Orientation constraints in diffusion-limited macromolecular association. The role of surface diffusion as a rate-enhancing mechanism.

Ligand association to a reactive site on a macromolecular surface could be very slow if the site is small. The effective capture radius of the reactive site can be significantly increased if the ligand can bind weakly to the nonspecific surface around the site and then slide in a two-dimensional diffusion along the surface. In this model, the diffusion along the surface has to be properly coupled with the free diffusion in solution and the effective bimolecular association rate constant to the reactive site can be calculated as a function of the nonspecific affinity. This is carried out both for a plane and spherical surface, modeling the association to a membrane receptor or to the catalytic site on an enzyme. The result of these calculations can be used to assign reasonable values to the parameters in the quasichemical approximation of K. Solc and W. H. Stockmayer (1973, Int. J. Chem. Kinet., 5:733-752). In this way a simple analytical expression can be derived for the diffusion-limited association rate constant of two asymmetrically reactive molecules, with or without surface diffusion contributing.     

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.     

Fluorescent staining of sialidases in polyacrylamide gel electrophoresis and ultrathin-layer isoelectric focusing

Polyacrylamide gels were stained with the sialidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid showing the activity of Vibrio cholerae and Clostridium sordellii sialidases in the gels after electrophoresis. With this fluorogenic method minimum sialidase activities of 5 microU could be determined. The sensitivity of this staining is about 10,000-fold higher compared to protein-staining with Coomassie brilliant blue. For the visualization of other proteins than sialidases the specific sialidase staining could be followed by a protein-staining method in the same gel.     

Vitamin K-dependent carboxylase: the carboxylation of exogenous substrates in different systems

Two types of solid-phase carboxylase, SPC-II and SPC-X, have been prepared from the livers of warfarin-treated cows. Their enzymatic activities were compared with substrate-free carboxylase in microsomes from normal cows and substrate-bound carboxylase in microsomes from warfarin-treated cows. A number of exogenous substrates for carboxylase have been purified and tested. We found that large substrates, such as descarboxyprothrombin, are carboxylated only by substrate-free carboxylase and not by the substrate-bound enzyme. No differences in apparent Km values between solid-phase carboxylases II and X were observed.     

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.     

Retinol and retinyl esters in parenchymal and nonparenchymal rat liver cell fractions after long-term administration of ethanol

Chronic ethanol consumption reduces the liver retinoid store in man and rat. We have studied the effect of ethanol on some aspects of retinoid metabolism in parenchymal and nonparenchymal liver cells. Rats fed 36% of total energy intake as ethanol for 5-6 weeks had the liver retinoid concentration reduced to about one-third, as compared to pair-fed controls. The reduction in liver retinoid affected both the parenchymal and the nonparenchymal cell fractions. Plasma retinol level was normal. Liver uptake of injected chylomicron [3H]retinyl ester was similar in the experimental and control group. The transport of retinoid from the parenchymal to the nonparenchymal cells was not found to be significantly retarded in the ethanol-fed rats. Despite the reduction in total retinoid level in liver, the concentrations of unesterified retinol and retinyl oleate were increased in the ethanol fed rats. Hepatic retinol esterification was not significantly affected in the ethanol-fed rats. Since our study has demonstrated that liver uptake of chylomicron retinyl ester is not impaired in the ethanol-fed rat, we suggest that liver retinoid metabolism may be increased.     

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.     

Effect of hypothermia on cell kinetics and response to hyperthermia and X rays

Hyperthermia is a potent radio enhancer. Studies using hypothermia in combination with irradiation have given confusing results due to lack of uniformity in experimental design. This report shows that hypothermia might have potential significance in the treatment of malignant cells with both thermo- and radiotherapy. Reuber H35 hepatoma cells, clone KRC-7 were used to study the effect of hypothermia on cell kinetics and subsequent response to hyperthermia and/or X rays. Cells were incubated at 8.5 degrees C or between 25 and 37 degrees C for 24 hr prior to hyperthermia or irradiation. Hypothermia caused sensitization to both hyperthermia and X rays. Maximum sensitization was observed between 25 and 30 degrees C and no sensitization was found at 8.5 degrees C. At 25 degrees C maximum sensitization was achieved in approximately 24 hr, cell proliferation was almost completely blocked, and cells gradually accumulated in the G2 phase of the cell cycle. In contrast to the effect of hypothermia on either hyperthermia or X rays alone, thermal radiosensitization was decreased in hypothermically pretreated cells (24 hr at 25 degrees C) compared to control cells (37 degrees C). The expression of thermotolerance and the rate of development at 37 degrees C after an initial heating at 42.5 degrees C were not influenced after preincubation at 25 degrees C for 24 hr. The expression of thermotolerance for heat or heat plus X rays during incubation at 41 degrees C occurred in a significantly smaller number of cells after 24 hr preincubation at 25 degrees C. The enhanced thermo- and radiosensitivity in hypothermically treated cells disappeared in approximately 6 hr after return to 37 degrees C.     

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.