Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.     

Structural and functional alterations induced in the mitochondrial cytochrome b-c1 complex by N,N'-dicyclohexylcarbodiimide

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstituted mitochondrial cytochrome b-c1 complex. In proteoliposomes containing b-c1 complex DCCD inhibits equally electron flow and proton translocation catalyzed by the enzyme. In both isolated and reconstituted systems the inhibitory effect is accompanied by structural alterations in the polypeptide pattern of the enzyme consistent with cross-linking between subunits V and VII. The kinetics of inhibition of enzymic activity correlates with that of the cross-linking, suggesting that the two phenomena may be coupled. Binding of [14C] DCCD to both isolated and reconstituted enzyme was also observed, though it was not correlated kinetically with the inhibition.     

The role of cations and other factors on the apparent energy of activation of (Na + K)-ATPase

Data concerning the temperature dependence of ouabain-sensitive (Na⁺ + K⁺)-activated ATPase have enabled estimates of the apparent activation energies of this process to be obtained. Arrhenius plots show a point of inflection at about 20 °; at higher temperatures the activation energy is about 13.5 kcal/mole while below this temperature the value increases to 28.5 kcal/mole. Storage at −5 ° or reduction in total cation concentration without alteration of the Na⁺:K⁺ ratio causes no significant change in these values, although the specific activity is markedly reduced. Reduction in the sodium concentration alone, however, increases the apparent activation energy at lower temperatures. These results support the hypothesis that two independent processes are involved in ATP hydrolysis, one operating above the critical temperature and one operating below this temperature. Storage, or reduction in the concentrations of both sodium and potassium ions, appears to reduce the number of functional ATPase units, without significantly altering the properties of those which can still hydrolyze ATP. Reduction in the sodium concentration alone, however, may also cause some inhibition of all units. This is more marked at lower temperatures, and may arise from competition by potassium for sodium-binding sites.     

Rumen contents as a reservoir of enterohemorrhagic Escherichia coli

Abstract We investigatedthe role of the rumen fermentation as a barries to the foodborne pathogen, Escherichia coli O157:H7. Strains of E. coli , including several isolates of O157:H7, grew poorly in media which simulated the ruminal environment of a well-fed animal. Strains of E. coli O157:H7 did not display a superior tolerance to ruminal conditions which may facilitate their colonization of the bovine digestive tract. Unrestricted growth of E. coli was observed in rumen fluid collected from fasted cattle. Growth was inhibited by rumen fluid collected from well-fed animals. Well-fed animals appear less likely to become reservoirs for pathogenic E. coli . These results have implications for cattle slaughter practices and epidemiological studies of E. coli O157:H7.     

Characterization and classification of virulent lactococcal bacteriophages isolated from a Cheddar cheese plant

C.N. CASEY, E. MORGAN, C. DALY AND G.F. FITZGERALD, 1993. Twenty-two bacteriophages, isolated from cheese-vat whey samples over a period of 4 years, were found to be active against one or more of four different strains of Lactococcus lactis subsp. cremoris used in a defined strain starter system in an Irish Cheddar cheese factory. All the phages were small isometric-headed with non-contractile tails, a baseplate and a collar; they had genome sizes of approximately 30 kb, and belonged to a single DNA hybridization group. All 22 phages could be classified into four distinct groups based on restriction analysis which overlapped perfectly with those based on host range. Each group of phage examined showed cross-reactive host ranges. None of the phage DNAs hybridized to the chromosomes of any of the seven cultures used in the factory during the four cheesemaking seasons, and neither could phages be induced from any of the strains by mitomycin-C or ultraviolet light treatment.     

Artificial nanovesicles for dsRNA delivery in spray-induced gene silencing for crop protection

Spray-induced gene silencing (SIGS) is an innovative and eco-friendly technology where topical application of pathogen gene-targeting RNAs to plant material can enable disease control. SIGS applications remain limited because of the instability of RNA, which can be rapidly degraded when exposed to various environmental conditions. Inspired by the natural mechanism of cross-kingdom RNAi through extracellular vesicle trafficking, we describe herein the use of artificial nanovesicles (AVs) for RNA encapsulation and control against the fungal pathogen, Botrytis cinerea. AVs were synthesized using three different cationic lipid formulations, DOTAP + PEG, DOTAP and DODMA, and examined for their ability to protect and deliver double stranded RNA (dsRNA). All three formulations enabled dsRNA delivery and uptake by B. cinerea. Further, encapsulating dsRNA in AVs provided strong protection from nuclease degradation and from removal by leaf washing. This improved stability led to prolonged RNAi-mediated protection against B. cinerea both on pre- and post-harvest plant material using AVs. Specifically, the AVs extended the protection duration conferred by dsRNA to 10 days on tomato and grape fruits and to 21 days on grape leaves. The results of this work demonstrate how AVs can be used as a new nanocarrier to overcome RNA instability in SIGS for crop protection.     

Aspects of early postnatal development of cortical neurons that proceed independently of normally present extrinsic influences

To examine the contribution of local versus extrinsic influences on postnatal development of cortical neurons, we compared the maturation of deep (infragranular) layer neurons in isolated slices of neocortex grown in organotypic culture to a similar population of neurons developing in vivo. All slice cultures were prepared from sensorimotor cortices of newborn mice (P0) and neurons in these cultures were examined at daily intervals during the first 9 days in vitro (DIV). The maturational state of neurons developing in vivo over this same time period was assessed in acute slices prepared from animals of equivalent postnatal age, P1–P9. Electrophysiological recordings were obtained from neurons in both cultured and acute slices, using Lucifer yellow filled whole-cell recording electrodes, enabling subsequent morphometric analysis of the labeled cells. We report significant changes in both cellular morphology and electrical membrane properties of these deep layer cortical neurons during the frist week in culture. Morphological maturation over this time period was characterized by a two- to three-fold increase in cell body size and total process length, and an increase in dendritic complexity. In this same population of cells a three-fold decrease in input resistance and changes in the action potential waveform, including a two-fold decrease in the AP duration, also occur. The degree of morphological and electrophysiological differentiation of individual neurons was highly correlated across developmental ages, suggesting that the maturational state of a cell is reflected in both cellular morphology and intrinsic membrane properties. A remarkably similar pattern of neuronal maturation was observed in neurons in layers V, VI/SP examined in acute slices prepared from animals between P1–P9. Because our culture system preserves many aspects of the local cortical environment while eliminating normal extrinsic influences (including thalamic, brainstem, and callosal connections), our findings argue that this early phase of neuronal differentiation, including the rate and extent of dendritic growth and development of AP waveform, results from instructive and/or permissive local influences, and appears to proceed independently of the many normally present extrinsic factors. © 1993 John Wiley & Sons, Inc.     

Origin of deoxycorticosterone sulfate (DOC-SO4) in plasma of pregnant women: Pregnenolone-3,21-disulfate is a placental precursor of DOC-SO4

The plasma levels of deoxycorticosterone sulfate (DOC-SO₄) in near-term pregnant women are 100 times those in plasma of men or non-pregnant women. Yet, neither the tissue site of synthesis nor the precursor of DOC-SO₄ that enters maternal plasma is known. Several potential sources have been excluded: plasma DOC-SO₄ is not derived from plasma DOC; and the secretion of C₂₁-steroids (other than aldosterone) from the maternal adrenals during human pregnancy is not increased. Similarly, the transfer of DOC-SO₄ from fetal plasma cannot account for the high level of DOC-SO₄ in the maternal compartment, and a reduced clearance of plasma DOC-SO₄ during pregnancy cannot account for the high levels of DOC-SO₄. Indeed, the rate of clearance of DOC-SO₄ from plasma is 10–100 times that of most other steroid sulfates. To address this question further, we evaluated the possibility that fetal plasma pregnenolone-3,21-disulfate serves as a precursor for DOC-SO₄ formation in the placenta. The preferential hydrolysis of the 3β-sulfate of pregnenolone-3,21-disulfate in placenta would give rise to pregnenolone-21-monosulfate, which, if acted upon by placental 3β-hydroxysteroid dehydrogenase/Δ^{5 → 4} isomerase, could give DOC-SO₄. [³H]Pregnenolone-3,21-disulfate was incubated with minces of human placental tissue for 5, 20, 60 and 120 min. Radiolabelled DOC-SO₄, DOC, and pregnenolone-21-monosulfate were isolated from the incubation media and quantified. After a 5 min incubation, 7.5% of substrate was converted to DOC-SO₄; and after 20, 60 and 120 min 30% of the [³H]pregnenolone-3,21-disulfate was recovered from the media of these incubations as [³H]DOC-SO₄. [³H]DOC was also present in the incubation media and the concentrations of this product increased as a function of incubation time. Therefore, pregnenolone-3,21-disulfate, which is present in very high concentrations in fetal plasma (1000 ng/ml), is metabolized in the placenta to DOC-SO₄. Because of the fetal and maternal vascular arrangements of the hemochorioendothelial placenta of human pregnancy, steroids produced in syncytiotrophoblasts preferentially enter the intervillous space; thus, fetal plasma pregnenolone-3,21-disulfate may serve as a placental precursor of maternal plasma DOC-SO₄.