A late role for a subset of neurogenic genes to limit sensory precursor recruitments in Drosophila embryos

Summary In Drosophila, mutations in a class of genes, the neurogenic genes, produce an excess of neurons. This neural hyperplasia has been attributed to the formation of more than the normal number of neuronal precursor cells at the expense of epidermal cells. In order to find out whether the neurogenic genes only act at this intial step of neurogenesis, we studied the replication pattern of the sensory organ precursor cells by monitoring BrdU incorporation in embryos mutant for Notch (N), Delta (Dl), mastermind (mam), almondex (amx), neuralized (neu), big brain (bib) and the Enhancer of split-Complex (E(spl)-C). Using temperature sensitive alleles of two of the neurogenic genes, DI and N, we also induced an acute increase of replicating sensory precursors by shifting briefly to the restricted temperature. We have found that the loss of function of all the seven neurogenic loci that were tested causes an increase in replicating sensory precursor cells, consistent with the model that these neurogenic genes normally participate in the process of restricting the number of neuronal precursors. Whereas the temporal pattern of replication appeared normal in mutants of five of the seven neurogenic loci, in N and mam embryos replicating PNS cells are present beyond the time when they normally undergo replication. Experiments with colchicine suggest that many of these late replicating cells may be newly emerging precursors and probably not additional cell divisions of already recruited precursors. Thus, different neurogenic genes may be required over different periods of time for the specification of sensory precursor cells. Correspondence to: R. Bodmer     

The influence of prey consumption and demographic stochasticity on population growth rate of Isle Royale wolves Canis lupus

The relationship between the rates of prey capture and predator population growth is a fundamental aspect of predation, yet it is rarely measured for vertebrate predators. For the isolated wolf population on Isle Royale, annual variation in kill rate explains 22% of the variation in wolf population growth rate. From the slope of this relationship, we estimate that the production efficiency (ratio of production to respiration) of wolves is between 0.5% and 1.5%. More generally, we assess the relative extent to which wolf population growth rate is affected by density dependence, prey availability (moose, Alces alces ), winter weather, and demographic stochasticity. Prey availability explains the most variation in wolf growth rate (42%), but this is only recognized after accounting for the influence of a disease-induced population crash and age structure of the prey population (i.e. number of vulnerable moose, >9 years of age). Demographic stochasticity accounts for approximately 30% of the variation in wolf growth rate. This recognition is important, but not surprising, given that the average population size of Isle Royale wolves is 22. Previous work indicates that the effect of winter climate, as mediated through prey vulnerability and kill rates, is substantial. This work indicates that the direct effect of winter climate is weak, and explains only about 4% of the variation in wolf growth rate (P=0.10).     

Tumor necrosis factor-alpha converting enzyme is processed by proprotein-convertases to its mature form which is degraded upon phorbol ester stimulation.

Tumor necrosis factor-alpha converting enzyme (TACE or ADAM17) is a member of the ADAM (a disintegrin and metalloproteinase) family of type I membrane proteins and mediates the ectodomain shedding of various membrane-anchored signaling and adhesion proteins. TACE is synthesized as an inactive zymogen, which is subsequently proteolytically processed to the catalytically active form. We have identified the proprotein-convertases PC7 and furin to be involved in maturation of TACE. This maturation is negatively influenced by the phorbol ester phorbol-12-myristate-13-acetate (PMA), which decreases the cellular amount of the mature form of TACE in PMA-treated HEK293 and SH-SY5Y cells. Furthermore, we found that stimulation of protein kinase C or protein kinase A signaling pathways did not influence long-term degradation of mature TACE. Interestingly, PMA treatment of furin-deficient LoVo cells did not affect the degradation of mature TACE. By examination of furin reconstituted LoVo cells we were able to exclude the possibility that PMA modulates furin activity. Moreover, the PMA dependent decrease of the mature enzyme form is specific for TACE, as the amount of mature ADAM10 was unaffected in PMA-treated HEK293 and SH-SY5Y cells. Our results indicate that the activation of TACE by the proprotein-convertases PC7 and furin is very similar to the maturation of ADAM10 although there is a significant difference in the cellular stability of the mature enzyme forms after phorbol ester treatment.     

Regional ventilation in statically and dynamically hyperinflated dogs

Hubmayr, Rolf D., and Susan S. Margulies. Regionalventilation in statically and dynamically hyperinflated dogs.J. Appl. Physiol. 81(4):1815-1821, 1996.Using the parenchymal marker technique innormal anesthetized dogs, we compared the dynamics of regional lungexpansion between two ventilation strategies designed to increase meanthoracic volume. Dynamic hyperinflation (DH) was produced byventilating the lungs at a rate of 50 breaths/min and with a duty cycleof 0.5. Static hyperinflation (SH) was produced throughthe application of extrinsic positive end-expiratory pressure while thelungs were ventilated at a rate of 15 breaths/min and with a duty cycleof 0.15. Regional tidal volume(VT,r), regional functionalresidual volume, and the time delay between regional expansion and the flow signal at the common airway were computed for upto 100 regions/lobe in 5 animals. Ventilation strategy had no effect onthe overall variance of VT,rwithin lobes. Although the VT,rmeasured during SH correlated withVT,r measured during DH, theaverage correlation coefficient was only 0.69. Ventilation rate-relateddifferences in VT,r and regionalfunctional residual capacity varied with the regional time delay inways qualitatively consistent with parallel inhomogeneity of unit timeconstants. However, a large component of frequency-dependent behaviorremains unexplained by established mechanisms. We conclude that DH and SH should not be considered equivalent lung unit recruitmentstrategies.

     

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wa6, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5 ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extrachromosomal genetic traits.     

Definition of constitutive gene expression in plants: the translation initiation factor 4A gene as a model

The NeIF-4A10 gene belongs to a family of at least ten genes, all of which encode closely related isoforms of translation initiation factor 4A. The promoter region of NeIF-4A10 was sequenced, and four mRNA 5 ends were determined. Deletions containing 2750, 689 and 188 bp of untranscribed upstream DNA were fused to the GUS reporter gene and introduced into transgenic tobacco. The three constructs mediated GUS expression in all cells of the leaf, stem and shoot apical meristem. Control experiments using in situ hybridization and tissue printing indicated that the observed GUS expression matches the expression patterns of NeIF-4A mRNA and protein. This detailed analysis at the level of mRNA, protein and reporter gene expression shows that NeIF-4A10 is an ideal constitutively expressed control gene. We argue that inclusion of such a control gene in experiments dealing with specifically expressed genes is in many cases essential for the correct interpretation of observed expression patterns.     

Calcitonin gene-related peptide and its receptor in the thymus

Calcitonin gene-related peptide (CGRP), a 37-amino acid residue neuropeptide, was immunostained in rat thymus at two sites: a subpopulation of thymic epithelial cells, namely subcapsular/perivascular cells, were heavily stained besides some nerve fibers surrounding arteries and arterioles. The administration of nanomolar concentrations of rat -CGRP dose-dependently raised intracellular cyclic adenosine monophosphate (cAMP) levels in isolated rat thymocytes (half-maximum stimulation 1 nM) but not in cultured rat thymic epithelial cells. Peptides structurally related to CGRP (i.e., rat calcitonin or amylin) had no effect. CGRP(8–37), an N-terminally truncated form, acted as an antagonist. Peripheral blood lymphocytes did not respond to CGRP, suggesting that receptors are present only on a subpopulation of thymocytes but not on mature T cells. This was substantiated by visualization of CGRP receptors on single cells by use of CGRP-gold and -biotin conjugates of established biological activity: only a small proportion of isolated thymocytes was surface labeled. In situ, the CGRP conjugates labeled receptors on large thymocytes residing in the outer cortical region of rat thymus pseudolobules. Thus, immunoreactive CGRP is found in subcapsular/perivascular thymic epithelial cells and acts via specific CGRP receptors on thymocytes by raising their intracellular cAMP level. It is suggested that CGRP is a paracrine thymic mediator that might influence the differentiation, maturation, and proliferation of thymocytes.     

Measurement of 15N-1H coupling constants in uniformly 15N-labeled proteins: Application to the photoactive yellow protein

A modified HNHB experiment is presented that allows thedetermination of J(NH) coupling constants directly from the ratio ofcross-peak to diagonal-peak intensities. The experiment was applied to thephotoactive yellow protein (PYP) and yielded the magnitude of 117³J(NH) coupling constants. In addition, 29³J(NH^(i–1)) coupling constantscould be measured, providing information about the backbone angle .These data, in conjunction with the magnitudes of the³J(H^NH) coupling constantsobtained from the HNHA spectrum, effectively discriminate the twopossibilities for the stereospecific assignment of theH resonances in glycine residues. For all eight glycineresidues in PYP that were not subject to conformational averaging and hadnon-degenerate H resonance frequencies, the J-couplingdata, together with limited NOE data, yielded the stereospecific assignmentof the H resonances for these residues. In addition,reliable and precise , dihedral constraints were also derived forthese residues from the J-coupling data.     

Stable carbon isotopes in tree rings of beech: climatic versus site-related influences

 Stable carbon isotopes in tree rings are a promising tool in palaeoclimate research, provided attempts are made to disentangle climatic from local effects (e.g. soil properties, competition, light). The ¹³C/¹²C variations in cellulose of tree rings of beech (Fagus sylvatica) were determined at several sites in the Swiss Central Plateau covering the last 50 years. We chose sites which differ in moisture conditions and sampled cores from four to six trees per site. The mean ¹³C/¹²C series from the different dry sites (distant by up to 40 km) are closely interrelated suggesting a common external cause. Correlation analysis with climate data proved the total precipitation in the months May, June and July to have the strongest effect on the carbon isotopes (r =  – 0.73). This result is in agreement with the commonly used model which relates the isotope discrimination to the water use efficiency. On the other hand, the isotope series of the wet sites are not as well correlated to the climate. At two of the sites (a dry and a humid) tree ring width suddenly increased. We used this effect as a test-case to study the influence of local growth conditions on the climate-isotope relationship. Received: 17 April 1996 / Accepted: 2 September 1996