Fractionation of immunoglobulins by liquid-liquid partition chromatography in aqueous two-phase systems

In this paper we show that although immunoglobulins are easily precipitated in solutions containing polyethylene glycol (PEG), especially at pH's where the conformation of the proteins should be close to native, human and rabbit IgG can be solubilized in aqueous dextran/PEG two-phase systems containing glycine and sodium chloride at pH 7.0 and that human IgA and IgM can be solubilized in such systems if the pH is increased to 9.0. Liquid-liquid partition chromatography (LLPC) on Li-ParGel was used to separate immunoglobulins into subfractions. Human IgG, IgM, and IgA all gave three peaks in the system used. These results indicate the possibility of separating different classes of immunoglobulins with this method. Specific IgG antibodies isolated from a rabbit antiserum against human serum proteins gave only two peaks in the LLPC system while the total IgG population gave three, as did human IgG. Thus, partitioning of immunoglobulins seems to be related to antibody activity.     

δ and κ Opiate receptors in primary astroglial cultures from rat cerebral cortex

The effects of , , and receptor-agonists on forskolin stimulated cyclic adenosine-3, 5-monophosphate (cAMP) formation were examined in astroglial enriched primary cultures from the cerebral cortex of newborn rats. Intracellular cAMP accumulation was quantified by radioimmunoassay. Morphine was used as a -receptor agonist, D-Ala-D-Leu-Enkephalin (DADLE) as a -receptor agonist and dynorphine 1–13 (Dyn) as a -receptor agonist. Basal cAMP levels were unaffected by either the opiate agonists or the antagonists used. In the presence of the cAMP stimulator forskolin, morphine had no significant effect on the cytoplasmic cAMP levels. DADLE caused a dose related inhibition of the forskolin stimulated cAMP accumulation. The effects of this receptor stimulation was blocked with the selective antagonist ICI 174.864. In the presence of Dyn, the forskolin stimulated cAMP accumulation was inhibited in a dose related manner. This receptor stimulation was blocked with the selective antagonist MR 2266. Co-administration of DADLE and Dyn resulted in a non additive inhibition of the forskolin stimulated accumulation of cAMP. These findings indicate that astroglial enriched cultures from the cerebral cortex of rats express and -receptors co-localized ont he same population of cells, and that these receptors are inhibitory coupled to adenylate cyclase.     

Immunohistochemical localization of insulin-like growth factor I in the adult rat

Rabbit antisera against native human insulin-like growth factor I (IGF-I; somatomedin C) or a synthetic tetradecapeptide, representing the carboxyterminal amino acids 57-70 of human IGF-I, were used to map immunohistochemically the distribution of IGF-I immunoreactive material in adult rats. Both antisera were specific for IGF-I, as characterized by immunoabsorption, immunoblotting and radioimmunoassay. There was no cross-reactivity to IGF-II, relaxin or pro-insulin; substances having a high degree of structural homology with IGF-I. High IGF-I immunoreactivity was observed in spermatocytes of the testis; in oocytes, granulosa and theca interna cells of the ovary during early stages of follicle development; in some lymphocytes and in reticular cells of lymphoid and hematopoietic organs; in salivary gland duct cells; in the adrenal medulla, the parathyroid gland and the Langerhans' islets. Chondrocytes in the epiphyseal and rib growth plates and at articular surfaces showed strong IGF-I immunoreactivity. Brown but not white fat cells were stained. Nerve cells in the peripheral and autonomic nervous system showed faint to intense IGF-I immunoreactivity. In contrast, neurons and neuroglial cells in the central nervous system were generally negative; motor neurons being an exception. Erythropoietic, thrombocytopoietic and myeloic cells in the bone marrow showed IGF-I immunoreactivity, but only at defined developmental stages. Hepatocytes showed faint IGF-I immunoreactivity, but became more intensely stained after pretreatment with colchicine. The present results suggest that IGF-I is synthetized by cells in several tissues and organs in the adult rat. There was an apparent association between the localization of IGF-I and cell differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

MHC nonrestricted cytotoxic T cell clones with selective specificity from patients with transitional cell carcinoma (TCC) of the urinary bladder

Summary Lymphocytes from patients with transitional cell carcinoma (TCC) of the urinary bladder are more cytotoxic to bladder tumor cells than to a variety of control cells. This disease-related cytotoxicity has previously been shown to involve several mechanisms and different types of effector cells. To analyze further the nature of the effector cells operative in this system, peripheral blood lymphocytes from eight TCC patients were stimulated in vitro with TCC extract and cultured in the presence of interleukin 2 and allogeneic feeder cells. When tested for cytotoxicity in vitro on a target cell panel including both adherent and nonadherent cell lines, the lymphocytes killed a broad spectrum of targets in a major histocompatibility complex (MHC)-unrestricted fashion. When cloned by limiting dilution, clones were obtained which displayed a more restricted pattern of target cell killing. Some of the clones were highly but not exclusively selective for TCC-derived target cells. Phenotypically, these cells resembled mature T cells of CTL-type (CD8⁺/CD4^–). They also expressed the CD3/5 T cell antigen receptor complex but target cell killing was not MHC-restricted. The results of various inhibition experiments suggested that the CD3/TCR complex was involved in the cytotoxicity exhibited by these effector cells. However, its precise role in target cell recognition and the identification of the tumor cell structures recognised by the effector cells require further studies.     

Assignment of the gene encoding the catalytic subunit Cβ of CAMP-dependent protein kinase to the p36 band on chromosome

Summary A cDNA for the human catalytic subunit (C) of cAMP-dependent protein kinase (PKA) has been cloned from a testis cDNA library. In the present study, we have determined the chromosomal localization of this gene using a cDNA for C as a probe. Southern blot analysis of genomic DNA from human/mouse cell hybrids revealed that the presence or absence of a 20-kbXbaI fragment, which hybridized with the C probe, was concordant with the presence of human chromosome 1.In situ hybridization to metaphase chromosome confirmed the somatic cell hybrid data and regionally mapped the C gene of PKA to the p36 band on chromosome 1.     

Production of ψ-linolenic acid by the fungus Mucor rouxii on cheap nitrogen and carbon sources

Summary The production of -linolenic acid (GLA) by the fungus Mucor rouxii CBS 416.77 was studied on low budget nitrogen and carbon sources, i.e. rape meal, cocos expeller and two types of yeast extract (nitrogen sources), and starch, starch hydrolysate, beet molasses and cocos expeller (carbon sources). As references, Difco yeast extract and glucose were used. In flask cultivations the three yeast extracts were fully interchangeable, while the Difco yeast extract (the most expensive of those tested) gave a higher productivity of GLA in fermentor cultures (14 mg·l^–1·h^–1). The yield of lipids and GLA were increased in the order yeast extract < rape meal < cocos expeller. Thus the amount of lipid increased from 0.56 to 2.8 g·l^–1, and that of GLA from 0.15 to 0.33 g·l^–1. Use of beet molasses or cocos expeller as carbon sources gave poor growth. Starch and starch hydrolysate resulted in better productivity of GLA than glucose (4.7 and 4.9 compared to 3.4 mg·l^–1·h^–1). Offsprint requests to: A.-M. Lindberg     

Sex pheromone perception in male pine sawflies,Neodiprion sertifer (Hymenoptera; Diprionidae)

Summary Electroantennographic and single sensillum recordings were performed on male pine sawfly, Neodiprion sertifer, antennae. Responses to the sex pheromone component (2S, 3S, 7S)- 3,7-dimethyl-2-pentadecenyl (diprionyl) acetate (SSS:OAc), to the behavioral inhibitor (2S, 3R, 7R)-diprionyl acetate (SRR:OAc), to the six other enantiomers of diprionyl acetate, and to the biosynthetic precursor diprionol were recorded. Responses to trans-perillenal, a monoterpene identified in female gland extracts and to (2S, 3S, 7S)-diprionyl propionate (SSS:OPr), a field attractant for N. sertifer and some related sawfly species were also recorded.EAG recordings demonstrated a high antennal sensitivity to SSS:OAc and to SSS:OPr. A somewhat lower response was elicited by SRR:OAc.Single sensillum recordings revealed 8–12 different cells firing in each sensillum, corresponding to the number of cells observed in earlier morphological investigations. Out of these cells all, except one, responded to SSS:OAc and to SSS:OPr. No differences in the response to the two components could be observed. The largest amplitude cell in each sensillum was specifically tuned to the behavioral antagonist, SRR:OAc. The pheromone perception system encountered in male pine sawflies thus differs clearly from that observed in moths.Abbreviation EAG electroantennogram - OAc acetate - OPr propionate     

A simple vertical gill net system for variable current conditions

A new technique for suspending vertical gill nets is described. The system is designed to minimize the effects of currents, which otherwise could lift the lead line off the bottom or pull the floating line down.For more than two years, 30 m deep gill nets have been used in a Baltic Sea coastal region and have been found to work well in current, wave and wind conditions characteristic of this area. Setting a net takes about 5 minutes and retrieving it 10 minutes, even under relatively windy conditions (10–15 ms^–1). No special equipment is required to set or to haul a net, and we have used a variety of boats, from a small rowing boat to a 16 m long research vessel. The total material cost per net is about US $100.     

A polarized epithelial cell mutant deficient in translocation of UDP-galactose into the Golgi complex

Two lectin-resistant mutants derived from a polarized epithelial cell line have been described (Meiss, H.K., Green, R.F., and Rodriguez-Boulan, E.J. (1982) Mol. Cell. Biol. 2, 1287-1294). One of these mutants, the Madin-Darby canine kidney strain II cell line resistant to Ricinus communis agglutinin (MDCKII-RCAr), has been further characterized, and the biochemical defect leading to its altered phenotype has been determined. MDCKII-RCAr cells are shown to be enriched in cell-surface glycoconjugates bearing terminal N-acetylglucosamine residues by in vitro exogalactosylation and by labeling with fluorescent lectins. Binding assays with a sialic acid-specific lectin reveal a 70-75% reduction in sialylation of cell-surface glycoconjugates. The defect is pleiotropic in nature, affecting glycoproteins as well as glycosphingolipids. Analysis of glycosphingolipids shows a strong reduction of galactose-containing glycosphingolipids. Almost 90% of the glycosphingolipids are identified as glucosyl-ceramide. The mutant is not deficient in galactosyl- and sialytransferase activities. However, Golgi vesicles isolated from MDCKII-RCAr cells translocate UDP-galactose at only 2% of the rate observed for vesicles from wild-type MDCKII cells. The deficiency is specific, because translocation rates of UDP-N-acetylglucosamine and CMP-sialic acid are comparable for vesicles isolated from MDCKII-RCAr cells and wild-type cells. Despite the inability to translocate UDP-galactose into the lumen of the Golgi apparatus, MDCKII-RCAr cells are able to form monolayers with normal apical and basolateral polarity as shown by plasma membrane domain-restricted exogalactosylation.