Protein kinase C inhibition by calmodulin and its fragments

Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10^–5 M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC₅₀ of 6 µ M compared to 1 µ M of intact calmodulin. They were identified as Ser³⁸-Arg⁷⁴ and His¹⁰⁷-Lys¹⁴⁸. Each of the inhibiting fragments contains an intact Ca²⁺-binding domain with complete helix-loop-helix structure (EF hand). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.     

The 76 kD cell-adhesion factor from crayfish haemocytes promotes encapsulation in vitro

Summary Semigranular cells from the crayfish, Pacifastacus leniusculus, were separated by Percoll gradient centrifugation and were used to study the encapsulation of foreign particles. The semigranular cells were found strongly to encapsulate glass beads coated with haemocyte lysate in which the prophenoloxidase-activating system had been activated with laminarin or with a low concentration of calcium ions. The granular cells only weakly encapsulated these particles. The encapsulationpromoting factor was purified from haemocyte lysates and found to be a 76 kD protein which was recognized by an antiserum to the previously described 76 kD cell-adhesion factor. After the last step in purification (Con A-Sepharose chromatography), the flowthrough consisted of several proteins, which had some, but less, encapsulation-promoting activity and contained a 30 kD band that was also recognized by the antiserum to the 76 kD cell-adhesion factor. If the haemocyte lysate prepared in low [Ca²⁺] was incubated with a -1,3-glucan prior to purification, no 76 kD protein could be isolated but only a 30 kD protein. The 30 kD protein thus seems to be a degradation product of the 76 kD cell-adhesion factor. We conclude that the 76 kD protein which is released from degranulating haemocytes, and to a lesser extent its 30 kD fragment, can promote encapsulation. Phenoloxidase did not have any encapsulation-promoting activity.     

Effects of endogenous and exogenous cholesterol on the ultrastructure and steroid secretion of undifferentiated rat adrenocortical cells in primary culture

Summary The present study was undertaken to define the effects of lipoprotein-derived cholesterol and endogenous, de novo synthesized cholesterol on the ultrastructure and function of undifferentiated rat adrenocortical cells [lipoprotein (HDL₃ and LDL) receptor-negative, zona glomerulosa-like adrenocortical cells] in primary culture. For this purpose human plasma high density lipoprotein (HDL₃) or low density lipoprotein (LDL) was added to culture medium devoid of cholesterol. Steroid secretion remained at the low basal level even after addition of lipoproteins, and the amount of intracellular lipid droplets did not increase. When mevinolin (0.96 µg/ml), an inhibitor of cholesterol synthesis, was added to the culture medium, a low secretion of corticosterone was measured both in serum-free and serum-containing media. Ultrastructurally, lipid droplets disappeared after treatment with mevinolin in both media used. At this concentration of mevinolin cell proliferation was similar to that in the controls, but at higher concentrations (4.8 or 9.6 µg/ml) proliferation was inhibited to 42% and 26% in serum-free medium, and 20% and 12% in serum-supplemented medium, respectively. This study demonstrates that cell proliferation and synthesis of corticosterone by undifferentiated rat adrenocortical cells is identical in the absence or presence of exogenous lipoprotein cholesterol. Inhibition of de novo cholesterol synthesis by mevinolin over a period of 7 days does not inhibit corticosterone secretion or proliferation of cells but decreases the amount of intracellular lipid droplets, thus suggesting utilization of intracellular cholesterol esters. However, higher concentrations of mevinolin inhibit proliferation of cells both in serum-free and serum-containing media.     

The morphology of suboesophageal ganglion cells innervating the nervus corporis cardiaci III of the locust

Summary The nervus corporis cardiaci III (NCC III) of the locust Locust migratoria was investigated with intracellular and extracellular cobalt staining techniques in order to elucidate the morphology of neurons within the suboesophageal ganglion, which send axons into this nerve. Six neurons have many features in common with the dorsal, unpaired, median (DUM) neurons of thoracic and abdominal ganglia. Three other cells have cell bodies contralateral to their axons (contralateral neuron 1–3; CN 1–3). Two of these neurons (CN2 and CN3) appear to degenerate after imaginal ecdysis. CN3 innervates pharyngeal dilator muscles via its anterior axon in the NCC III, and a neck muscle via an additional posterior axon within the intersegmental nerve between the suboesophageal and prothoracic ganglia. A large cell with a ventral posterior cell body is located close to the sagittal plane of the ganglion (ventral, posterior, median neuron; VPMN). Staining of the NCC III towards the periphery reveals that the branching pattern of this nerve is extremely variable. It innervates the retrocerebral glandular complex, the antennal heart and pharyngeal dilator muscles, and has a connection to the frontal ganglion.Abbreviations AH antennal heart - AN antennal nerves - AO aorta - AV antennal vessel - CA corpus allatum - CC corpus cardiacum - CN1, CN2, CN3 contralateral neuron 1–3 - DIT dorsal intermediate tract - DMT dorsal median tract - DUM dorsal, unpaired, median - FC frontal connective - FG frontal ganglion - HG hypocerebral ganglion - LDT lateral dorsal tract - LMN, LSN labral motor and sensory nerves - LN+FC common root of labral nerves and frontal connective - LO lateral ocellus - MDT median dorsal tract - MDVR ventral root of mandibular nerve - MVT median ventral tract - NCA I, II nervus corporis allati I, II - NCC I, II, III nervus corporis cardiaci I, III - NR nervus recurrens - NTD nervus tegumentarius dorsalis - N8 nerve 8 of SOG - OE oesophagus - OEN oesophageal nerve - PH pharynx - SOG suboesophageal ganglion - T tentorium - TVN tritocerebral ventral nerve - VLT ventral lateral tract - VIT ventral intermediate tract - VMT ventral median tract - VPMN ventral, posterior, median neuron - 1–7 peripheral nerves of the SOG - 36, 37, 40–45 pharyngeal dilator muscles     

Hydrolysis ofγ-glutamyl linkages byFusobacterium nucleatum

The cell extracts of two human oral strains (FN2 and FN3) ofFusobacterium nucleatum displayed exceptionally high-glutamylpeptidase activity as determined withN--l-glutamyl-2-naphthylamine as substrate. This activity was so dominant that the hydrolysis of otherN-aminoacyl-2-naphthylamines progressed at a rate <10% of the former. Two major enzymes (I and II) were partially purified from FN2. I had a molecular weight of 115,000 and did not hydrolyze-glutamylcysteinylglycine (glutathione). II had a molecular weight of 70,000 and rapidly liberated only glutamic acid from glutathione. Strain FN3 contained several enzymes hydrolyzing-glu-2NA. Direct anion exchange chromatography of FN3 cell extracts separated one enzyme that liberated both glutamic acid and glycine from glutathione, one that was inactive against glutathione (but hydrolyzed-glu-2NA), and one that liberated only glutamic acid. Although-glu-2NA was a good synthetic substrate, glutathione was hydrolyzed at least 500 times faster by an enzyme present in both strains. These results indicate that the presence of-glutamylpeptidase activity is very characteristic of theseF. nucleatum strains.     

An improved method for the detection of Dcm methylation in DNA molecules

The intrinsic insensitivity of EcoRII recognition sites in RF DNAs of phage M13 and vector M13mp18 towards this restriction endonuclease can be overcome by adding site-specific oligodeoxyribonucleotide duplexes to the restriction sample. Since Dcm- DNA but not Dcm(+)-methylated DNA becomes susceptible under these conditions, this procedure constitutes an improvement of the Dcm methylation assay.     

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.     

EMA,an epithelial membrane-associated antigen during early development and morphogenesis ofXenopus laevis

Summary We raised monoclonal antibodies against a membrane fraction ofXenopus neurulae in order to detect tissue-specific cell-surface markers. Here we describe a monoclonal antibody that recognizes an epithelial membrane-associated antigen (EMA) in immunohistological stainings. The tissue-specific and membrane-associated antigen detected in immunohistological stainings could serve as useful marker in epithelium differentiation and membrane organization of the early embryo. In tadpoles and adults EMA was found in specific epithelial tissues derived from different germ layers such as kidney, skin, gut, pancreas, epiphysis and choroid plexus. In the cleaving embryo this antibody stained newly formed membranes between blastomeres from the two-cell stage onwards. Cytoplasmic staining in large oocytes and early embryos was also observed. The possibility that the cytoplasmic signal represents a maternal store of membrane material is discussed.