The rep region of pR plasmid regulates the expression of SOS system

Summary By using an artificial hybrid between phage and the pR plasmid, we have shown that the rep region of the pR plasmid encodes a function which regulates the expression of the muc genes (plasmid genes that are under the negative control of lexA and responsible for an increased rate of spontaneous mutagenesis and resistance to UV and chemicals). Expression of the muc genes was monitored by a fusion between the muc promoter and the lacZ structural gene. When E. coli cells containing such a fusion are infected by the hybrid pR phasmid, -galactosidase activity is enhanced, indicating that pR encodes an antagonist of lexA. By deletion mapping we have located the gene encoding the antagonist of lexA (bat) in the rep region of the plasmid. The bat gene product can also antagonize the cI repressor as shown by the observation that pR phasmids are virulent on a homoimmune lysogen. We have exploited this latter property to carry out genetic and functional analysis of the bat region. This region is organized as a classical operon where the expression of the bat structural gene is negatively regulated by a repressor gene that encodes a proteic product.     

Behavior of L-threonine-degrading enzymes during liver regeneration

We examined the effects of a two-thirds hepatectomy in the adult rat on the activities of the three L-threonine-degrading enzymes, L-threonine dehydratase, L-threonine aldolase and L-threonine dehydrogenase. Noticeable variations were observed which did not occur in either sham-operated or turpentine-treated rats and were not linked to food intake. They were considered specific to the regenerating liver. When the reactions were followed in vitro, L-threonine deaminase and L-threonine aldolase were significantly lower for the first 12-24 h: L-threonine dehydrogenase decreased only after 48 h. These results are linked to a decrease in the enzyme concentration in the tissue. L-Serine and L-threonine liver concentrations increased 2-3-fold during the same periods. When the activities were evaluated in vivo, the levels of the first two enzymes remained constant for 24 h, but increased after 48 h; L-threonine dehydrogenase increased between 12 and 48 h. The in vivo activity of the enzymes was reflected by total L-threonine degradation, which had a single sharp peak at 48 h. The asynchronous variations in enzyme activity are related to the differences in protein metabolism which occur in the regenerating liver, and are the consequence of a new transient differential control. The changes observed are significant in liver regeneration; they regulate the consumption and the serum and liver levels of L-serine and L-threonine, setting them aside for protein synthesis. They minutely control the flux of amino acids toward gluconeogenesis, since, during the first 48 h after partial hepatectomy, the production of glucose is ensured principally by lactate; the contribution of L-threonine seems to be more significant only at 48 h. These findings are useful in the study of the regulation of the enzymes involved in amino acid metabolism during liver regeneration.     

Molecular aspects of the removal of ferritin-bound iron by DL-dihydrolipoate

The removal of ferritin-bound iron by the physiologic dithiol DL-dihydrolipoate was studied over the pH range 5.5-9.0. A novel method was devised for the determination of iron removal, making it possible to study the actual release of iron from ferritin, regardless of the oxidation state or complexation form. The overall iron-removal process appears to depend upon a balance between the deprotonation of the dithiol and the protolytic dissolution of the iron core inside the ferritin molecule. The amount of iron removed at equilibrium increases with the pH, at any of the dihydrolipoate/ferritin iron ratios tested. The formation of the binuclear iron-dithiol complex [Fe2(dihydrolipoate)3]-3 is not strictly required for iron mobilization, but it seems to affect the efficiency of the dithiol in iron mobilization by providing a stable complexation form for the released iron outside the ferritin protein shell. Comparison of the release of ferritin-bound iron by free and immobilized dihydrolipoate indicates that mobility of the dithiol is mandatory for the removal process to take place.     

Voltage-dependent channels permeable to K+ and Na+ in the membrane ofAcer pseudoplatanus vacuoles

Summary The patch-clamp technique in whole-cell configuration was used to study the electrical properties of the tonoplast in isolated vacuoles fromAcer pseudoplatanus cultured cells. In symmetrical KCl or K₂ malate solutions, voltage- and time-dependent inward currents were elicited by hyperpolarizing the tonoplast (inside negative), while in the positive range of potential the conductance was very small. The specific conductance of the tonoplast at –100 mV, in 100mm symmetrical KCl was about 160 S/cm². The reversal potentials (E _rev) of the current, measured in symmetrical or asymmetrical ion concentrations (cation, anion or both) were very close to the values of the K⁺ equilibrium potential. Experiments performed in symmetrical or asymmetrical NaCl indicate that Na⁺ too can flow through the channels. NeitherE _rev nor amplitude and kinetics of the current changed by replacing NaCl with KCl in the external solution. These results indicate the presence of hyperpolarization-activated channels in tonoplasts, which are permeable to K⁺ as well as to Na⁺. Anions such as Cl^– or malate seem to contribute little to the channel current.     

Tricyclic Imipramine Modification of the Circadian Rhythms of Hypothalamic Serotonin, its Precursors and Acid Catabolite in Individually Housed Rats

Circadian rhythms of serotonin (5HT), its precursors tryptophan (TP) and 5-hydroxy-tryptophan (5HTP) and its acid catabolite 5-hydroxy-indoleacetic acid (5HIAA), were determined in the hypothalamus of control rats and rats which had been treated continuously with subcutaneous imipramine (10 mg/kg/day) for 2 weeks.

Rats were individually housed and entrained to LD12:12. Controls showed the 5HT and TP peaks in the light and dark periods respectively, as reported in the literature, but no inverted correlation (antiphase) between SHT and 5HIAA rhythms.

Imipramine significantly modified circadian rhythm characteristics: the 5HT acrophase was advanced, that of TP and 5HIAA was delayed. Imipramine also significantly increased hypothalamic SHT and TP concentrations.     

Enzymic synthesis of the 4Fe-4S clusters of Clostridium pasteurianum ferredoxin

Ex novo enzymic synthesis of the two 4Fe-4S clusters of Clostridium pasteurianum ferredoxin has been achieved by incubation of the apoprotein with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, DL-dihydrolipoate and ferric ammonium citrate. This enzymic reconstitution procedure was compared to a chemical one, in which the enzyme was replaced by sodium sulfide. A further comparison was made with the results previously obtained in the enzymic synthesis of the 2Fe-2S cluster of spinach ferredoxin, allowing the following conclusions to be drawn. The nature of the cluster to be inserted into the reconstituted iron-sulfur protein is determined by the apoprotein itself. The refolding of the structure of the iron-sulfur proteins around the newly inserted cluster is the rate-limiting step in both chemical and enzymic reconstitution. Rhodanese appears to play a role in the recovery of the native architecture of the reconstituted iron-sulfur protein(s). The extension to the 4Fe-4S centers of the rhodanese-based biosynthetic system allows this enzymic route to be proposed as a general way to the in vivo synthesis of iron-sulfur structures.