Inhibition of ceruloplasmin and other copper oxidases by thiomolybdate

The dietary antagonism between copper and molybdate salts prompted a study of the inhibition of copper enzymes by thiomolybdate (TM). TM strongly inhibited the oxidase activity of five copper oxidase with I50% values in the 1-5 microM range. The mechanism of the TM effect on the copper oxidase, ceruloplasmin (Cp) (E.C. 1.16.3.1), was studied in detail. In Vmax vs. E plots, TM gave parallel data suggesting irreversibility but a large number of TM molecules per Cp were required. The inhibition of Cp by TM could not be reversed by dialysis. Isolation of TM-inhibited Cp on Sephadex G-10 did not yield any active Cp molecules. Cu(II) did not restore any inhibited oxidase activity. Gel electrophoresis supported the covalent binding of Cp by TM without any extensive change in protein structure. EPR results confirmed that Cu(II) is reduced to Cu(I) after reaction with TM. However, the Mo(VI) in MoS4(2-) did not change in oxidation number. Analysis of the TM-Cp compound accounted for all six Cu atoms as found in native Cp. The data suggest the covalent binding of sulfide to Cp copper. TM also inhibited the activity of ascorbate oxidase, cytochrome oxidase, superoxide dismutase, and tyrosinase. However, no inhibition of carbonic anhydrase, a zinc enzyme, was observed at 1 mM TM.     

Generation of a transmembrane electric potential during respiration by Azotobacter vinelandii membrand vesicles.

Membrane vesicles isolated from Azotobacter vinelandii strain O by lysis of spheroplasts in potassium of sodium phosphate buffer develop a transmembrane electric potential during respiration. The magnitude of this potential was determined by three independent methods: (i) fluorescence of 3,3'-dipropylthiodicarbocyanine and 3,3'-dihexyloxacarbocyanine; (ii) uptake of 86Rb+ in the presence of valinomycin; and (iii) uptake of [3H]triphenylmethyl phosphonium. In method (i), the relative fluorescence of these cyanine dyes in the presence of intact cells or derived vesicles is quenched during oxication of electron donors. A linear relationship between this quenching and a potassium diffusion potential was employed to calibrate the probe response. In method (ii), the steady-state concentration ratio of rubidium across the vesicle membrane during oxidation of L-malate was converted to potential by the Nernst equation. In method (iii), the steady-state concentration ratio of this lipophilic cation was likewise converted to a potential. With the exception of 3,3'-dihexyloxacarbocyanine fluorescence, these methods gave good agreement for the potential developed during L-malate oxidation by membrane vesicles. A value of 75 to 80 mV (inside negative) was obtained for vesicles prepared in potassium phosphate, and 104 mV (inside negative) was obtained for vesicles prepared in sodium phosphate. Electrogenic expulsion of hydrogen ion was observed during L-malate oxidation, and the amount of proton exodus was greater in potassium rather than the sodium-containing vesicles. This indicates the presence of a sodium-proton antiport mechanism. In addition, D-glucose uptake was observed during development of a potassium diffusion potential that was artificially imposed across the vesicle membrane. These observations suggest the presence of a glucose-proton symport mechanism in accordance with the principles of Mitchell.     

Effect of culture conditions on synthesis of L-asparaginase by Escherichia coli A-1.

The nutritional requirements and culture conditions affecting biosynthesis of L-asparaginase in a mutant of Escherichia coli HAP designated strain A-1 were studied. Asparaginase activity was increased by the addition of L-glutamic acid, L-glutamine, or commercial-grade monosodium glutamate. The rate of enzyme synthesis was dependent on the interaction between the pH of the culture and the amount of oxygen dissolved in the medium. A critical oxygen transfer rate essential for asparaginase formation was identified, and a fermentation procedure is described in which enzyme synthesis is controlled by aeration rate. Enhancement of L-asparaginase activity by monosodium glutamate was inhibited by the presence of glucose, culture pH, chloramphenicol, and oxygen dissolved in the fermentation medium.     

A sensitive and specific radiometric method for the measurement of plasma histamine in normal individuals

A radioenzymatic method for assaying histamine using histamine-N-methyltransferase from guinea pig brain was modified to increase its sensitivity, precision, and specificity. This was achieved by incorporation of a thin-layer chromatography step and use of N_α-methyl histamine as an internal standard in each sample. No prior extraction of the plasma samples is required, permitting the assay of 30 samples in 1 working day. Histamine was detected in the plasma of 17 normal volunteers, at a level of 3.4 ± 0.69 nmol/liter (range 0.82 to 4.7 nmol/liter). In 19 asthmatics, a low-peak expiratory flow rate was found to be associated with a plasma histamine concentration above this “normal” range.     

A silver carbonate method for cell counts of neurons and glial elements on paraffin embedded brain tissue.

A modification of the Del Rio-Hortega method for the demonstration of central nervous system elements is presented. This silver impregnation technique is particularly useful for the classification of cell types for quantitative differential cell counts. Formalin fixed paraffin sections are immersed in formol-ammonium bromide for 1 1/2 hours; this solution is an excellent mordant for various silver nitrate stains. The samples are stained for 20 to 60 minutes in a silver carbonate solution (25 ml of 25% silver nitrate combined with 200 ml of 5% sodium carbonate) and then reduced in a 1% formaldehyde solution to which 20 drops of acetic acid have been added. Finally, the slides are fixed in sodium thiosulfate, rinsed in tap water, dehydrated, cleared, and mounted. This procedure will enable this investigator to identify neurons, oligodendroglia, and astrocytes on the basis of their nuclear staining as well as to demonstrate the laminae of brain tissue since the method allows differentiation of cell layers and fiber tracts.     

Dark adaptation,sensitivity, and rhodopsin level in the eye of the lobster,Homarus

Summary Dark adaptation of living lobsters was measured by recording the ERG at several temperatures in the range 5–20 °C following adapting flashes that convert about 70% of the rhodopsin to metarhodopsin. Recovery of log threshold is rapid, and at 10–20° is nearly complete in 10 min. Only at 5 °C is dark adaptation significantly slowed. Comparison of dark adaptation with data on regeneration of pigment (Bruno et al., 1977) is consistent with the hypothesis that as rhodopsin concentration rises and falls, its only effect on sensitivity is to alter the probability of quantum catch. This interpretation is further bolstered by observations on winter lobsters that have a 70% deficiency of rhodopsin without the concomitant increase in metarhodopsin that accompanies light adaptation. No effect of metarhodopsin on sensitivity was detected. These experiments support the growing body of evidence indicating that the relationship between rhodopsin concentration and log threshold is fundamentally different in the rhabdomeric photoreceptors of invertebrates and the rods and cones of vertebrates.This work was supported by USPHS research grant EY 00222 to Yale University. S.N.B. was aided by NIH Postdoctoral Fellowship EY 52378, by funds made available through the Unidel Foundation, and by a grant from the University of Delaware Research Foundation.     

Some observations in tetraclita squamosa rufotincta Pilsbry

Aspects of the biology of Tetraclita squamosa rufotincta Pilsbry were investigated. Although a tropical species, on the shores at Elat, Israel, its breeding is virtually restricted to the colder winter months. The causal factors are considered and the results of some experimental work relative to temperature control given.For a cirripede, T.s. rufotincta has a remarkably large ‘egg’ — unusual in a tropical or even warm-water species. The egg size is compared with that of other species and the survival value of large size considered; excess nutrients are carried over from the embryo to at least the first planktonic larva, and may aid survival in a relatively nutrient poor environment.The animals do not become sexually mature until the second year after their settlement. Growth takes place largély in the colder winter months and is seasonally separated from the period when gonadal tissue is developed.     

Inhibition of sugar uptake in adenosine-treated 3T3 cells

Addition of 5 to 250 micromolar adenosine to the culture medium resulted in a 30–80% inhibition of the rate of uptake of 2-deoxyglucose or 3–0-methylglucose by sparse or confluent 3T3 cells within three hours. The inhibition of deoxyglucose uptake could be reversed partially by changing the cells to medium without adenosine for two hours and could be prevented completely by the addition of persantin, an inhibitor of nucleoside uptake. The adenosine effect is not due to inhibition of pyrimidine synthesis, since it is not prevented by uridine. It is not seen in 3T6 cells lacking adenosine kinase. The inhibition could be observed on confluent cells whose deoxyglucose uptake was stimulated by insulin, epidermal growth factor (EGF), calf serum or calcium phosphate. Although the percentage stimulation over control by these factors varied, the percentage inhibition by addition of adenosine of the stimulated rates, as well as the unstimulated rate, was relatively constant. EGF, insulin and calcium phosphate caused little or no stimulation of deoxyglucose uptake by sparse cells, whether adenosine treated or untreated. The results suggest that adenosine acts intracellularly after phosphorylation to regulate sugar uptake through a mechanism which is independent of the regulation by hormones and cell density.     

Spawning of hermatypic corals in Bermuda: a pilot study

This study investigates spawning of 4 hermatypic coral species from the subtropical environment of Bermuda. Laboratory evidence of spawning behaviour is supported by synchronous field observations. Development of scleractinian planulae to postlarval stages is recorded. Diploria strigosa, D. labyrinthiformis, Montastrea annularis and M. cavernosa shed highly buoyant, pigmented eggs (300–440 µm diam.) during July to September 1986. Brief spawning periods, synchronous between conspecific colonies, were recorded for M. annularis (July and August) and M. cavernosa (August) within 1 d of the last quarter of the lunar cycle. In August, there were overlaps amongst the spawning dates of D. strigosa and the Montastrea species. Nocturnal spawning periods differed between M. annularis and M. cavernosa. This constitutes the first evidence from an Atlantic community of overlapping spawning dates amongst several faviid species, and of the accumulation of scleractinian eggs and planulae in surface slicks.     

More is not better: brood size and population growth in a self-fertilizing nematode.

The normal form of the nematode Caenorhabditis elegans is a self-fertilizing hermaphrodite, which produces from the same germ-line tissue first a limited number of sperm and then a larger number of oocytes. Self-progeny brood sizes are determined by the number of sperm, and most of the oocytes remain unfertilized. Therefore it might seem selectively advantageous to increase the number of sperm, and hence the size of the brood. A mutation that leads to a 50% increase in sperm production allows a comparison of population growth rates between the wild type (mean brood 327 progeny) and the mutant (mean brood 499 progeny). Wild-type populations grow faster, as measured by food consumption, indicating that increased brood size is not advantageous. The mutant appears to be at a disadvantage because the additional spermatogenesis leads to a delay in the onset of oogenesis, and hence to an increase in the minimum generation time. In support of the notion of an optimal brood size, it was found that different natural isolates of this species have self-fertilities similar to that of the standard laboratory strain, in the range 250-350 progeny per worm.