Heterogeneity of rat TCGF defined by Mono P isoelectric focusing.

Interleukin-2 (IL-2) prepared from Con A-activated rat spleen cells was partially purified using hydroxylapatite chromatography (HTP) and chromatofocusing on Mono P. IL-2 eluted in a major peak between 0.1 and 0.25 M NaCl in PBS (purification factor 36-fold) and in a second peak in the high salt elution (purification factor 5-fold). When analysed on Mono P, the major peak was found to resolve into four components with apparent pI values in the range of 7.05-5.80; further activity eluted in the high salt fraction. Similar patterns were observed using high salt eluted activity with minor variations in the apparent pI values. Neuraminidase treatment caused a shift in IL-2 charge towards more basic pI values. This analysis of the multiple species of IL-2 suggests that part of the heterogeneity may be due to variation in the degree of sialylation of the peptide chain.

Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay.

Precursor forms of the alpha-subunit of inhibin are abundant in human follicular fluid and possibly plasma, although their function is uncertain. We now describe the development of a new enzyme-linked immunosorbent assay to measure inhibin forms containing both the pro and alpha C regions of the alpha-subunit. The assay has a detection limit for purified human pro-alpha C of 0.5 pg/mL and less than 0.02% cross-reaction with recombinant forms of inhibin, activin, and follistatin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of follicular fluid extracts demonstrated that the assay is likely to detect pro-containing precursor forms of both the free alpha-subunit and intact dimeric inhibin. The serum concentration was measured in normal men (446 +/- 28 pg/mL), postmenopausal women (45.8 +/- 3.8 pg/mL), and women treated with FSH before in vitro fertilization (1827 pg/mL). Pooled human follicular fluid contained 488 ng/mL. The mean serum concentration in the female menstrual cycle rose from 150.6 +/- 26.1 pg/mL in the early follicular phase to 692.2 +/- 113 pg/mL in the midluteal phase. This assay offers a useful tool for investigation of the role of inhibin-related proteins in human reproduction. There may be particular clinical value under circumstances in which other assays for inhibin forms have insufficient sensitivity.

A calcium pump at the higher plant nuclear envelope?

Evidence for a Ca2+-pump at the nuclear envelope (NE) in plant cells has been obtained using confocal and electron microscope immunocytochemistry and antibodies raised to a plant homologue of the mammalian SERCA pump. This is the first evidence suggesting an NE Ca2+-pump in plants. In addition to being localised with the NE in interphase, the antigen was localised to membrane derived from the NE and associated ER during mitosis, correlating with known Ca2+-pools. The work suggests that a SERCA pump is present at the NE of plant as well as animal cells.

8-Cl-adenosine is an active metabolite of 8-Cl-cAMP responsible for its in vitro antiproliferative effects on CHO mutants hypersensitive to cytostatic drugs.

8-Cl-cAMP has been undergoing clinical trials as a potential chemotherapy agent, but there is much discussion in the literature as to whether the active agent is 8-Cl-cAMP itself, or its major metabolite, 8-Cl-adenosine. 8-Cl-cAMP is susceptible to the action of serum enzymes such as phosphodiesterases, and its metabolism when administered to cancer patients raises questions as to the mechanism of action of 8-Cl-cAMP. The stability of 8-Cl-cAMP when incubated with serum, and the effects of both 8-Cl-cAMP and 8-Cl-adenosine on the proliferation of variant lines of CHO cells hypersensitive to 8-Cl-cAMP were investigated. A solid-phase extraction (SPE) purification protocol and the HPLC method previously developed were used to determine 8-Cl-cAMP and 8-Cl-adenosine. Heat treatment of serum inactivated the enzymes in the culture medium responsible for activating 8-Cl-cAMP. Under these conditions 8-Cl-cAMP remained stable and there were no traces of its metabolite, 8-Cl-adenosine. Cell culture experiments showed that 8-Cl-cAMP only affected cell growth in medium that contained untreated serum. In contrast, 8-Cl-adenosine was shown to be growth inhibitory in medium containing either heat-treated or untreated serum. HPLC analysis of the culture medium from the cell culture experiments supported the hypothesis that 8-Cl-cAMP was only effective in inhibiting cell growth after metabolism to 8-Cl-adenosine. Thus further studies of this drug and its mechanism of action should focus on 8-Cl-adenosine.

The preparation of human intrinsic factor-cobalamin complex from human gastric juice by immunoadsorption.

Immune complexes of human intrinsic factor were prepared by mixing gastric juice saturated with vitamin B12, and sera from patients with pernicious anaemia that had a high proportion of binding (Type II) antibody. The complexes were isolated by sodium sulphate precipitation followed by Sephadex G-150 gel filtration. Acid conditions dissociated the immune complexes and allowed separation of specific antibody and purified antigen bound to vitamin B12 by Sephadex G-200 gel filtration. Specific antibody was covalently attached to Protein A Sepharose CL-4B by coupling with water soluble carbodiimide which allowed intrinsic factor-B12 complex to be purified directly from gastric juice. The intrinsic factor obtained after iodination, ran as a single band on SDS-polyacrylamide gel electrophoresis and was biologically active.

Production and characterization of separate monoclonal antibodies to human brain and erythrocyte acetylcholinesterases.

Four murine monoclonal antibodies (MAbs) of the IgM class were raised against human acetylcholinesterase (AChE; Ec 3.1.1.7). The MAbs BMS-3E4, BMS-7G10, and BMS-9F4 all recognized human erythrocyte AChE, while BMS-6D6 bound specifically to human soluble brain AChE, on the basis of immunobinding assays. Dose-response studies gave an ELISA ED50 titer of 4.5 x 10(-4) M for BMS-6D6, while BMS-3E4 gave the best titer at 8.8 x 10(-4) M. Sucrose density gradients demonstrated sedimentation of antigen-antibody complexes, consistent with earlier findings (i.e., BMS-6D6 bound with brain AChE while BMS-3E4 preferred erythrocyte (AChE). No cross-reactivity between the two MAbs against the two antigens was noted.

Preliminary characterisation of a murine embryonal carcinoma cell derived growth promoting activity.

Embryonal carcinoma cell conditioned medium is shown to contain growth promoting molecules whose properties, both biological and biochemical, distinguish them from certain other known growth factors.

Cell-mediated immunity to a synthetic gliadin peptide resembling a sequence from adenovirus 12.

Cell-mediated immunity to a synthetic peptide, which has a 12-residue sequence from A-gliadin analogous to part of an early-region protein (Elb) from adenovirus 12, was investigated in patients with coeliac disease, healthy subjects, and disease controls by means of an indirect leucocyte-migration-inhibition assay. Patients with coeliac disease being treated with a gluten-free diet showed a significantly greater response than healthy subjects (p less than 0.001) or patients with inflammatory bowel disease. This cellular immune response was dependent on antigen concentration and was not present in untreated coeliac patients.

Comparison of 5-aminosalicylic acid and N-acetylaminosalicylic acid uptake by the isolated human colonic epithelial cell.

Isolated human colonic epithelial cell suspensions were incubated with either 0.1 mM 5-aminosalicylic acid (5-ASA) or 0.1 mM acetylaminosalicylic acid (Ac-ASA) for up to two hours. Intra- and extracellular 5-ASA and Ac-ASA were measured by high performance liquid chromatography. Mean 5-ASA uptake in one hour was 160.5 nmol/g dry weight, compared with an Ac-ASA uptake of only 5.75 nmol/g dry weight. No unchanged 5-ASA was detected inside the cell. Repeated washing had no effect on the intracellular Ac-ASA concentration. This discrepancy in drug uptake may explain why Ac-ASA seems to be ineffective when given to patients with ulcerative colitis.

Production of epithelial cell growth factors by lamina propria mononuclear cells.

The effects of lamina propria mononuclear cell culture supernatant on epithelial cell DNA synthesis were studied using cells isolated from patients with inflammatory bowel disease and normal controls. Supernatants from resting and phytohaemagglutinin stimulated cells were studied and supernatants that strongly promoted DNA synthesis were pooled, and growth factor activity partially characterised. The effects of recombinant interleukins-1 beta,2,3,interferon-gamma, and granulocyte macrophage colony stimulating factor were tested in the same system. Resting lamina propria mononuclear cells produce factors that increase DNA synthesis. Production of these factors is increased by phytohaemagglutinin stimulation. No significant differences were found in production of these factors between patients with inflammatory bowel disease and normal controls. The molecular weight of the active factor(s) lies in the region 31-48 kD. Chromatofocusing produced two peaks of activity, one in the region pk 5.5 and one around pk 6.4. The activity was heat and acid pH labile. Activity was not destroyed, however, by 0.05% trypsin. Recombinant granulocyte macrophage colony stimulating factor was a weak stimulus to epithelial DNA synthesis, interleukin-1 beta was weakly inhibitory but other cytokines tested did not have any effect. Granulocyte macrophage colony stimulating factor is probably important in controlling epithelial cell growth.

Development of a gas chromatography mass spectrometry assay for the two eosinophil chemotactic factors of anaphylaxis.

Gas chromatography mass spectrometry has been used for over a decade for the determination of the amino acid sequences of fragment peptides derived from larger parent molecules. The majority of these fragments have from four to seven residues and several different methods of derivatization have been devised. Few reports have been published in which similar techniques have been used for the quantification of such peptides, but there is a growing list of small peptides which have been shown to have biological activity in their own right. This report is concerned with the development of a gas chromatographic mass spectrometric assay for the two eosinophil chemotactic peptides, Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu, which appear to have a role to play in the course of the inflammatory process in skin disorders.

The separation and sequencing of permethylated peptides by mass spectrometry directly coupled to gas-liquid chromatography.

The use of g.l.c. coupled to mass spectrometry to separate and sequence permethylated acetyl- and trifluoroacetyl-peptides in a single operation is described. Both electron impact and chemical ionization were used to induce fragmentation, and the latter was found to be more sensitive. Chromatographic retention data are presented which suggest that peptide derivatives of molecular weight of at least 750 are accessible to the technique. The application of our methods to the determination of the primary sequence of proteins is discussed.

Acetylation of 5-aminosalicylic acid by isolated human colonic epithelial cells.

1. This study investigates the acetylation of 5-aminosalicylic acid by isolated human colonic epithelial cells. 2. After incubation of intact cells with 0.1 mmol/l 5-aminosalicylic acid, N-acetyl-5-aminosalicylic acid was detected in a concentration of 141 (+/- 23.8 SEM) nmol/g dry weight in the incubation medium, and 34.8 (+/- 5.5 SEM) nmol/g dry weight intracellularly. No unchanged 5-aminosalicylic acid was detected inside the cell. 3. Acetylation of 5-aminosalicylic acid by a cell homogenate was very poor, but the addition of 1 mmol/l acetyl-CoA resulted in complete conversion of 0.1 mmol/l 5-aminosalicylic acid to N-acetyl-5-aminosalicylic acid. 4. N-Acetyltransferase activity was detected in the cytosol, with a mean of 3.3 nmol min-1 mg-1 of protein. There was no N-acetyltransferase activity in the brush border. There was no difference in enzyme activity between epithelial cells derived from normal, Crohn's disease or ulcerative colitis patients. 5. Preliminary characterization of the N-acetyltransferase enzyme suggests a molecular weight of 150,000.

T lymphocyte responses to a synthetic peptide from the human intestinal adenovirus 12 in coeliac disease.

Peripheral blood mononuclear cells of treated coeliac patients are known to liberate leucocyte migration inhibition factor when challenged with a synthetic dodecapeptide of the E1b protein of adenovirus 12 sharing sequence homology with A gliadin. This study has compared the response of the unfractionated peripheral blood mononuclear cells with a highly purified T cell population. Coeliac mononuclear cells and T lymphocytes showed identical leucocyte migration inhibition activity. The migration indices for the coeliac group were significantly different than for the control group irrespective of the cell population tested. This work justifies the use of the indirect leucocyte migration inhibition assay using mononuclear cell supernatants as a test of in vitro cell-mediated immunity to clearly defined antigens in coeliac disease. It also offers further evidence for the possible implication of adenovirus 12 in the pathogenesis of coeliac disease.

Antigenic reactivity of peptides derived from wheat gluten with sera from patients with coeliac disease.

Fraction B from a peptic-tryptic digest of gluten from Scout 66 wheat has already been shown to cause histological damage to the jejunal mucosa of coeliac patients. Peptide fractions, designated P1-P4, have been prepared from it by a combination of gel filtration (producing an intermediate fraction pseudo-B2: psi B2) and reverse-phase high pressure liquid chromatography. An enzyme-linked immunosorbent assay (ELISA) has been used to measure IgG antibodies to fraction B in sera from untreated coeliac patients, patients with inflammatory bowel disease (IBD) and healthy individuals. The coeliac group had significantly higher (P less than 0.05) antibody levels to fraction B than either of the control groups [medians: coeliac disease (n = 21), 0.247; IBD (n = 17) 0.019; healthy controls (n = 13) 0.020]. Five coeliac sera which gave high absorbance values in the ELISA were chosen and preincubated with fraction B in a range of concentrations, before assay by ELISA: a dose-dependent inhibition of binding was found. Two sera which gave high ELISA values were preincubated with fractions B2 and P1-P4. B2, P1, P2 and P4 gave a dose-dependent inhibition, with P1 being the most potent. Absolute values were different for the two sera but the same relative pattern of reactivity was observed for each. With the serum giving the higher ELISA value the concentration of fraction (microgram/ml) giving a 50% inhibition of binding when 0.5 ml was added to 0.5 ml of a 1/500 dilution of the serum (IC50) was 2.6 for fraction B, 61 for P1, 155 for B2 and 285 and 295 for P4 and P2 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)