Carbon isotope fractionation by ribulose-1,5-bisophosphate carboxylase from various organisms

Carbon isotope fractionation by structurally and catalytically distinct ribulose-1,5-bisphosphate carboxylases from one eucaryotic and four procaryotic organisms has been measured under nitrogen. The average fractionation for 40 experiments was −34.1 ‰ with respect to the δ¹³C of the dissolved CO₂ used, although average fractionations for each enzyme varied slightly: spinach carboxylase, −36.5 ‰; Hydrogenomonas eutropha, −38.7 ‰; Agmenellum quadruplicatum, −32.2 ‰; Rhodospirillum rubrum, −32.1 ‰; Rhodopseudomonas sphaeroides peak I carboxylase, −31.4 ‰; and R. sphaeroides peak II carboxylase, −28.3 ‰. The carbon isotope fractionation value was largely independent of method of enzyme preparation, purity, or reaction temperature, but in the case of spinach ribulose-1,5-bisphosphate carboxylase fractionation, changing the metal cofactor used for enzyme activation had a distinct effect on the fractionation value. The fractionation value of −36.5 ‰ with Mg²⁺ as activator shifted to −29.9 ‰ with Ni²⁺ as activator and to −41.7 ‰ with Mn²⁺ as activator. These dramatic metal effects on carbon isotope fractionation may be useful in examining the catalytic site of the enzyme.     

Serum estradiol, testosterone and dihydrotestosterone in male monkeys treated with testosterone propionate.

Serum estradiol (E2), testosterone (T) and dihydrotestosterone (DHT) were measured in juvenile (pre-pubertal) male rhesus monkeys injected with either 8 mg or 80 mg of testosterone propionate (TP). After one week, the three steroids were elevated and remained essentially unchanged for the duration of the study. There was little difference in serum E2 or DHT when comparing the two groups of steroid-treated monkeys. In contrast, T levels were consistently greater in the animals given the high dosage of TP.     

Gene-specific formation and repair of cisplatin intrastrand adducts and interstrand cross-links in Chinese hamster ovary cells

We have used three methods to study the formation and repair of intrastrand adducts and interstrand cross-links in the DNA of Chinese hamster ovary cells induced by the anticancer drug cis-diamminedichloroplatinum II (cisplatin). Using atomic absorption spectroscopy, we found that 21% of the total genomic cisplatin adducts were removed at 8 h and 42% at 24 h. We used ABC excinuclease digestion, coupled with out previously reported methodology to quantify DNA in specific genomic regions. These adducts were removed faster in the transcribed dihydrofolate reductase and c-myc genes compared to a noncoding fragment, a region containing the little or nontranscribed c-fos oncogene, and to the overall genome. Interstrand cross-links in specific sequences were quantified by Southern hybridization of denatured-renatured DNA separated on a neutral gel. We found that cross-links were removed more efficiently from the gene regions than intrastrand adducts and, at high levels of cross-linking, removal was similar from transcribed and from nontranscribed regions.     

Truncation of the extended carboxyl-terminal domain increases the expression and regulatory activity of the avian beta-adrenergic receptor.

A series of mutant avian beta-adrenergic receptors with progressively truncated carboxyl termini have been expressed in insect and mammalian cells. Removal of 18-124 amino acid residues caused multiple phenotypic changes in the receptor. Membranes from cells that expressed the truncated receptors displayed elevated basal (2- to 3-fold) and agonist-stimulated adenylylcyclase activities. Adenylylcyclase activity in these membranes also displayed greater stimulation in response to partial agonists. Activity was also markedly stimulated by beta-adrenergic ligands that are usually considered to be antagonists (alprenolol, greater than 4-fold; propranolol, approximately 2-fold). Wild type receptor did not mediate a response to these classical antagonists. After purification and reconstitution with Gs, the truncated receptors did not appear to be more active than the wild type. Guanine nucleotides modulated the affinity of agonist for the truncated receptors, whereas the affinity of agonist for the wild type receptor was not altered by guanine nucleotides. The truncated receptors were solubilized from the membrane more efficiently and were more susceptible to amino-terminal proteolysis than was the wild type protein. These results suggest interaction of the carboxyl terminus of the avian beta-adrenergic receptor with cellular regulatory or structural elements.     

A second gene product of the inositol-phospholipid-specific phospholipase C delta subclass.

Sequence analysis of a inositol-phospholipid-specific phospholipase C (PtdIns-PLC) purified from bovine brain has led to the isolation of a novel cDNA that encodes this protein. While this cDNA contains two introns, these appear to be removed upon transfection of the cDNA into COS-1 cells. The protein transiently expressed in COS-1 cells shows phosphatidylinositol 4,5-bisphosphate hydrolysing activity which distributes preferentially into the particulate fraction. Comparison of the predicted amino acid sequence of this PtdIns-PLC with other known PtdIns-PLCs reveals a high degree of similarity, throughout all of its sequence, with PtdIns-PLC delta. Thus, we believe that the identification of this cDNA represents evidence for multiple functional-gene products within the delta subclass of PtdIns-PLCs.     

Natural and man-caused factors affecting the abundance and cycling of dissolved organic substances in precambrian shield lakes

A study on nutrient regeneration processes and a measure of their fluxes at the sediment-water interface was carried out in two different stations of a shallow lagoon of the Po delta river (Italy). A few parameters on the solid fraction (grain-size, porosity, C, N) and pore water profiles of o-P, NH₃, NO _inf3 ^sup– , SiO₂, Tot-CO₂, SO _inf4 ^sup2– , Fe, Mn, Ca, Mg, pH, Eh were determined. At both stations the results were typical for fine sediments rich in organic matter. The ratio of variations of sulphate (SO _inf4 ^sup2– ) to total carbonate demonstrates the main role sulphate reduction plays on the organic matter decay. The use of the ratios of variations of sulphate (SO _inf4 ^sup2– ) to ammonia (NH₃) and of sulphate (SO _inf4 ^sup2– ) to phosphate (o-P) in pore waters enabled us to calculate the C/N/P of the decomposing organic matter. Obtained C/N/P indicated an enrichment of N and P with regard to C/N/P ratios of the solid fraction, due to the selective stripping of N and P during organic matter mineralization. This phenomenon decreases with depth, where organic matter becomes more refractory. Calculations on saturation degrees have shown the possibility of authigenic calcite, apatite and rhodochrosite precipitation in sediments. Nutrient fluxes were estimated for SiO₂, NH₃ and o-P by means of benthic chambers and modelling the pore water profiles. The model used for the calculation of fluxes allowed us to account for the bioturbation-irrigation influence near the interface, by means of a cumulative diffusion coefficient. Directly measured fluxes proved to be always significantly greater than the theoretical ones. These differences seem to be due to surface processes which do not affect pore water concentrations (degradation of fresh materials at the interface; micro-bioturbation by small gasteropoda such as Hydrobia ventrosa) and/or to the different concept of the two methods in time and space. Number, size and biomass of macrobenthic species living in the sediment underneath the benthic chambers were determined. The comparison between data on macrobenthic populations and flux values showed a good relationship between the number of organisms and benthic fluxes within each station. However, flux variations between stations are to be attributed mainly to the different arrangement of the tubes of the polychaetes Polydora ciliata in the sediment.