Improved analysis of hemagglutination assays for quantitation of lectin activity

A method to quantitate lectin activity based on hemagglutination assay in microtiter plates is described. In addition to the normal method of visual titer evaluation an electronic particle counter is used for counting of nonagglutinated cells in the microtiter wells; this allows a rapid, quantitative determination of the amount of lectin required to agglutinate 50% of the countable single cells. It is also recommended that counting results should be related to a standard curve of concanavalin A to improve the reproducibility of the assay.     

Altered surface glycoproteins in melanoma cell variants with reduced metastasizing capacity selected for resistance to wheat germ agglutinin

Variants of B 16-F 1 mouse melanoma cells selected for resistance to wheat germ agglutinin (WGA) toxicity $\text{in}\text{vitro}$ were found to have undergone a stable surface change correlated both to lectin resistance and reduced metastasizing potential. The surface alteration, as indicated by the increased electrophoretic mobilities of several lactoperoxidase-iodinated cell surface proteins in SDS-PAGE, was restricted to polypeptides able to interact with WGA. The availability of lectin-resistant melanoma cell variants having altered metastasizing behavior provides a promising approach to studies of the role of specific cell surface components in the metastasizing process.     

Growth of Fusarium moniliforme on n-Alkanes: Comparison of an immobilization method with conventional processes

Summary In submerged culture there was negligible growth of Fusarium moniliforme with either n-tetradecane or gasoil (C₁₃–C₁₉) as the only carbon and energy source. In surface culture the cell yield was about 0.25 g dm^–3 dry weight after four weeks incubation. Some oxidation products, mainly isomeric tetradecanones (4-one, 5-one, 6-one and 7-one), could be identified. However the cell yield in a trickle-flow column was about 3 g dm^–3 dry weight after 7 days. Only traces of oxidation products could be detected. In a fixed-bed reactor, filled with glass rings, cell yields were similar to those in the trickle-flow column and depended on the medium flow rate.After termination of growth in the fixed-bed reactor, similar amounts of gibberellic acid were produced in a nitrogen-free medium with either gasoil or glucose.     

Progesterone in the uterus. VIII. Uptake of corticosterone and incorporation of progesterone under concurrent injection of corticosterone into rat uterus

A study of the subcellular distribution of radioactivity in rat uterus after injection of labelled corticosterone showed that the radioactivity was observed in all fractions from 5 min. to 120 min. A maximum uptake was observed 10 min. after application of the labelled steroid. Competitive uptake of radioactive progesterone and unlabelled corticosterone was assayed 10 min. after injection of the hormone mixture. The ratio between radioactive progesterone and unlabelled corticosterone was 1 : 1 and 1 : 2 (moles:moles), respectively. Compared with control experiments with rats which had received radioactive progesterone alone, the results gave evidence that progesterone found in all subcellular fractions and in the total homogenate was not depressed by unlabelled corticosterone. However, unlabelled progesterone reduced the tritiated progesterone in uterine tissue. This observation demonstrates that the uptake of progesterone by rat uterus is specific.     

Isolation and purification of bacterial membrane proteins by the use of organic solvents: The lactose permease and the carbodiimide-reactive protein of the adenosinetriphosphatase complex of escherichia coli

Techniques for the solubilization and fractionation of integral membrane proteins have been developed in recent years. A small portion of membrane protein (about 2%, proteolipid fraction) will partition into chloroform or 1-butanol, and, in several cases, these proteins retain functional activity. A virtually complete solubilization can be achieved at neutral pH by use of aprotic solvents, like hexamethylphosphoric triamide or N-methylpyrrolidone. At relatively low concentrations (< 3 M) aprotic solvents inhibited β-D-galactoside transport by whole cells and the derivative membrane vesicles of Escherichia coli, but this inhibition could be largely reversed by a simple washing procedure. At higher concentrations of aprotic solvent (5–6 M), 50–80% of the total protein of lactose transport-positive membrane vesicles was solubilized. When these extracts were added to intact lactose transport-negative membrane vesicles, lactose transport was reconstituted, the required energy being provided by either respiration (e.g., addition of D-lactate) or by a K⁺ diffusion potential established with the aid of valinomycin. The dicyclohexylcarbodiimide (DCCD)-reactive subunit of the E. coli ATPase complex was found to partition into chloroform, and to be amenable to further purification in organic solvent. Ether precipitation and chromatography on DEAE-cellulose and hydroxypropyl-Sephadex G-50 yielded an homogeneous polypeptide of an apparent molecular weight of 9,000. The purified and unlabeled DCCD-reactive protein was incorporated into K⁺-loaded liposomes, and a membrane potential was generated by the addition of valinomycin. There are indications that the DCCD-reactive protein alone made the membrane specifically permeable for protons.     

THE KINETICS OF CONTACT INHIBITION IN MAMMALIAN CELLS

To elucidate the process of contact inhibition in mammalian cells, we investigated the kinetics of growth arrest in [³H]thymidine labelled embryonic chinese hamster (Cricetulus griseus L.) cells after the addition of various concentrations of unlabelled cells. It was observed that after the contact inhibition concentration had been reached, the cells grew undisturbed for one more generation. In the following 24 hr the concentration fell back to the level at the beginning of the experiment and stayed there.     

Recombinant human [Cys281]insulin-like growth factor-binding protein 2 inhibits both basal and insulin-like growth factor I-stimulated proliferation and collagen synthesis in fetal rat calvariae.

It is recognized that insulin-like growth factors (IGFs) are bound to specific high-affinity insulin-like growth factor-binding proteins (IGFBPs). The role of IGFBPs in bone metabolism is not well established. The effect of recombinant human [Cys281]IGFBP-2 ([Cys281]rhIGFBP-2) on bone formation in 21-day-old fetal rat calvariae was investigated. [Cys281]rhIGFBP-2 was expressed in and purified from conditioned medium of a clonal Chinese hamster ovary cell line. IGF-I-stimulated cell proliferation was inhibited dose dependently by [Cys281]rhIGFBP-2, with half-maximal inhibition observed at 2 x 10(-8) M. Suppression of the IGF-I-stimulated DNA synthesis was observed at an apparent dose ratio of 1:10. [Cys281]rhIGFBP-2 (10(-6) M) also inhibited the basal incorporation of [3H]thymidine into DNA by up to 45%. Insulin-stimulated cell proliferation was not affected in the presence of the binding protein. In addition, [Cys281]rhIGFBP-2 inhibited bone collagen synthesis under basal and IGF-I-stimulated conditions. In contrast, [Cys281]rhIGFBP-2 did not alter the parathyroid hormone-stimulated bone cell proliferation rate. In conclusion, binding of hIGF-I to rhIGFBP-2 results in an inhibition of the actions of free IGF-I on bone cell replication and matrix synthesis. Parathyroid hormone-stimulated cell proliferation is not mediated by an increase in free IGFs.     

Fractionation of Primary Cultured Cerebellar Neurons: Distribution of Sialyltransferases Involved in Ganglioside Biosynthesis

Primary cultured neurons were fractionated using sucrose density gradients. The activities of four sialyltransferases (GM3, GD3, GD1a, and GT1a synthase) involved in ganglioside biosynthesis were assayed in the collected fractions. The distribution of GM3 synthase coincided with that of mannosidase II, an enzyme assumed to be a cis-Golgi marker. Both enzymes were mainly associated with the more dense fraction. GD1a and GT1a synthase activities, on the other hand, were mainly recovered in the less dense fraction. Moreover, they were colocalized with thiamine pyrophosphatase, an enzyme assumed to be a marker of the late Golgi (trans-Golgi and trans-Golgi network). GD3 synthase activity was equally distributed between both fractions. These results are integrated in a model of ganglioside biosynthesis.     

Molecular cloning of the Ig-α subunit of the human B-cell antigen receptor complex

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M74721.