Freeze-fracture study of water-soluble, standard proteins and of detergent-solubilized forms of sarcoplasmic reticulum Ca2+-ATPase

Conventional freeze-fracturing electron microscopy was used to study water-soluble proteins and different forms of Ca2+-ATPase-detergent complexes. Freeze-fracture images of solutions containing proteins larger than myoglobin showed the presence of distinct, randomly dispersed particles on smooth fracture surfaces. The distribution of sizes of these particles was closely to Gaussian, with a mean size which was correlated to the Stokes diameter. Monomeric Ca2+-ATPase from sarcoplasmic reticulum, solubilized by deoxycholate or a non-ionic detergent, showed a bimodal distribution of particle sizes. Even more complex distributions were found for dimeric and trimeric preparations of Ca2+-ATPase. The results can be interpreted on the assumption that the Ca2+-ATPase molecule is elongated, with an overall length of about 110 A and a width in its largest part of about 75 A. It is concluded on the basis of the presented results that freeze-fracture electron microscopy can be successfully used for morphological studies of protein molecules in solution.     

Substrate specificity and adenosine triphosphatase activity of the ATP-dependent deoxyribonuclease of Bacillus subtilis

Studies on the specificity of the ATP-dependent DNase of Bacillus subtilis 168, carried out with pure enzyme at the optimal conditions for its action, have shown that the substrate is double-stranded linear DNA. Linear single-stranded DNA (separated strands of B. subtilis DNA and linear phage fd DNA) is not attacked, neither are there any circular forms (supercoiled or nicked simian virus 40 and circular single-stranded fd DNAs). The double-stranded DNA can be completely hydrolysed, the limit products being, almost exclusively, mononucleotides. The presence of terminal phosphate residues in the substrate (either at the 3' or the 5' end) is not necessary for enzyme action. This DNase appears therefore to be an exonuclease processively liberating mononucleotides from both strands of the native linear DNA. ATP (indispensable for the DNase reaction) is also hydrolysed by the enzyme, to ADP and inorganic orthophosphate (Pi) in the presence of DNA. The apparent Km for ATP, in the ATPase reaction, is 0.15 mM. At high ATP concentrations, which inhibit the DNase activity, there is activation of the ATPase reaction. Three molecules of ATP are consumed for each DNA phosphodiester bond split, at optimal conditions for DNase activity.     

Active K+ transport in Mycoplasma mycoides var. Capri. Net and unidirectional K+ movements.

Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K+ concentration (Ki: 200--300 mM) which greatly exceeds that of the growth medium, and a low Na+ concentration (Na+i: 20 mM). Unlike Na+i,K+i varies with cell aging. The K+ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K+ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, K+i decreases and is partially compensated by a gain in Na+. This substitution completely reverses when metabolic substrate is added (K+ reaccumulation process). Kinetic analysis of K+ movement in cells with steady K+ level shows that most of K+ influx is mediated by an autologous K+-K+ exchange mechanism. On the other hand, during K+ reaccumulation by K+-depleted cells, a different mechanism (a K+ uptake mechanism) with higher transport capacity and affinity drives the net K+ influx. Both mechanisms are energy-dependent. Ouabain and anoxia have no effect on K+ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg2+ -dependent ATPase activity.     

INTERFERENCE OF SEX-RELATED FACTORS IN THE RESPONSE OF LIVER CELLS TO EXPERIMENTAL MITOTIC STIMULI

Stimulation of liver cell multiplication was obtained under two different experimental conditions.

• 1 A single injection of casein solution resulted in (a) an identical synchronized mitotic wave response in 10-day old male and female rats and (b) a significantly lower response in adult male rats compared to females, a difference which was reduced by castration of males at birth but essentially maintained if animals were operated when 10 days old.
• 2 Partial hepatectomy shortly after puberty resulted in active hepatocyte multiplication occurring 3 hr earlier in females than in males. This difference was suppressed when females were ovariectomized at birth and significantly reduced when they were spayed at a later age. Hepatocytes of castrated females entered actively into S phase 2 hr later than the sham-operated controls. Unilateral ovariectomy on the other hand indicated that during compensatory and/or hyper-compensatory activity of the single ovary there was a maximum difference between the male and female rate of [³H]thymidine uptake in liver nuclei 20 hr after hepatectomy. A further kinetic study (t= 25, 30, 40, 65, 90 hr) indicated no significant sex-related difference in the number of S phases per 10,000 cells.

The DNA content of regenerating versus control livers was comparable in both sexes at t= 22 and 90 hr but higher in females at t= 40 and 65 hr. A possible early postnatal interference of certain hormonal mechanisms in the receptivity to mitotic stimuli is postulated and discussed.     

Displacements of Simulium damnosum and strategy of control against onchocerciasis.

Regular aerial treatment of 14 000 km of watercourses has achieved and maintained, over an area of 700 000 km2 of West African savannah, a very high degree of control of the larvae of Simulium damnosum sensu stricto and S. sirbanum, the vectors of onchocerciasis in this area. However, particular and relatively restricted parts of this area, mainly in northern Ivory Coast and neighbouring parts of Upper Volta, experience regular and prolonged reinvasions by parous female vectors, which have already taken bloodmeals (and many of them carrying the parasites) and arrive from unknown sources probably hundreds of kilometres away, from directions probably between southwest and north. This reinvasion, now experienced in three successive years, represents the outstanding scientific, epidemiological and logistic problem still facing the WHO Onchocerciasis Control Programme. An outline is presented of the multidisciplinary investigations being undertaken to find a solution.     

Increase of cyclic AMP-dependent protein kinase type II as an early event in hormone-dependent mammary tumor regression.

Primary, 7,12-dimethylbenz(α)anthracene (DMBA)-induced mammary carcinoma in the rat contains cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent and -independent forms of protein kinase. When growth of DMBA-induced tumors was arrested by either ovariectomy or N⁶,O²′-dibutyryl cAMP treatment of the host, the activity of cAMP-dependent protein kinase type II markedly increased in the tumor cytosol, as shown by DEAE-cellulose chromatography and autophosphorylation. The increase in activity of cAMP-dependent protein kinase was also demonstrable in the tumor cytosol and nuclei following $\text{in}$$\text{vitro}$ incubation of tumor slices with cAMP. These results suggest that protein kinase type II is involved in the regression of hormone-dependent mammary tumors.     

DNA bifunctional intercalators. I. Synthesis and conformational properties of an ethidium homodimer and of an acridine ethidium heterodimer.

An ethidium homodimer and an acridine ethidium heterodimer have been synthesized. The ethidium and the acridine chromophore were introduced in such bifunctional intercalators in order to allow the fluorometric study of the interaction of such molecules with DNA, which is reported in the companion paper (Gaugain, B., Barbet, J., Capelle, N., Roques, B.P., & Le Pecq, J.B.(1978) Biochemistry 17 (following paper in this issue)). In the preparation of the acridine-ethidium dimer, we report the use of acetyl groups as new protecting agents in the phenanthridine series. Conformational studies of these molecules by visible absorption and NMR spectroscopy indicate that these dimers exist in equilibrium between folded and unfolded conformations and that this equilibrium is pH and temperature dependent. Models for the geometry of the folded forms are proposed.