Purification to homogeneity of rat liver dinucleoside tetraphosphatase by affinity elution with adenosine 5'-tetraphosphate

Starting from a partially purified dinucleoside tetraphosphatase (Np4Nase; EC 3.6.1.17), we developed an affinity elution purification protocol involving the strong competitive inhibitor adenosine 5'-tetraphosphate. Np4Nase bound to Cibacron Blue F3G-A-Sepharose 4B or to Reactive Blue 2-Sepharose CL-6B was specifically eluted with 10 microM adenosine 5'-tetraphosphate and 5 mM MgCl2, but not by either of them separately. The final Np4Nase preparation was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Coomassie blue or silver staining. The protein band showed an apparent 18 kDa molecular mass. The specific activity of the homogeneous Np4Nase was about 150 units/mg, meaning a 45,000-fold increase and a 10% recovery with respect to the crude extract. After preparative polyacrylamide gel electrophoresis, protein visualization with KCl, fragmentation of the gel lane, and extraction, all the renatured Np4Nase activity was found associated to the 18 kDa band. The renatured enzyme showed the same Km value for diadenosine 5',5"'-P1,P4-tetraphosphate as the partially purified or the native homogeneous Np4Nase.     

Enzyme composition and the taxonomy of Acanthamoeba

The enzyme activities of 37 representative strains of Acanthamoeba against 19 substrates have been examined. A total of 13 enzyme complements were identified, which could be arranged in six larger groups. There was good agreement between these groupings and the arrangement of the strains that was suggested by the electrophoresis patterns of their esterases and acid phosphatases. A numericlature is described which provides an unequivocal numerical label for each enzyme complement.     

Particulate diadenosine 5',5"'-P1,P3-triphosphate hydrolases in rat brain: two specific dinucleoside triphosphatases and two phosphodiesterase I-like hydrolases

Rat liver and brain differ in the distribution pattern of the total hydrolytic activity on diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) between the soluble and particulate fractions. The Ap3A-hydrolase activity in both the soluble and particulate liver fractions and in the brain soluble fraction had been previously studied in detail. We report now on the brain particulate fraction which, unlike liver, showed a low unspecific phosphodiesterase I-like (PDEaseI, EC 3.1.4.1) activity relative to the specific dinucleoside triphosphatase (Ap3Aase, EC 3.6.1.29). Two PDEaseI-like forms (PDEaseI-A and PDEaseI-B), with different apparent Mrs and kinetic properties, and two Ap3Aases (Ap3Aase-alpha and Ap3Aase-beta) were solubilized with 0.5% Triton X-100 from the particulate fraction. Ap3Aase-alpha resembled the cytosolic Ap3Aase (Ap3Aase-c), a known situation in liver. Comparative to Ap3Aase-alpha, Ap3Aase-beta showed a slightly higher Km (35 vs. 15 micron) and lower isoelectric point (5.25 vs. 5.45); Ap3Aase-beta was absent from the soluble fraction, and its recovery was unaffected by proteinase inhibitors, strongly arguing for distinct soluble and particulate turnover pathways for dinucleoside polyphosphates.     

Early post-fire regeneration of a Pinus halepensis forest on Mount Párnis,Greece

Abstract. The post-fire regeneration of a 45-yr-old Pinus halepensis (Aleppo pine) forest, burned in July 1989, has been studied on Mount Párnis, Attiki, Greece. Four experimental plots at various slopes and exposures were established at altitudes of 400 - 450 m, and monitored for 3 yr at 3-month intervals. Early regeneration took place abundantly, through both resprouting and seed germination of mostly hard-seeded herbs and shrubs; the floristic richness was high with 80 taxa. Pine seedling emergence took place during the winter of the first post-fire year. The mean pine seedling density by the end of the recruitment period (March 1990) was 5–6 seedlings/m². This density decreased slightly during late spring and considerably during summer. During the second post-fire year only a relatively slight decline was observed; thereafter the density was stabilized to 1 - 2 seedlings/m². Mortality follows a negative exponential curve that levels off at ca. 20 %. Height distributions throughout the three post-fire years were all positively skewed as a result of the presence of few very tall saplings. A considerable fraction (20 %) of very short (5–15 cm) saplings were still alive 39 months after the fire; these may constitute the sapling bank. Based on the analysis of height distribution curves, it is concluded that the taller seedlings survived significantly better than the shorter ones.     

Differential responsiveness of late passage C-6 glial cells and advanced passages of astrocytes derived from aged mouse cerebral hemispheres to cytokines and growth factor: Glutamine synthetase activity

In this study, we were interested to compare the responsiveness to growth factors, NGF, b-FGF and EGF and cytokines, IL1β, and TNF-α, in late passages (74–79) C6 glial cells committed astrocytes and astrocytes of advanced passages (26–28) in cultures derived from aged mouse cerebral hemispheres (MACH). Cultures were grown in either DMEM or chemically defined medium (CDM/TIPS) in order to test the effects of growth factors or cytokines. The activity of glutamine synthetase (GS), a marker for astrocytes, was used as a test parameter. We found that treatment with growth factors increased GS activity in both glial cell culture systems with the exception of EGF in C-6 glial cells. Treatment with cytokines markedly decreased GS activity in the late passage C6 glial cells whereas only TNF-α had a similar effect on MACH astrocytes. In view of the generally opposite effects of growth factors and cytokines on GS activity, we-speculate that these molecules which are also endogenously present in glial cells may play a role in the maintenance of cellular homeostasis.     

Developmental studies in Drosophila

A study of the puffing patterns of the salivary gland chromosomes of D. pseudoobscura was carried out through several larval, prepupal, and pupal stages of development. A total of 176 puffs were found, 111 of which changed during the stages studied. As described in previous investigations with other Drosophila species there are two major peaks of puffing activity. These two peaks occur during puparium formation and pupation. Additionally, a minor activity-peak occurs during mid-prepupal life. Attempts have been made to establish correlations between the puffing data and those obtained from electrophoretic and ultrastructural studies.Supported in part by grants GM-16736-03 and FR-05426-09 from the U.S. Department of Health, Education, and Welfare. Ann Jacob Stocker was a holder of a University of Texas predoctoral fellowship.     

Studies of a homologous gene-enzyme system,Esterase C,in Drosophila melanogaster and Drosophila simulans

Electrophoretic variants of nonspecific esterases (Est-6 and Est-C) of various populations of Drosophila melanogaster and Drosophila simulans from Northern Greece were studied by means of starch gel electrophoresis, and the results are compared with those obtained from standard stocks. Two new alleles of the Est-6 locus of D. melanogaster and two new alleles of the Est-6 locus of D. simulans are described. The position of the Est-C locus in D. simulans is estimated. Evidence is presented for the genetic homology of the Est-C locus of D. melanogaster and the Est-C locus of D. simulans.     

The genetics ofDrosophila subobscura populations

Summary The inversion polymorphism in a population ofDrosophila subobscura from Holland is examined. The high frequency of the Standard gene arrangement in all five chromosomes, the high IFR value, and the low mean number of inversions show a low polymorphism expected in submarginal northern populations within the area of the geographic distribution of the species. The study of this population further confirms the existence of North-South clines for inversion types in every chromosome of this species.     

A comparative chromosome study in the incipient species of the Drosophila paulistorum complex

The gene arrangements in the chromosomes of the races or incipient species of the Drosophila paulistorum complex have been compared in the interracial hybrids. The results are correlated with those obtained by Dobzhansky and Pavlovsky (1962), and also with data on the intraracial polymorphisms, to be published elsewhere. A Standard strain was chosen arbitrarily, the Palmira stock of the Transitional race, and other races and strains described in terms of comparison with the Standard. The minimal number of inverted sections differentiating the Andean race from the Standard is 1, Centroamerican 5, Orinocan 3, Amazonian 6. Little chromosome pairing takes place in the hybrids between the Standard and the Guianan strains. These strains may well be regarded as belonging to a full-fledged species distinct from D. paulistorum complex. The results of the present study furnish little support to the Mayr-Carson hypothesis, according to which diverging incipient species are expected to share few or no intrapopulational polymorphisms.The work reported in this article has been carried under Contract No. AT (30-1)-3096-10, U.S. Atomic Energy Commission.     

Typing of Proteus mirabilis from clinical sources by computerized analysis of electrophoretic protein patterns

B. HOLMES, M. COSTAS AND A.C. WOOD. 1991. Seventy-five strains of Proteus mirabilis (mainly of human origin) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries; 24 were from urine, 17 from faeces (including one from a dog and one from a monkey), 12 from the respiratory tract (including nine from sputum), four from a cerebral abscess, 12 from miscellaneous sources and six from unknown sources. The protein patterns, which contained 45 to 50 discrete bands, were highly reproducible. The patterns of the 75 P. mirabilis cultures plus those of the type strains of six Proteus and Providencia species were used as the basis for two numerical analyses. In the first, which included all the protein bands, the P. mirabilis cultures formed nine clusters at the 85% S level. In the second analysis, in which the principal protein bands (in the 34.0–44.6 kDa range) were excluded, 74 of the 75 cultures of P. mirabilis formed a single cluster at the 77% S level distinct from the six Proteus and Providencia reference strains. The P. mirabilis strain which failed to cluster with the others had a background band pattern typical of the species in the lower molecular weight region but appeared to be less typical in the heavier bands. It is concluded that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of P. mirabilis. Reference strains of each of the nine PAGE types identified are available from NCTC for inclusion in future studies.