Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

Subtypes of non-transformed human mammary epithelial cells cultured in vitro: histo-blood group antigen H type 2 defines basal cell-derived cells

Abstract. Normal (non-transformed) human mammary epithelial cell lines derived from reduction mammoplasties were analyzed by immunocytochemistry with more than 80 monoclonal antibodies (mAbs) and other specific reagents to tissue-specific and developmentally regulated antigens at different passage levels. A subpopulation of poorly differentiated, proliferating epithelial cells, corresponding to the 'selected' cell type of late passages, is shown to be characterized by a new marker, the histo-blood group antigen H type 2, probably carried on a membrane-bound glycolipid. These cells also express a number of other onco-developmental carbohydrate antigens [Le^y, Le^x, sialosyl-Le^a, precursor of Thomsen Friedenreich antigen (T_n), but not Thomsen-Friedenreich antigen and sialosyl-T_n]. Their cytokeratin (CK) phenotype, as assessed by reactivity with monospecific mAbs and two-dimensional gel electrophoresis, is CK 5, 6, 14 and 17, with CK 19 being consistently absent, and varying minor amounts of CK 7, 8 and 18, as well as 15 and 16. The reactivity of these cells with a panel of 11 mAbs specific for CK 18 varies considerably even after cloning, indicating heterogeneity of epitope expression or accessibility. Our data strongly suggest that the H type 2⁺ cells develop from the basal cell layer of the mammary gland.     

Heterogeneity in the mouse epidermal cell cycle analysed by computer simulations

Abstract. Different sets of cell kinetic data obtained over many years from hairless mouse epidermis have been simulated by a mathematical model including circadian variations. Simulating several independent sets of data with the same mathematical model strengthens the validity of the results obtained. The data simulated in this investigation were all obtained with the experimental system in a state of natural synchrony. The data include cell cycle phase distributions measured by DNA flow cytometry of isolated epidermal basal cells, fractions of tritiated thymidine ([³H]TdR) labelled cells within the cell cycle phases measured by cell sorting at intervals after [³H]TdR pulse labelling, bivariate bromodeoxyuridine (BrdUrd)/DNA data from epidermal basal cells isolated at intervals after pulse labelling with BrdUrd, mitotic rate and per cent labelled mitosis (PLM) data from histologic sections. The following main new findings were made from the simulations: the second PLM peak observed at about 35 h after pulse labelling is hardly influenced by circadian variations; the peak is mainly determined by persisting synchrony of a rapidly cycling population with a G₁-duration (T_G1) of 20 h to 30 h; and there is a highly significant population of slowly cycling G₁-cells (G_1σ). However, no significant circadian variations were found in the number of these cells.     

Age-related changes in responsiveness of rat Leydig cells to hCG

The responsiveness of decapsulated testes and isolated Leydig cell preparations from rats (30-80 days of age) to a constant dose of 3 ng hCG/2 ml was assessed by comparison of the production of testosterone and "total 17beta-hydroxy androgen" (17beta-HA). When testosterone secretion was used as the index of response, there was a marked increase in the production with age by decapsulated testes and also by equal numbers of Leydig cells. When 17beta-HA was taken as the response parameter this increase was only marginal for the decapsulated testes and there was an age-dependent decrease when expressed per 10(6) cells. These differences probably reflect changes in the metabolism of testosterone to 5alpha-reduced products with increasing age because 80% of androgen secreted at 30 days is 3alpha-androstanediol and 86% is secreted as testosterone at 80 days. We conclude that for studies on hCG responsiveness and the steroidogenic capacity of immature rat Leydig cells (a) testosterone is an inappropriate response parameter and (b) this response undergoes a decrease rather than an increase during prepubertal development.     

Effects of bleomycin on the epidermal content of growth-regulatory substances (chalones).

Hairless male mice were given 2 mg Bleomycin i.p. on two successive days. At different time intervals from 1 to 10 days after the last Bleomycin injection, groups of animals were killed and water extracts of hemogenized skin were made. These extracts, supposed to contain the epidermal G1 and G2 chalones, were injected into female hairless mice, and their growth inhibitory potency determined by two methods. 5 mg of lyophilized crude skin extract were injected i.p. together with Colcemid, and the animals killed 4 hr later. The number of Colcemid-arrested mitoses was determined, and was considered to be a measure of the G2 inhibitor present in the skin extracts. 10 mg of the same extracts were injected i.p., and these animals also got 3H-TdR i.p. 12 hr later, and were killed after a subsequent 30 min. The epidermal LI was determined, and was considered to be a measure of the epidermal G1 factor in the skin extracts. The results obtained were compared to the effect of Bleomycin alone and to the effects of skin extracts from non-Bleomycin-treated animals. The results show that Bleomycin provoked slight alterations in the growth-inhibitory potency of the G1 chalone, whereas significant effects were seen in the G2 chalone, There was an increased amount of growth-inhibiting factors on days 2 and 3, and on days 8-10. The results are discussed and it is concluded that the most probable hypothesis is that Bleomycin, in addition to its known inhibition by accumulation of cells with high growth inhibitory potency. An initial, additional direct effect of Bleomycin on the chalone system cammot be excluded.     

Estimation of levels of IgG to multiple sclerosis specific brain antigens in the cerebrospinal fluid of MS patients

The binding of partially purified multiple sclerosis (MS) specific brain antigens (MSG2) and of the corresponding antigens of non-MS brains (KG2) to cerebrospinal fluid IgG of patients with MS and other neurological diseases was assayed employing sandwich enzyme linked immunosorbent assay (ELISA). Assay of the antigen-antibody binding revealed that the concentration of MSG2 required for the optimum binding to IgG in the undiluted MS CSFs was lower than that of KG2 in all cases. The index for IgG binding capacity of an antigen (IgBC) was expressed as a ratio of the optical density of the enzymic products in ELISA at the optimal antigen-antibody binding to the lowest concentration of the antigen required for the optimal binding. The IgBC of MSG2 was found to be linearly correlated with the IgG concentration in the CSF of MS patients. These results indicate that IgG with specificity to MSG2 may be present in the CSF of MS patients.     

Electrogenic bicarbonate secretion in the turtle bladder: Apical membrane conductance characteristics

Summary We have recently shown that stimulation of electrogenic HCO ₃ ^– secretion is accompanied by a simultaneous increase in short-circuit current (I _sc, equivalent to HCO ₃ ^– secretion rate under these conditions), apical membrane capacitance (C _a , proportional to membrane area), and apical membrane conductance (G _a , proportional to membrane ionic permeability). The current experiments were undertaken to explore the ionic basis for the increase inG _a and the possibility that the rate of electrogenic HCO ₃ ^– secretion is regulated by changes inG _a . Membrane electrical parameters were measured using impedance-analysis techniques before and after stimulation of electrogenic HCO ₃ ^– secretion with cAMP in three solutions which contained different chloride concentrations. In another series of experiments, the effects of an anion channel blocker, anthracene-9-carboxylic acid (9-AA), were measured after stimulation of electrogenic HCO ₃ ^– secretion with cAMP. The major conclusions are: (i) a measurable apical Cl^– conductance exists in control hemibladders; (ii) the transport-associated increase inG _a includes a Cl^–-conductive component; (iii)G _a also appears to reflect a HCO ₃ ^– conductance; (iv) the relative magnitudes of the apical membrane conductances to Cl^– and HCO ₃ ^– are similar; (v) 9-AA reducesG _a andI _sc appear cAMP-stimulated hemibladders; and (vi) alterations inI _sc appear to be mediated by changes inG _a .     

Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuc alpha 1----2Gal alpha 1----3-N-acetylgalactosaminyltransferase from human lung tissue

The soluble histo-blood group A glycosyltransferase (Fuc alpha 1----Gal alpha 1----3-N-acetylgalactosaminyltransferase) was purified approximately 600,000-fold to homogeneity from human lung tissue. The enzyme was solubilized in 1% Triton X-100, partially purified by affinity chromatography on Sepharose 4B, and eluted with UDP. Final purification was obtained by twice repeated fast protein liquid chromatography ion exchange (Mono STM) with NaCl gradient elution and reverse-phase chromatography (proRPC) with acetonitrile gradient elution. Identity of the purified protein was established by (i) demonstration of the putative A transferase protein only in affinity-purified extracts of A but not O individuals, and (ii) specific immunoprecipitation of enzyme activity and putative protein with monoclonal antibodies. Sodium dodecyl sulfate electrophoresis revealed a single protein band with apparent Mr of approximately 40,000 under both reducing and nonreducing conditions. Digestion with N-glycanase yielded a reduction in Mr of approximately 6,000 (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), suggesting that the A transferase is a glycoprotein with N-linked carbohydrate chains. Amino acid composition and N-terminal amino acid sequence of the intact transferase, as well as of peptides released by endolysyl peptidase digest or cyanogen bromide cleavage, are presented.