Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

The disposition of ethiofos (WR-2721) in the isolated perfused rat liver

We have investigated the disposition of ethiofos (20 mg, 4 microCi [14C]ethiofos) in the isolated perfused rat liver preparation to determine the hepatic contribution to the poor oral bioavailability of the drug. Ethiofos clearance (10.6 +/- 3.3 ml h-1) was only a small fraction (1.2 +/- 0.03%) of the perfusate flow rate. The elimination half-life was calculated at 7.1 +/- 1.9 h. The area under curve, AUC0-4 h, for ethiofos (2858 +/- 314 nM h ml-1) was not significantly different from that of 14C (3038 +/- 692 nM h ml-1) or total material convertible to WR-1065 (total WR-1065, 3324 +/- 612 nM h ml-1), indicating a low level of metabolism. The AUC0-4 h for free WR-1065 (37.5 +/- 23.3 nM h ml-1) was less than 2% of ethiofos. Biliary elimination of ethiofos, WR-1065, and 14C was below 1%. At 4 h postdose, 7.9 +/- 1.9% of the dose of radioactivity remained in the liver. Less than 1.5% could be identified as ethiofos (0.12 +/- 0.09%) or total WR-1065 (1.09 +/- 0.05%). Ethiofos, 14C, and total WR-1065 were approximately evenly distributed between the 10,000-g pellet and supernatant. However, significantly more ethiofos, WR-1065, and 14C were recovered from the 105,000-g supernatant compared with the pellet. In summary, both the metabolism and biliary elimination of ethiofos and its derivatives were sparing. Hence it is likely that in the rat, the contribution of the liver to the presystemic biotransformation and poor bioavailability of ethiofos is relatively minor.     

Inhibited enzyme electrodes. Part 3: A sensor for low levels of H2S and HCN

It is shown that an inhibited enzyme electrode, using cytochrome oxidase, will respond to H₂S, HCN and azide ion. For all three inhibitors the kinetics of the inhibition and recovery processes have been analysed using the theoretical model presented previously (Albery et al., 1990a). Rearrangement of the differential equation describing inhibition and the development of the necessary software has enabled us to obtain values of the concentration of inhibitor in a matter of seconds after exposure of the sensor. The sensor will measure concentrations of H₂S down to 1 ppm in the gas phase and concentrations of HCN and azide ion down to 0·4 μmol dm⁻³ in the solution     

The life cycle ofAmblyomma parvum Aragao, 1908 (Acari: Ixodidae) under laboratory conditions

A colony ofAmblyomma parvum was started with engorged females collected from cattle in the Province of Salta (25°01 S, 63°56 W), Argentina. The progeny of those ticks were fed on rabbits and the non-parasitic stages maintained at 27±1°C, 83–86% RH in darkness. The life cycle (prefeeding period not evaluated) had a mean duration of 99.6 days. The mean time (days) for the different phases of the cycle were as follows: feeding period of females, 8.0; pre-oviposition period, 5.7; oviposition period, 17.5; minimum incubation period of the eggs, 31.8; feeding period of larvae, 3.2; premoult period to nymphs, 10.9; feeding period of nymphs, 4.7; premoult period to adults, 17.8. The oviposition pattern was typical of an ixodid tick, including a linear relationship between weights of engorged females and the number of eggs laid (r=0.8659). The males increased 18% in weight after feeding on hosts (P<0.01). The mean recovery rates of larvae, nymphs and females were 28.2%, 95.3% and 90.7%, respectively. The nymphs moulting to females were heavier (6.8±0.69 mg) than those moulting to males (3.2±0.29 mg) (P<0.01). A comparison of biological values ofA. parvum with American and non-AmericanAmblyomma species is presented.     

The early life of Octopus vulgaris (Cephalopoda: Octopodidae) in the plankton and at settlement: a change in lifestyle

Newly hatched young of the benthic, coastal-living octopod, Octopus vulgaris , enter the plankton and remain there for perhaps eight weeks. At hatching the arms are short and bear a few, large, primary suckers. The buccal mass is relatively large in proportion to the size of the animal. The eyes are large. The central nervous system has fairly well-defined lobes, some of which develop earlier than others. We shall follow the development of several features of O. vulgaris from hatching, through its life in the plankton until settlement and correlate them with changes in the brain and behaviour.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

The mandibular movements of Octopus vulgaris

The movements of the isolated buccal mass of Octopus vulgaris have been investigated. The beaks undergo rhythmic cycles of activity in the absence of applied stimulation and after electrical stimulation of the inter-buccal connective. Initial opening, closing, retraction and re-opening phases of movement are described. This cycle of movements is taken to resemble those in the intact animal. Anatomical and electrical evidence identifies the superior mandibular muscle as being partly responsible for the closing and retraction phases of movement. The inferior buccal ganglion determines the sequence of these buccal movements, but modification by sensory feed-back from the musculature is also implied. The preparation will allow a closer comparison of the control of movement in cephalopods and gastropods.     

The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.