A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 2. Effect of VIP on cAMP production and on cell-surface VIP-binding sites

Vasoactive intestinal peptide (VIP) stimulated in a dose-dependent manner the accumulation of cAMP in human melanoma-derived cell line IGR39. The maximal effect (about 100 times the basal level) was observed with 10 nM VIP. Half-maximum cAMP production was obtained at 0.78 nM VIP. VIP-related peptides were also potent in stimulating the cAMP production in IGR39 cells. The order of potency was VIP much greater than peptide histidine-methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin greater than glucagon. Using the same conditions, IGR37 cells, a metastasic counterpart of IGR39 cells, displayed a weak stimulation of cAMP production. After exposure of IGR39 cells to 10 nM VIP, the cAMP response to a new stimulation by VIP was strongly reduced. This desensitization of IGR39 cells to VIP was rapid (t1/2 less than 2 min) and homologous. Preincubation of IGR39 cells in the presence of native VIP induced disappearance of the VIP-binding sites at the cell surface. This phenomenon was dependent on time and VIP concentration. Maximum effect (loss of 80% of binding capacity) was obtained after exposure of the cells at 37 degrees C with a VIP concentration of 1 microM. The t1/2 of maximum disappearance was less than 2 min and the concentration of VIP giving half-maximum decrease in binding of mono[125I]iodinated VIP (125I-VIP) was 8 nM. This phenomenon was also reversible since 85% of the VIP-binding capacity could be restored in less than 1 h by incubating IGR39 cells in a VIP-free medium. The IGR39 cell line should be a useful model for further study of the structure and function of the human VIP receptor.     

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 1. Molecular characterization of the binding site

Using mono[125I]iodinated vasoactive intestinal peptide (125I-VIP), a very high number of specific binding sites for VIP were identified at the surface of the human melanoma cell line IGR39. The Scatchard analysis of competitive displacement experiments between native VIP and 125I-VIP was consistent with the existence of two classes of VIP-binding sites. IGR39 cells possess 0.54 x 10(6) high-affinity sites with a dissociation constant (Kd) of 0.66 nM and 1.3 x 10(6) sites of moderate affinity with a Kd of 4.7 nM. Pharmacological studies indicated that the order of potency in inhibiting 125I-VIP binding of the VIP/secretin family peptides was VIP much greater than peptide histidine methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin. Glucagon has no effect on the binding of the labelled peptide. By means of photoaffinity labelling a polypeptide of Mr 63,000 was characterized. The labelling of this species was completely abolished by native VIP. The order of potency of VIP-related peptides in inhibiting 125I-VIP cross-linking to its receptor was the same as in the competition experiments. The glycoprotein nature of the VIP-binding sites of IGR39 cells has been investigated by affinity chromatography on wheat-germ-agglutinin-Sepharose.     

Characterization of the interleukin-1-induced tyrosine phosphorylation of a 41-kDa plasma membrane protein of the human tumor cell line K 562

A 41-kDa protein, which was specifically phosphorylated upon incubation with natural purified murine interleukin 1, was recently identified by us [Martin, M., Lovett, D. H. and Resch, K. (1986) Immunobiology 171, 165-169] in highly purified plasma membranes from the human tumor cell line K 562. An in vitro assay was used to investigate and characterize the phosphorylation induced by interleukin 1, possibly involved in signal transduction and generation. Plasma membranes were incubated with radiolabeled ATP in the presence of purified natural murine interleukin 1, or recombinant human interleukin 1 alpha and the pattern of phosphoproteins was studied after separation by SDS/PAGE and subsequent autoradiography. A 41-kDa protein (pp41) was specifically phosphorylated on a tyrosine residue in the presence of interleukin 1 in a dose- and time-dependent manner. The protein showed a weak background phosphorylation in the absence of monokine. Phosphorylation took place very efficiently at 0 degrees C, whereas phosphatases were not active at that temperature. At 37 degrees C, a rapid dephosphorylation was observed which was inhibited specifically by Zn2+ and vanadate. The interleukin-1-specific induction of the phosphorylation could also be observed after detergent solubilization of the plasma membranes. Affinity labeling with an ATP analogue revealed an ATP-binding and cleaving site at 41 kDa. Interleukin 1 did not induce the phosphorylation of p41 in plasma membranes obtained from a subclone of K 562, which did not respond to interleukin 1 with growth inhibition, as was reported recently for the K 562 mother line [Lovett, D. H., Kozan, B., Hadam, M., Resch, K. and Gemsa, D. (1986) J. Immunol. 136, 340-347]. These data suggest that the interleukin-1 receptor is functionally linked to a protein-tyrosine kinase, which is implicated in its biological function.     

A cytogenetic investigation of the effects of cryopreservation on human sperm.

The effects of cryopreservation on the frequency and type of chromosome abnormalities in human sperm have been investigated for the first time. With a technique which enables direct visualization of human sperm chromosomes following in vitro penetration of hamster oocytes, sperm samples from 13 normal men were examined before and after being frozen in liquid nitrogen. The overall abnormality frequencies of 17.8% for fresh semen and 13.4% for previously frozen semen were not significantly different (chi 2(1) df = 3.04, p = 0.08). When specific abnormality types were analyzed, only the category of hypohaploidy was significantly different (chi 2(1) df = 6.75, p = 0.009) before (7.5%) and after (3.4%) freezing. Hypohaploidy was significantly higher than hyperhaploidy both prefreeze and postfreeze, and chromosome loss was random. Because the observed excess of hypohaploid cells may be attributable to technical artifact, the aneuploidy levels were estimated by doubling the number of hyperhaploid cells. Neither the adjusted numerical abnormality frequencies (1% prefreeze vs. 0% postfreeze) nor the overall abnormality frequencies (11.8% prefreeze vs. 10.4% postfreeze) were significantly different. The types and distributions of karyotypically abnormal sperm complements (numerical, structural, or combined) observed before and after freezing were not different. Interdonor variability in sperm chromosome abnormality frequencies and a possible donor-dependent response to cryopreservation were suggested by the data. The sex ratios were not affected by cryopreservation and did not differ significantly from the theoretical 50%. It is concluded that cryopreservation does not affect the type or frequencies of chromosome abnormalities or alter the sex ratio in human sperm.     

Calyculin A and okadaic acid: inhibitors of protein phosphatase activity

Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme.     

Chromobindin A, a Ca2+- and ATP-dependent chromaffin granule-binding protein, is found in a variety of tissues and in yeast

Chromobindin A is a ring shaped, multisubunit protein which exhibits Ca2+ and ATP-dependent binding to chromaffin granule membranes. Here we report biochemical and immunochemical evidence for the presence of chromobindin A in a surprisingly broad range of tissues: The protein is abundant in bovine skeletal muscle, pancreas, adrenal cortex and in brain gray and white matter; it is present in low quantities in the parotid, intestine and spleen and is undetectable in lung. Interestingly, chromobindin A was also detected in extracts of yeast. Electron micrographs of the yeast protein reveal a morphology virtually identical to the mammalian protein. These results suggest that chromobindin A is an important protein in a wide variety of cell types and that it has been highly conserved through evolution.     

Long-term phorbol ester treatment dissociates phospholipase D activation from phosphoinositide hydrolysis and prostacyclin synthesis in endothelial cells stimulated with bradykinin

Bovine pulmonary artery endothelial cells (BPAEC) were prelabeled with [3H]choline or [3H]myristic acid to selectively label endogenous phosphatidylcholine. BPAEC were stimulated with ATP and bradykinin (BK), and phospholipase D (PLD) activation was detected as a 4-fold increase in [3H]choline in cells prelabeled with [3H]choline or as a 2- to 3-fold increase in [3H]phosphatidylethanol in cells prelabeled with [3H]myristic acid and stimulated in the presence of ethanol. Pretreatment of BPAEC with 0.1 microM phorbol 12-myristate 13-acetate (PMA) for 22 hr completely inhibited agonist-induced PLD activation, whereas prostacyclin synthesis and [3H]phosphoinositide ([3H]PIns) hydrolysis were enhanced in pretreated cells. Long-term PMA treatment thus dissociates agonist-induced PLD activation from [3H]PIns hydrolysis, and agonist-induced prostacyclin synthesis is not dependent upon PLD activation.     

Secondary structure and orientation of a chemically synthesized mitochondrial signal sequence in phospholipid bilayers

A pre-sequence of 25 amino acids is required for import of yeast cytochrome oxidase subunit IV into mitochondria. Structure and orientation of the 25 amino acids synthesized peptide (p25) in a lipid bilayer were investigated by infrared attenuated total reflection spectroscopy. This method allowed to overcome the difficulties related to the optical turbidity due to the light scattering on membrane fragments which prevents the use of circular dichroism. We demonstrate here that incubation of the peptide with DOPC (dioleoylphosphatidylcholine) and DOPC-CL (dioleoylphosphatidylcholine - cardiolipin) liposomes is accompanied by an increase in alpha-helical content as compared to beta structure. Polarisation measurements indicate that the amphipathic helical segment is inserted parallel to the lipid acyl chains in cardiolipin containing liposomes.     

Seasonal variations in the loosely sorbed phosphorus fraction of the sediment of a shallow and hypereutrophic lake

Material cycling

Seasonal variations in the loosely sorbed phosphorus fraction of the sediment of a shallow and hypereutrophic lake     

Chlorierte Kohlenwasserstoffe in Brutv?geln aus dem Binnenland Niedersachsens

Zusammenfassung Rückstände chlorierter Kohlenwasserstoffe in Eiern und Lebern von im Binnenland Niedersachsens brütenden Vogelarten — Feldsperling, Mehlschwalbe, Weißstorch, Graureiher, Saatkrähe, Stockente und andere Arten — werden angegeben und deren Abhängigkeit von Brutort, Nahrung und Zugverhalten diskutiert.

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Chlorinated hydrocarbons of some bird species breeding in the inland of Lower Saxony (FRG)

Summary Residues of chlorinated hydrocarbons in eggs and livers of some bird species — Tree Sparrow, House Martin, White Stork, Heron, Rook, Mallard, and further species — are presented. The dependence on place of breeding, food web, and migration is discussed.