Clustering of phosphorylated amino acid residues in neurofilament proteins as revealed by 31P NMR

The state of phosphorylation in neurofilament (NF) proteins is studied by the 31P NMR technique. The 31P NMR spectrum of intact NF proteins at pH 7.0 is comprised of a major resonance at 4.18 ppm and a minor resonance at 3.53 ppm. The chemical shifts of the major and minor resonances are strongly dependent on pH and have pKa values for phosphoserine of 5.85 and for phosphothreonine of 6.00, respectively. 31P NMR spectra of isolated NF polypeptides show nonequivalent phosphoserine clusters in NF150 and in NF200. Their chemical shifts are very similar in both polypeptides, but the intensities of homologous resonances are different. NF68 has no detectable 31P resonance signal. Phosphate-specific monoclonal antibodies to NF200 can distinguish phosphates of various clusters. Microtubule proteins can also produce specific alteration of the 31P resonances of NF200. NF proteins digested by calcium-activated neutral protease (CANP) show relatively little change in 31P resonances.     

An assay for inhibitors of nucleoside transport based upon the use of 5-[125I]iodo-2'-deoxyuridine as permeant

5-[125I]Iodo-2'-deoxyuridine (IdUrd) has been shown to serve as a permeant for the nucleoside transport system of human erythrocytes and to be matabolically inert in these cells. Linear initial velocities were obtained at 20 degrees C for 125IdUrd transport, yielding a Km of 73 +/- 18 microM (n = 6). Low-affinity inhibitors of 125IdUrd transport, such as adenosine (Ki = 32 +/- 2 microM, n = 2), could be characterized by Michaelis-Menten kinetics. However, high-affinity inhibitors, such as 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, caused nonlinear initial velocities when added to the cells simultaneously with 125IdUrd. Conditions were defined (viz., 20-min pretreatment of cells with test compound followed by 5.0-min incubation with 1.0 microM 125IdUrd, all at 20 degrees C) whereby high-affinity inhibitors of IdUrd transport can be identified and evaluated according to their 50% inhibitory concentrations. The use of 125IdUrd as permeant greatly expedites the testing of compounds as inhibitors of nucleoside transport by allowing the cell pellets generated in these assays to be monitored directly in a gamma spectrometer, thereby circumventing the solubilization and decolorization of cell pellets required by assays that use 3H- or 14C-labeled nucleoside permeants.     

Human endothelial cells inhibit granulocyte aggregation in vitro

Granulocyte aggregation in response to circulating or locally released inflammatory mediators may cause vascular injury. The factors that regulate the granulocyte aggregation response and prevent its occurrence are not defined. We found that primary monolayers of human endothelial cells (EC) derived from umbilical veins released products that inhibited granulocyte aggregation. When polymorphonuclear leukocytes (PMN) and EC were incubated together, the subsequent aggregation response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) was inhibited by 40 to 60%, depending in part on the duration of incubation and the concentration of the agonist. Suspension of the granulocytes in albumin-containing buffer that had been rocked with EC monolayers had a similar effect, demonstrating that the EC release a soluble product that modulates the aggregation response. The fMLP concentration-response curve was shifted downward and to the right by EC. Incubation of the granulocytes with endothelial monolayers for various times indicated that the inhibition was maximal at 2 to 3 min, and the PMN responsiveness returned to control over the next 15 min. The inhibiting effect was not selectively directed against fMLP, because incubation of PMN with EC or suspending the PMN in supernatants from endothelial monolayers also inhibited aggregation stimulated by platelet-activating factor, leukotriene B4, and C5a desarg. Release of the inhibitory activity by EC was attenuated by indomethacin, suggesting that the activity is in part due to a cyclooxygenase pathway product. Prostacyclin (PGI2), an eicosanoid produced by EC via the cyclooxygenase pathway, inhibited granulocyte aggregation; however, PGI2 was much less potent as an inhibitor of PMN aggregation than of platelet aggregation. Furthermore, the concentration of PGI2 in buffer that had been incubated with EC was not sufficient to account for the magnitude of the PMN inhibition. The concentration of prostaglandin E2 (PGE2) was also insufficient to completely account for the inhibition. EC that had been treated with indomethacin or aspirin, which blocked the release of PGI2 and PGE2, retained the partial ability to release an activity that blunted granulocyte aggregation; this inhibiting activity was stable at 37 degrees C for 60 min. The results indicate that human EC have the biologic potential to modulate granulocyte aggregation stimulated by inflammatory mediators, and the activity is only partly due to PGI2 and other cyclooxygenase products of arachidonic acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

von Willebrand factor. A reduced and alkylated 52/48-kDa fragment beginning at amino acid residue 449 contains the domain interacting with platelet glycoprotein Ib

We have purified a reduced and alkylated tryptic fragment of von Willebrand factor (vWF) which migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 52/48-kDa doublet, but behaved as a single 46-kDa species after partial deglycosylation. After extensive treatment with denaturants, the 52/48-kDa polypeptide retained its ability to inhibit ristocetin-induced platelet aggregation in the presence of native vWF, as well as aggregation induced by desialylated vWF alone. Therefore, the 52/48-kDa polypeptide interacts with the platelet glycoprotein Ib receptor even in the absence of ristocetin. Both the 52/48- and the 46-kDa species inhibited ristocetin-induced binding of the intact molecule to platelets, but did not affect thrombin-induced binding. Determination of the NH2-terminal sequence of both members of the doublet gave identical results: VTLNPSDPEHCQ. This provided additional evidence that differences between the doublet constituents were only of carbohydrate composition and established the position of this peptide within the vWF polypeptide chain of approximately 2050 amino acid residues as beginning with the residue tentatively designated 449. These studies suggest that native conformation is not necessary for binding of vWF to platelets at the glycoprotein Ib receptor and that a linear amino acid sequence following residue 449 defines a domain responsible for this interaction.     

Methanogenesis in an Upflow Anaerobic Sludge Blanket Reactor at pH 6 on an Acetate-Propionate Mixture

High-rate anaerobic digestion can be applied in upflow anaerobic sludge blanket reactors for the treatment of various wastewaters. In upflow anaerobic sludge blanket reactors, sludge retention time is increased by a natural immobilization mechanism (viz. the formation of a granular type of sludge). When this sludge is cultivated on acid-containing wastewater, the granules mainly consist of an acetoclastic methanogen resembling Methanothrix soehngenii. This organism grows either in rods or in long filaments. Attempts to cultivate a stable sludge consisting predominantly of Methanosarcina sp. on an acetate-propionate mixture as substrate by lowering the pH from 7.5 during the start-up to approximately 6 failed. After 140 days of continuous operation of the reactor a filamentous organism resembling Methanothrix soehngenii prevailed in the sludge. The specific methanogenic activity of this sludge on acetate-propionate was optimal at pH 6.6 to 6.8 and 7.0 to 7.2, respectively.     

Cell cycle traverse and protein metabolism in human NHIK 3025 cells: the role of anchorage

We have studied the effect of cell anchorage on the human cell line NHIK 3025 in vitro, to see whether the growth regulating effect of cell anchorage primarily affected DNA division cycle or mass growth cycle. It was found that cell to cell anchorage had the same effect on cell cycle progression as anchorage to a solid surface, which indicates that it is anchorage per se and not cell shape that is important for growth control in NHIK 3025 cells. When NHIK 3025 cells were grown without attachment to a solid surface, both G1 and cell cycle duration was prolonged by 6 h, which means that the prolonged cell cycle was due to a prolonged G1. During the first part of the cell cycle the rate of protein synthesis and degradation was constant, and at the same level in cells grown with and without attachment. This means that the prolonged G1 was not due to a reduced protein accumulation or mass growth. Towards the end of the cell cycle protein accumulation was reduced. This effect was either due to a size control before cell division or a secondary effect of the prolonged G1. We therefore conclude that cell anchorage as a growth regulator primarily affects the DNA/cell division cycle.     

An Immunoblot Study of Neurofilament Degradation In Situ and During Calcium-Activated Proteolysis

The degradation of neurofilament (NF) proteins was examined by immunoblot methods to identify, characterize, and monitor the appearance of immunoreactive breakdown products during the loss of NF triplet proteins. Individual NF proteins and their breakdown products were identified using polyclonal and monoclonal antibodies to NF proteins. NF degradation was compared during calcium-activated proteolysis of isolated rat NF, during an experimental influx of calcium into excised rat spinal nerve roots, and during NF breakdown in transected rat peripheral nerve. These different experimental conditions produced similar patterns of NF fragmentation, including the transient appearance of NF immunobands between Mr 150,000-200,000 and 110,000-120,000 as well as the appearance and accumulation of NF immunobands between Mr 45,000 and 65,000. Most immunoreactive NF fragments remained Triton-insoluble. Low levels of the same immunoreactive fragments were present in control neural tissues, suggesting that calcium-activated proteolysis may be operative in the turnover and/or processing of NF proteins in vivo. Very similar patterns of NF degradation during experimental calcium influxes into different CNS and PNS tissues are indicative of the widespread distribution of calcium-activated NF protease in neural tissues.     

Modulation of [3H]Diazepam Binding in Rat Cortical Membranes by GABAA Agonists

GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23 degrees C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]gamma-aminobutyric acid [( 3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA greater than isoguvacine greater than 3-aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) greater than 4,5,6,7-tetrahydroisoxazolo (4,5,6)-pyridin-3-ol (THIP) = taurine greater than piperidine 4-sulphonic acid (P4S). At 37 degrees C, the potencies of agonists remained unchanged, but isoguvacine, 3 APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist-induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor-receptor interaction under near-physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABA receptor ligands.     

Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis

To study plasmid instability in Bacillus subtilis the pUB110-derived hybrid plasmid pLB2 (3.6 kb) and the bifunctional replicon pLB5 (5.9 kb), able to replicate in B. subtilis and Escherichia coli, were constructed. In both vectors homologous B. subtilis, or heterologous E. coli DNA fragments of various lengths were inserted. Irrespective of the source of the cloned DNA, the segregational stability of the recombinant plasmids in B. subtilis was severely affected by the DNA inserts. In contrast, no instability was observed in E. coli. In B. subtilis a steep inverse relationship existed between the size of the inserts and the level of stability. Increased size of the pLB plasmids resulted in strongly reduced copy numbers. This seems to be the primary cause of the size-dependent segregational instability.     

An immunocytochemical study of the optic ganglia of the crayfish Astacus leptodactylus (Nordmann 1842) with antisera against biologically active peptides of vertebrates and invertebrates

Summary The peptidergic system in the optic ganglia of Astacus leptodactylus is characterized by the immunocytochemical application of 15 antisera raised against biologically active peptides of vertebrates and invertebrates. Positive reactions were found with anti-FMRFamide, antiMSH, anti-vasotocin, anti-gastrin, anti-CCK, anti-oxytocin, anti-secretin, anti-glucagon and anti-GIP. Based on immunochemical reaction and localization it is possible to distinguish 30 cell groups. Only part of these cell groups is found in the known classical neurosecretory cell regions. This observation demonstrates a more extensive peptidergic system than formerly recognized. The morphology of this peptidergic system suggests that one part is neurohormonal and the other part neurotransmitter-like or neuromodulatory.