Are dendrites in Drosophila homologous to vertebrate dendrites?

Dendrites represent arborising neurites in both vertebrates and invertebrates. However, in vertebrates, dendrites develop on neuronal cell bodies, whereas in higher invertebrates, they arise from very different neuronal structures, the primary neurites, which also form the axons. Is this anatomical difference paralleled by principal developmental and/or physiological differences? We address this question by focussing on one cellular model, motorneurons of Drosophila and characterise the compartmentalisation of these cells. We find that motorneuronal dendrites of Drosophila share with typical vertebrate dendrites that they lack presynaptic but harbour postsynaptic proteins, display calcium elevation upon excitation, have distinct cytoskeletal features, develop later than axons and are preceded by restricted localisation of Par6-complex proteins. Furthermore, we demonstrate in situ and culture that Drosophila dendrites can be shifted from the primary neurite to their soma, i.e. into vertebrate-like positions. Integrating these different lines of argumentation, we propose that dendrites in vertebrates and higher invertebrates have a common origin, and differences in dendrite location can be explained through translocation of neuronal cell bodies introduced during the evolutionary process by which arthropods and vertebrates diverged from a common urbilaterian ancestor. Implications of these findings for studies of dendrite development, neuronal polarity, transport and evolution are discussed.     

Strong binding of myosin heads stretches and twists the actin helix

Calculation of the size of the power stroke of the myosin motor in contracting muscle requires knowledge of the compliance of the myofilaments. Current estimates of actin compliance vary significantly introducing uncertainty in the mechanical parameters of the motor. Using x-ray diffraction on small bundles of permeabilized fibers from rabbit muscle we show that strong binding of myosin heads changes directly the actin helix. The spacing of the 2.73-nm meridional x-ray reflection increased by 0.22% when relaxed fibers were put into low-tension rigor (<10 kN/m(2)) demonstrating that strongly bound myosin heads elongate the actin filaments even in the absence of external tension. The pitch of the 5.9-nm actin layer line increased by approximately 0.62% and that of the 5.1-nm layer line decreased by approximately 0.26%, suggesting that the elongation is accompanied by a decrease in its helical angle (approximately 166 degrees) by approximately 0.8 degrees. This effect explains the difference between actin compliance revealed from mechanical experiments with single fibers and from x-ray diffraction on whole muscles. Our measurement of actin compliance obtained by applying tension to fibers in rigor is consistent with the results of mechanical measurements.     

SP-A binds alpha1-antitrypsin in vitro and reduces the association rate constant for neutrophil elastase

Background

α1-antitrypsin and surfactant protein-A (SP-A) are major lung defense proteins. With the hypothesis that SP-A could bind α1-antitrypsin, we designed a series of in vitro experiments aimed at investigating the nature and consequences of such an interaction.

Methods and results

At an α1-antitrypsin:SP-A molar ratio of 1:1, the interaction resulted in a calcium-dependent decrease of 84.6% in the association rate constant of α1-antitrypsin for neutrophil elastase. The findings were similar when SP-A was coupled with the Z variant of α1-antitrypsin. The carbohydrate recognition domain of SP-A appeared to be a major determinant of the interaction, by recognizing α1-antitrypsin carbohydrate chains. However, binding of SP-A carbohydrate chains to the α1-antitrypsin amino acid backbone and interaction between carbohydrates of both proteins are also possible. Gel filtration chromatography and turnover per inactivation experiments indicated that one part of SP-A binds several molar parts of α1-antitrypsin.

Conclusion

We conclude that the binding of SP-A to α1-antitrypsin results in a decrease of the inhibition of neutrophil elastase. This interaction could have potential implications in the physiologic regulation of α1-antitrypsin activity, in the pathogenesis of pulmonary emphysema, and in the defense against infectious agents.     

Identification of a D-glycero-D-manno-heptosyltransferase gene from Helicobacter pylori

We have identified a Helicobacter pylori d-glycero-d-manno-heptosyltransferase gene, HP0479, which is involved in the biosynthesis of the outer core region of H. pylori lipopolysaccharide (LPS). Insertional inactivation of HP0479 resulted in formation of a truncated LPS molecule lacking an alpha-1,6-glucan-, dd-heptose-containing outer core region and O-chain polysaccharide. Detailed structural analysis of purified LPS from HP0479 mutants of strains SS1, 26695, O:3, and PJ1 by a combination of chemical and mass spectrometric methods showed that HP0479 likely encodes alpha-1,2-d-glycero-d-manno-heptosyltransferase, which adds a d-glycero-d-manno-heptose residue (DDHepII) to a distal dd-heptose of the core oligosaccharide backbone of H. pylori LPS. When the wild-type HP0479 gene was reintegrated into the chromosome of strain 26695 by using an "antibiotic cassette swapping" method, the complete LPS structure was restored. Introduction of the HP0479 mutation into the H. pylori mouse-colonizing Sydney (SS1) strain and the clinical isolate PJ1, which expresses dd-heptoglycan, resulted in the loss of colonization in a mouse model. This indicates that H. pylori expressing a deeply truncated LPS is unable to successfully colonize the murine stomach and provides evidence for a critical role of the outer core region of H. pylori LPS in colonization.     

Identification of genes involved in the glyoxylate regeneration cycle in Methylobacterium extorquens AM1, including two new genes, meaC and meaD

The glyoxylate regeneration cycle (GRC) operates in serine cycle methylotrophs to effect the net conversion of acetyl coenzyme A to glyoxylate. Mutants have been generated in several genes involved in the GRC, and phenotypic analysis has been carried out to clarify their role in this cycle.     

Methylotrophic autotrophy in Beijerinckia mobilis

Representatives of the genus Beijerinckia are known as heterotrophic, dinitrogen-fixing bacteria which utilize a wide range of multicarbon compounds. Here we show that at least one of the currently known species of this genus, i.e., Beijerinckia mobilis, is also capable of methylotrophic metabolism coupled with the ribulose bisphosphate (RuBP) pathway of C1 assimilation. A complete suite of dehydrogenases commonly involved in the sequential oxidation of methanol via formaldehyde and formate to CO2 was detected in cell extracts of B. mobilis grown on CH3OH. Carbon dioxide produced by oxidation of methanol was further assimilated via the RuBP pathway as evidenced by reasonably high activities of phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Detection and partial sequence analysis of genes encoding the large subunits of methanol dehydrogenase (mxaF) and form I RubisCO (cbbL) provided genotypic evidence for methylotrophic autotrophy in B. mobilis.     

Molecular mechanisms involved in robustness of yeast central metabolism against null mutations

Adaptive strategies employed by the yeast Saccharomyces cerevisiae provide robustness and adaptability of its central metabolism. Since central metabolism in yeast has been well studied at the enzymatic and genetic levels, it represents an excellent system for evaluating the relative roles of duplicate genes and alternative metabolic pathways as possible mechanisms for the stability of central metabolism against null mutations. Yeast appears to employ a variety of mechanisms to ensure functional robustness of its central metabolism. Uninterrupted flow of energy and precursor metabolites through the pathways of central metabolism via glycolysis (EMP), pentose phosphate shunt (PPS), and the tricarboxylic acid (TCA) cycle are ensured by a variety of adaptive mechanisms. One of the most significant mechanisms appears to be gene duplication events that have produced a number of isozymes functioning under variable environmental and physiological conditions. Alternative pathways represent another important mechanism for increasing the robustness of the system. The robustness of the pathways of central metabolism is apparently higher than that of the other parts of metabolism, because of its exceptional importance to the organism's vitality. The proportion of duplicated viable genes also is substantially larger in central metabolism than that in a pool of other metabolic genes.     

Mutational analysis of the stator subunit E of the yeast V-ATPase

Subunit E is a component of the peripheral stalk(s) that couples membrane and peripheral subunits of the V-ATPase complex. In order to elucidate the function of subunit E, site-directed mutations were performed at the amino terminus and carboxyl terminus. Except for S78A and D233A/T202A, which exhibited V(1)V(o) assembly defects, the function of subunit E was resistant to mutations. Most mutations complemented the growth phenotype of vma4Delta mutants, including T6A and D233A, which only had 25% of the wild-type ATPase activity. Residues Ser-78 and Thr-202 were essential for V(1)V(o) assembly and function. The mutation S78A destabilized subunit E and prevented assembly of V(1) subunits at the membranes. Mutant T202A membranes exhibited 2-fold increased V(max) and about 2-fold less of V(1)V(o) assembly; the mutation increased the specific activity of V(1)V(o) by enhancing the k(cat) of the enzyme 4-fold. Reduced levels of V(1)V(o) and V(o) complexes at T202A membranes suggest that the balance between V(1)V(o) and V(o) was not perturbed; instead, cells adjusted the amount of assembled V-ATPase complexes in order to compensate for the enhanced activity. These results indicated communication between subunit E and the catalytic sites at the A(3)B(3) hexamer and suggest potential regulatory roles for the carboxyl end of subunit E. At the carboxyl end, alanine substitution of Asp-233 significantly reduced ATP hydrolysis, although the truncation 229-233Delta and the point mutation K230A did not affect assembly and activity. The implication of these results for the topology and functions of subunit E within the V-ATPase complex are discussed.     

The copper-transporting ATPases, menkes and wilson disease proteins, have distinct roles in adult and developing cerebellum

Copper is essential for brain metabolism, serving as a cofactor to superoxide dismutase, dopamine-beta-hydroxylase, amyloid precursor protein, ceruloplasmin, and other proteins required for normal brain function. The copper-transporting ATPases ATP7A and ATP7B play a central role in distribution of copper in the central nervous system; genetic mutations in ATP7A and ATP7B lead to severe neurodegenerative disorders, Menkes disease and Wilson disease, respectively. Although both ATP7A and ATP7B are required, their specific roles and regulation in the brain remain poorly understood. Using high-resolution imaging and functional assays, we demonstrate that ATP7A and ATP7B show cell-specific distribution in adult cerebellum, have distinct enzymatic characteristics, and are regulated differently during development. ATP7B is continuously expressed in Purkinje neurons (PN) where it delivers copper to the ferroxidase ceruloplasmin. ATP7A is a faster copper transporter than Wilson disease protein as evidenced by faster rates of catalytic reactions. The expression of ATP7A switches during development from PN to Bergmann glia, the cells supporting PN function in adult brain. Inactivation of ATP7B (Wilson disease protein) by gene knock-out induces a striking shift in the expression of the ATP7B target protein, ceruloplasmin, from PN to Bergmann glia, where ATP7A (Menkes disease protein) is present. The induced cell-specific change in expression restores copper delivery to ceruloplasmin via ATP7A. Overall, the results provide evidence for distinct functions of ATP7A and ATP7B in the cerebellum and illustrate a tight link between copper homeostasis in PN and Bergmann glia.