Centrifugation of 2-cell mouse ova: cytoplasm stratification and recovery

Summary Two-cell mouse ova, which were centrifuged for l h at 70 000–90 000xg, showed a precise stratification of the cytoplasm and an elongation of the nucleus. The ova were fixed at different times and observed by light and electron microscopy using cytochemical methods and detergent extractions. Within 40 min after centrifugation the normal-looking morphology was recovered except for the persisting lipid caps at the centripetal poles of the blastomeres. Cleavage, compaction and blastulation were not prevented by centrifugation. Treatments with colcemid or cytochalasin D delayed but did not impair recovery. These results suggest that a resilient cytoskeletal structure may be involved in this kind of embryonic regulation.     

Mutational specificity of a proof-reading defective Escherichia coli dnaQ49 mutator

Summary The dnaQ (mutD) gene product which encodes the -subunit of the DNA polymerase III holoenzyme has a central role in controlling the fidelity of DNA replication because both mutD5 and dnaQ49 mutations severely decrease the 3–5 exonucleolytic editing capacity.It is shown in this paper that more than 95% of all anaQ49-induced base pair substitutions are transversions of the types G:C-T:A and A:T-T:A. Not only is this unusual mutational specificity precisely that observed recently for a number of potent carcinogens such as benzo(a) pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), which are dependent on the SOS system to mutagenize bacteria, but it is also seen for the constitutively expressed SOS mutator activity in E. coli tif-1 strains as well as for the SOS mutator activity mediated gap filling of apurinic sites. Because the G:C-T:A and A:T-T:A transversions can either result from the insertion of an adenine across from apurinic sites or arise due to the incorporation of syn-adenine opposite a purine base, we postulate that the DNA polymerase III holoenzyme also has a reduced discrimination ability in a dnaQ49 background.The introduction of a lexA (Ind^-) allele, which prevents the expression of SOS functions, led to a significant reduction in the dnaQ49-caused mutator effect.Both, the mutational specificity observed and the partial lexA ⁺ dependence of the mutator effect provoke a reanalysis of the hypothesis that the DNA polymerase III holoenzyme can be converted into the postulated but until now unidentified SOS polymerase.     

Steady state terrestrial ecosystems and the global carbon cycle

The assumption that landscapes dominated by mature vegetation are presently in carbon steady state with the atmosphere is challenged. Evidence suggests that the vegetation and soils of these landscapes are frequently disturbed and over short time periods (<300 yr) slowly sequester atmospheric carbon. The critical consideration in this argument is the time interval used to evaluate a steady state. Current models of carbon flux through the terrestrial biota limit their time considerations to 120 yr, a short and inadequate time interval for realistic assumptions about steady state in the carbon cycle of vegetation.Research performed under subcontract 19B-07762C with S. Brown and 19X-43326C with the Center for Energy and Environment Research of the University of Puerto Rico (A. E. Lugo) under Martin Marietta Energy Systems, Inc., contract DE-AC05-840R21400 with the U.S. Department of Energy.     

Nutrients and mass in litter and top soil of ten tropical tree plantations

The importance of litter to nutrient and organic matter storage and the possible influence of species selection on soil fertility in ten stands each consisting of a separate tree species were examined in this study. The plantations had been grown under similar conditions in an arboretum in the Luquillo Experimental Forest, Puerto Rico. The species involved were: Anthocephalus chinensis, Eucalyptus × patentinervis, E. saligna, Hernandia sonora, Hibiscus elatus, Khaya nyasica, Pinus caribaea var. hondurensis, P. elliottii var. densa, Swietenia macrophylla, and Terminalia ivorensis. After 26 yr, litter mass ranged from 5 mg ha^-1 in the H. sonora stand to 27.2 Mg ha^-1 in the P. caribaea stand. Nutrients in the litter (N, P, K, Ca, and Mg) also varied widely, but stands were ranked in different order when ranked by nutrients in the litter than then ranked according to accumulation of mass. Only E. saligna and A. chinensis stands were ranked similarly in accumulation of both nutrients and mass, and the stand of H. elatus was ranked higher with respect to nutrient accumulation than to accumulation of mass. The nutrient concentration in standing leaf litter generally increased in the order of recently fallen <old intact< fragmented. Nutrient concentration of standing leaf litter appears to increase with age and depth in the litter layer. The amount of nutrients stored in the litter compartment of these plantations was in the same order of magnitude as the quantity of available nutrients in the top 10-cm of mineral soil. Total litter mass was negatively correlated with the mass-weighted concentration of N, K, and Mg. The same relationship was found for Ca in the leaf litter and N in the fine wood litter compartments. In some stands (notably P. caribaea, P. elliottii, and E. saligna), leaf litter derived from species other than the species planted in that particular stand had higher nutrient concentration than leaf litter from the planted species. Soils of the 10 stands were classified in the same soil series and had similar texture (clay soils). However, significantly different chemical characteristics were found. Results obtained by analysis of covariance and by limiting comparisons to adjacent stands with similar soil texture, indicate that different species have had different influences on the concentration of available nutrients in soil.     

Electrostatic attraction by surface charge does not contribute to the catalytic efficiency of acetylcholinesterase.

Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.     

The Surface Exclusion System of RP1: Investigation of the Roles of trbJ and trbK in the Surface Exclusion, Transfer, and Slow-Growth Phenotypes

The contiguous trbJ and trbK genes of RP1 were cloned individually to study their effects. Surface exclusion was conferred only by trbK and only when gene dosage was high or when trbJ was also present in cis or in trans. This suggests that in the low-copy-number RP1, surface exclusion is due to a two-gene interaction in which trbK is the dominant partner. Among surface exclusion genes, trbJ is novel in yielding a periplasmic product that is also essential for conjugal transfer. This cellular location and the disturbed membrane function that accompanies TrbJ-processing probably accounts for the retarded growth caused by trbJ⁺ clones in Pseudomonas aeruginosa strain PAO.     

Regulation of DOPA Decarboxylase Activity in Brain of Living Rat

Abstract: To test the hypothesis that l -DOPA decarboxylase (DDC) is a regulated enzyme in the synthesis of dopamine (DA), we developed a model of the cerebral uptake and metabolism of [³H]DOPA. The unidirectional blood-brain clearance of [³H]DOPA ( K ^D₁) was 0.049 ml g⁻¹ min⁻¹. The relative DDC activity ( k ^D₃) was 0.26 min⁻¹ in striatum, 0.04 min⁻¹ in hypothalamus, and 0.02 min⁻¹ in hippocampus. In striatum, 3,4-[³H]dihydroxyphenylacetic acid ([³H]DOPAC) was formed from [³H]DA with a rate constant of 0.013 min⁻¹, [³H]homovanillic acid ([³H]HVA) was formed from [³H]DOPAC at a rate constant of 0.020 min⁻¹, and [³H]HVA was eliminated from brain at a rate constant of 0.037 min⁻¹. Together, these rate constants predicted the ratios of endogenous DOPAC and HVA to DA in rat striatum. Pargyline, an inhibitor of DA catabolism, substantially reduced the contrast between striatum and cortex, in comparison with the contrast seen in autoradiograms of control rats. At 30 min and at 4 h after pargyline, k ^D₃ was reduced by 50% in striatum and olfactory tubercle but was unaffected in hypothalamus, indicating that DDC activity is reduced in specific brain regions after monoamine oxidase inhibition. Thus, DDC activity may be a regulated step in the synthesis of DA.