A high affinity,calmodulin-responsive (Ca2+ + Mg2+)-ATPase in isolated bone cells

Although acute alterations in Ca²⁺ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca²⁺-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca²⁺-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca²⁺ of 280 nM, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg²⁺ could replace Ca²⁺ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg²⁺ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5′-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca²⁺-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca²⁺-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca²⁺ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function.     

Dopamine Uptake by Rat Striatal Synaptosomes: A Compartmental Analysis

Abstract: Dopamine (DA) uptake into synaptosomes from rat corpus striatum was studied in the presence of a monoamine oxidase (MAO) inhibitor and dithiothreitol, by means of a filtration technique. Under these conditions a steady state develops rapidly in which the synaptosomal DA content remains constant while the continuing DA uptake is counterbalanced by DA efflux from the synaptosome. Exchange of synaptosomal [³H]DA and [¹⁴C]DA was measured under these conditions. In timecourse experiments it was found that exchange could be described significantly better by a three-compartment model than by a two-compartment model. However, if synaptosomes from reserpine-pretreated animals were used, analysis according to a three-compartment model did not result in a significantly better fit compared with a two-compartment model. Subsequently, kinetic transfer parameters describing DA fluxes between compartments at different DA concentrations were calculated from the fitted exchange curves. A Michaelis-Menten kinetic analysis indicated that only the in-series three-compartment configuration, in which DA is taken up from the medium into one synaptosomal compartment, from which it can subsequently be transferred to a second compartment without direct access to the medium, gave kinetically acceptable results. Transfer parameters in synaptosomes from reserpine-treated rats were comparable to those parameters describing DA transport between the medium and the first intrasynaptosomal compartment as measured under control conditions. Morover, it was found that potassium depolarization of synaptosomes resulted in a release of DA in a quantity similar to that found in the second intrasynaptosomal compartment. It is suggested that the two intrasynaptosomal compartments found correspond to a cytoplasmatic and vesicular DA pool, respectively. The functional significance of these findings is discussed in terms of the regulation of DA levels within the nerve terminal.     

Restriction map of Tn7

Tn7, a transposon of 14 kb, encodes resistance to trimethoprim (Tp) and streptomycin (Sm). A cleavage site map of this transposon for twenty-two different restriction enzymes as determined by comparison of restriction enzyme cleavage patterns of the plasmids ColE1 and ColE1::Tn7 is presented. The precise localization of these sites was facilitated by the use of two deletion derivatives of ColE1::Tn7: pGB2 and ColE1::Tn7Δ6, and by the use of pOB14 and pOB15 which contain a part of Tn7 cloned into the plasmid pBR322. This map should aid in the study of the structural and genetic organization of this transposon.     

Recombinants between Clock Mutants of CHLAMYDOMONAS REINHARDI

Mutants affecting the period length of the biological clock in Chlamydomonas reinhardi have been isolated and a start has been made on analyzing the genetics of this system. In four mutants, the long period characteristic seems to be controlled by single genes at separate loci. Crosses between single mutants, as well as crosses involving three or four mutant genes, yielded progeny with periods characteristic of the parents as well as recombinant types, including normal period (wild type) and extra-long periods (double, triple and quadruple mutants). It was found that the period lengthening effect is additive; that is, the period of double mutants is lengthened by the sum of the period lengthening of the single mutants.     

In Vitro Effect of Rifampin on Mycobacteria

Rifampin inhibited 20 strains of Mycobacterium tuberculosis in concentrations of 0.005 to 0.02 mug/ml in 7H-9 broth with Tween 80 and killed all or nearly all of the inoculum in four to eight times greater concentrations. In the same medium without Tween 80, as well as on 7H-10 agar, about 16 to 64 times these amounts were required to produce the same effect. Rifampin was also active against M. kansasii and some of the nonchromogenic mycobacteria. The incidence of mycobacterial cells resistant to rifampin within the cultures studied was in the range of one to four per 10(8) to 10(9) colony-forming units with concentrations of 4 to 125 mug of rifampin per ml. Only one of the Battey cultures and that of M. fortuitum yielded cells resistant to rifampin at 125 mug/ml but not at 500 mug/ml. The same strains yielded more than double that number of organisms resistant to streptomycin and up to 100 times more organisms resistant to isoniazid. All three drugs stopped the growth or reduced the mycobacterial population in growing cultures after contact for 24 to 48 hr. Complete inhibition of growth was produced by rifampin at 1.0 mug/ml in an average of 6 days and by streptomycin at 5.0 mug/ml in 3 days. After an average contact of 10.7 days with rifampin, five of seven strains resumed growth and all strains began regrowth after exposure to streptomycin for 9.4 days. The marked susceptibility of M. tuberculosis and of atypical mycobacteria to rifampin in vitro and the relatively low incidence of resistant mutants suggests that this agent may have clinical usefulness in the treatment of tuberculosis and some other mycobacterioses.     

Bacterial Metabolism of Mevalonic Acid

Soluble cell-free extracts of actinomycete S4 grown on media containing mevalonate catalyze acetoacetate formation from mevalonate, mevaldate, and β-hydroxy-β-methylglutaryl-coenzyme A (CoA). Conversion of mevalonate to acetoacetate involves formation of free β-hydroxy-β-methylglutaryl-CoA, but not free mevaldate. The reaction favors mevalonate oxidation, and nicotinamide adenine dinucleotide, rather than nicotinamide adenine dinucleotide phosphate, acts as oxidant.     

Cholelithiasis and Cholecystitis in Childhood

Six cases of cholecystitis and cholelithiasis confirmed by x-ray examination and surgical operation were observed in a ten-year period. Due to the wide variability in signs and symptoms in children, cholecystitis and cholelithiasis can be diagnosed only with a high degree of clinical suspicion and roentgenological examination. Gallbladder disease is uncommon in childhood but should be considered in children with vague abdominal pains or bouts of unexplained jaundice. If a normal appendix is found at laparotomy in the “acute abdomen,” the surgeon would be wise to palpate other specific organs within the abdomen, including the liver and gallbladder.The treatment of choice is cholecystectomy. The prognosis for recovery is excellent if there is no complicating systemic disease.