotein of the human erythrocyte membrane. It is known to form, in SDS gels as well as in a membrane environment, homodimers, and also heterodimers with the homologous molecule Glycophorin B (GPB). It is shown in this report that the propensity of GPA to dimerize with GPB precludes satisfactory preparation with high yield of pure GPA using classical techniques including SEC and RPLC. It was demonstrated using multiple angle light scattering that GPA is eluted from RPLC columns as dimers. A convenient procedure was devised which allowed us to get pure GPA with high yield. This procedure consists of selectively blocking GPA-GPB heterodimer formation by selective modification of Cysteine 50 of GPB before RPLC.

A murine monoclonal antibody against Kx protein which reacts also with beta-spectrin.

Kx is a polytopic membrane protein of human erythrocytes carrying the Kx blood group antigen, which is deficient in rare patients with McLeod syndrome. Kx is disulphide bond linked to the Kell glycoprotein, which is a bitopic type II membrane protein carrying the Kell blood group antigen. Mice immunized with a synthetic peptide predicted to be located on the second external loop of Kx produced a monoclonal antibody called 3E12 which does not recognize red cells with common Kell phenotype by agglutination and flow cytometry. 3E12 recognizes the Kx protein and the spectrin beta-chain on western blots, the affinity for these two proteins being lowered with increasing ionic strength. Linear epitopes recognized by 3E12 are E116EIEKE121 and L484AQELEKE491 on the Kx protein and spectrin beta-chain, respectively. To quantify the relative amount of Kx in Empigen BB extracts of red cell membranes, an ELISA for Kx was set up which showed conclusively that (i) there is less Kx in membranes of K0 individuals (lacking the Kell glycoprotein) than in membranes of common individuals, and (ii) that all common individuals, typed as K+k-, K-k+ and K+k+, have the same amount of Kx on their red cell membranes. When an erythrocyte membrane detergent extract from one K0 individual was chromatographed on an immobilized 3E12 column, a minute amount of authentic Kell glycoprotein was recovered in acid eluted fractions, indicating that at least the K0 individual under study may still produce some Kell protein.

Immunopurification of the blood group RhD protein from human erythrocyte membranes.

Rh proteins are membrane proteins encoded by genes at the blood group RH locus. They are of paramount importance in transfusion medicine, but their function is still unknown. Biochemical and biophysical studies of these proteins are scarce since only minute amounts of the very hydrophobic Rh proteins, can be purified from human erythrocytes. Recently, a human monoclonal antibody (LOR-15C9) was described as having the unique property to recognize the Rh30 protein carrying the major blood group D specificity (RhD protein), either in a membrane detergent extract or when blotted on a membrane. In this report, we describe one-step purification of the RhD protein from detergent extracts of red cell membranes, based on immunoaffinity chromatography carried out with immobilized LOR-15C9 IgG. The technique yielded RhD protein with high purity which was devoid of other associated proteins (RhAG, CD47, LW and GPB) that comprise the Rh complex in the erythrocyte membrane. By contrast immunoprecipitation performed with the same antibody led to co-isolation of both RhD and RhAG.

Kx, a quantitatively minor protein from human erythrocytes, is palmitoylated in vivo.

Kx is a quantitatively minor blood group protein of human erythrocytes which is thought to be a membrane transporter. In the red cell membrane, Kx forms a complex stabilized by a disulfide bond with the Kell blood group membrane protein which might function as a metalloprotease. The palmitoylation status of these proteins was studied by incubating red cells with [3H] palmitic acid. Purification of the Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity demonstrated that the Kx but not the Kell protein is palmitoylated. Six cysteines in Kx are predicted to be intracytoplasmic and might be targets for palmitoylation. Three of these cysteines are present in a portion of sequence which is predicted to form an amphipathic alpha helix. Palmitoylation of one or several of these cysteines might contribute to anchor the cytoplasmic portion of the Kx protein to the inner surface of red cell membrane.

Screening of a large number of dyes for the separation of human immunoglobulin G2 from the other immunoglobulin G subclasses immunoglobulin G2 enrichment on immobilized Procion Yellow HE-4R.

Human immunoglobulins (IgG) are produced on a multi-ton scale for therapeutic applications. There is presently no available method to manufacture IgG preparations enriched with immunoglobulins from the IgG2 subclass although they might be useful for therapeutic purposes. By frontal chromatography, we have screened 69 immobilized dyes, among which, six display a different affinity for IgG2 and other subclasses. One (Procion Yellow HE-4R) was studied further. The screening of various mobile phase conditions allowed us to devise a procedure to prepare IgG2 enriched IgG solutions: The cumulative yield for IgG2 was 43% and IgG2/total IgG ratio in the final product was 67%.

Toxicity studies on native Procion Red HE-3B and released dye from affinity material exposed to degradative chemical conditions.

Leached ligands from chromatographic packing material submitted to drastic regeneration conditions can contaminate pure biological preparations. These contaminants could have adverse effects from a toxicology point of view that are very poorly documented in liquid chromatography for protein separation. Investigations on toxicity level have been made on released material from immobilized Procion Red HE-3B, after formal identification of the nature of the leached chemical material. Toxicity investigations in vitro involved a number of tests on living cells (eucaryotic and procaryotic) covering different aspects. Behaviour of cells in regular cultures, polyploïdia induction, genotoxicity as well as mechanisms of endocytosis have been studied. Results showed no toxic effects within the range of concentration of dye and dye derivatives studied. Genotoxicity studies in particular did not show any toxic effect over a range of concentration much higher than the regular level of dye leakage from the sorbent.

Toxicity studies on Reactive Blue-2 leached from affinity material exposed to extreme chemical conditions.

Toxicity effects related to leached ligands from affinity sorbents that can contaminate biological preparations were investigated in the particular case of immobilized Reactive Blue-2. Initially, identification of the real chemical structure of leached dye has been done by HPLC after incubation in extreme conditions. Toxicity investigations in vitro involving several well known tests showed no toxic effects within the studied range of dye concentration. Cell cultures behaved normally when the adhesion phase was successful; polyploidy induction in human cells by the native dye and its derivatives identified as possible leached material was very similar to standard cultures. Genotoxicity studies did not evidence any toxic effect in E. Coli cultures of dyes themselves or of the same dyes after metabolic activation.

Chromatography of human immunoglobulin G on immobilized drimarene rubine R/K-5BL. Study of mild, efficient elution procedures.

A range of substances were screened to find eluents for human immunoglobulin G (IgG) which are retained with a strong affinity by immobilized Drimarene Rubine R/K-5BL. The strong affinity of IgG for the dye is partly due to the presence of copper in the dye. This was suggested by the effect of substances able to make coordination bonds with metals that elute the IgG and also the effect of metal stripping from the immobilized dye. Several mobile phase conditions were found that allowed desorption of retained IgG on immobilized Drimarene Rubine R/K-5BL without using a protein denaturant. A procedure was also devised for separating IgG2 from other IgG subclasses using chromatography on immobilized Rubine R/K-5BL and column development with an AMP gradient.

Evaluation of several affinity chromatographic supports for the purification of maltose-binding protein from Escherichia coli.

To obtain affinity adsorbents with good mechanical resistance, suitable for the purification of maltose-binding protein (MBP) from Escherichia coli and genetically engineered proteins fused to MBP, a series of supports were prepared by grafting amylose on to agarose by different chemistries. Their capacities for MBP and their abilities to be used at relatively high flow-rates were examined. Efficient supports were most conveniently prepared by coupling amylose to epoxy-activated agarose in an aqueous-organic mixture.

Plasminogen receptors on rat colon carcinoma cells.

Cells from rat carcinoma cell lines PROb (giving progressive tumours) and REGb (giving regressive tumours) have cell surface receptors which bind specifically rat plasminogen and plasmin. Affinity for Pg was found to be higher in PROb (Kd = 10(-7) M) than in REGb cells (Kd = 5.10(-7) M) but with a concomitant decrease in the number of binding sites, 0.9 x 10(6)/cell (range from 0.6 to 1.2 x 10(6)) in PROb vs 3.6 x 10(6)/cell (range 1.2 to 6 x 10(6)) in REGb cells. The number and the affinity of binding sites varied in an opposite way in PROb and REGb cells. The difference in affinity parameters was unrelated to the degree of invasiveness of tumour cells in syngenetic rats. Bound plasmin retained its enzymatic activity, which indicates that its binding does not involve the catalytic active site. In cell solubilisates plasminogen receptor appeared as one major band situated in the area of 50-60 kDa.

High-performance hydrophobic interaction chromatography of proteins on reversed-phase supports coated with non-ionic surfactants of polyoxyethylene type. Purification of a fungal aspartic proteinase.

On coating reversed-phase supports with polyoxyethylene-type non-ionic surfactants, proteins are no longer retained on such supports at moderate or low ionic strength, but they are retained at high ionic strength and can be desorbed by a decreasing ionic strength gradient. These reversibly modified supports were used for hydrophobic interaction chromatography (HIC). The proteins probably interact with the polyoxyethylene tail of the non-ionic surfactant while the hydrophobic part of the surfactant anchors the surfactant to the reversed-phase support by interactions with its alkane coverage. Although the interactions between non-ionic surfactant and reversed-phase support are non-covalent and the HIC mobile phases contained no surfactant, the modified columns were stable and could be used repeatedly. A surfactant-modified reversed-phase column provided a rapid, efficient, one-step purification of a fungal aspartic proteinase from a commercial crude preparation.

New strategies for the screening of a large number of immobilized dyes for the purification of enzymes. Application to the purification of enzymes from human haemolysate.

A method is presented for screening immobilized dyes applicable to the purification of enzymes from haemolysate (haemolysate can be considered as a nearly pure solution of haemoglobin containing only marginal amounts of enzymes). Haemolysate is loaded on immobilized dye mini-columns until haemoglobin and the studied enzymes are found in the column eluate at the same concentrations as those present in the haemolysate. Such a frontal mode of screening allows those dyes to be selected which, displaying a higher affinity for the enzyme of interest than for haemoglobin, can be used to displace the unwanted protein (haemoglobin) from the column by the enzyme of interest (present at a much lower concentration).

Purification of human 6-phosphogluconate dehydrogenase from human haemolysate with chromatography on an immobilized dye as the essential step and use of automation. Simultaneous purification of lactate dehydrogenase.

The screening procedure described in the preceding paper allowed a practical purification procedure to be devised that was automated for human 6-phosphogluconate dehydrogenase. The purification needed only two chromatographic steps, first on immobilized Procion Blue HE-GN and then on Phenyl-Sepharose. This technique also gave purified lactate dehydrogenase. Both enzymes showed single bands in SDS polyacrylamide gel electrophoresis.

Protein precipitation induced by a textile dye. Precipitation of human plasminogen in the presence of Procion Red HE3B.

It was demonstrated that human purified plasminogen was precipitated in the presence of the textile dye Procion Red HE3B. The amount of precipitated plasminogen was dependent upon the molar ratio between the dye and protein and seemed to be independent of the protein concentration. A certain amount of dye was coprecipitated with the protein; this was shown also to be related to the dye-to-protein molar ratio. The precipitation of plasminogen induced by the dye was shown to have a pH optimum and to involve ionic and hydrophobic interactions. Procedures were devised which enabled the recovery of precipitated plasminogen in a soluble and non-denatured form totally free from dye. However, the precipitation of plasminogen by Procion Red HE3B could not be used as a single-step purification procedure from a heterogeneous starting material like plasma because of coprecipitation of other proteins. Nevertheless it is suggested that frequently there is a chance that a given protein will be precipitated by a given dye and therefore that protein precipitation by dyes could be a useful complementary method for the purification of proteins.

Bicarbonate-stimulated ATPase activity of bovine liver alkaline phosphatase.

A commercial preparation of bovine hepatic alkaline phosphatase was found to have a Mg2+-stimulated ATPase activity. The pH optimum was 8.5, the Km for ATP was 4.2 X 10(-5) M and the Vmax was 88.3 mumol Pi . h-1 . mg-1. HCO3- had a stimulatory effect on Mg2+ -ATPase activity. Other anions had no effect or an inhibitory effect while Na+, K+ and ouabain had no effect. Purification of the commercial preparation by gel filtration and affinity chromatography yielded a fraction with alkaline phosphatase and (Mg2+ + HCO3-)ATPase activities that had been enriched respectively 27-fold and 23-fold; both activities were inhibited by levamisole (93.1% and 93.8%, respectively) and the purified fraction was found to be a single protein on sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results suggest that alkaline phosphatase and (Mg2+ + HCO3-)ATPase may be properties of the same liver protein that might be involved in biliary HCO3- transport and bile secretion.

Automated purification of human erythrocytic 6-phosphogluconate dehydrogenase.

Human 6-phosphogluconate dehydrogenase (6PGD) was purified from hemolyzate by group affinity chromatography on 2',5'-ADP-Sepharose, followed by buffer exchange chromatography on Sephadex G-25 and finally salting-out chromatography on Sepharose 6B. An apparatus was assembled from commercially available elements, in which the purification procedure can proceed and be completed unattended and under fully automatic control. The weekly production of the set-up is at present 10 mg of pure 6PGD. The choice of the purification of procedure and the advantages of automation are discussed.