Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Ser 16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.

Morphology of the spermatozoa of the Microhylidae (Anura, Amphibia)

Microhylid spermatozoa show the autapomorphic condition of possessing a thin post-mitochondrial cytoplasmic collar. Their spermatozoa are apomorphic in several respects. They have lost the distinct nuclear shoulder, endonuclear canal and axial perforatorium observed in urodeles, caecilians and primitive frogs, possess a conical perforatorium and apomorphically lack any fibres associated with the axoneme. The spermatozoa of Cophixalus , however, differ in several respects from those of the other microhylids examined. Cophixalus spermatozoa are longer in almost all measurements, the acrosome vesicle is cylindrical and does not completely cover the putative perforatorium, the perforatorium is asymmetrical and composed of fine fibres, the nucleus is strongly attenuated and narrower, and the mitochondria are elongate. The absence of fibres associated with the axoneme is an apomorphic condition shared with the Ranidae, Rhacophoridae and Pipidae.

Spermatozoal morphology of the freshwater anomuran Aegla longirostri Bond-Buckup & Buckup, 1994 (Crustacea : Decapoda : Aeglidae) from South America

Unique sperm morphology is described for Aegla longirostri Bond-Buckup & Buckup, 1994. a representative of the freshwater anomuran family Aeglidae from South America. Comparisons of the spermatozoal ultrastructure of this species with that described for other anomurans indicate that A. longirostri has a distinct suite of spermatozoal characters. Within the Anomura, the aeglids share more spermatozoal characters with the superfamily Lomoidea. represented by the monotypic Australian endemic genus, Lomis, than to any previously described representative from the Galatheoidea, Hippoidea. or Paguroidea. A more basal ancestry, with an independent evolutionary lineage. within the Anomura is Postulated for the Aeglidae. A Superficial resemblance of the spermatozoal ultrastructure of A. longirostri to that described for a palinurid lobster, Jasus, and a thalassinidean mud shrimp, Neaxius, is also noted.

The crystallography and morphology of Mo2C in ferrite

The orientation relationships between hexagonal Mo2C precipitates (H) in ferrite (B) have been determined by electron diffraction to an accuracy of +/-2degrees. With one exception, the 19 results are consistent with the previously reported Pitsch and Schrader (P/S) orientation relationship. However, these more accurate determinations show clearly that there is a systematic deviation of up to 5.5degrees from the exact P/S relationship and that this deviation consists of a small rotation about the parallel close packed directions-[100](B)//[2 (1) over bar(1) over bar0](H). The long direction of the Mo2C needles has been determined unequivocally in terms of the orientation relationship to be [100](B)//[2 (1) over bar(1) over bar0](H). Moire fringes between precipitate and matrix have been used to improve the accuracy of the orientation relationship results and to determine the lattice parameters of the carbide precipitates investigated. The Moire fringe analysis has shown small systematic departures from the exact parallelism between [100](B) and [2 (1) over bar(1) over bar0](H) along the length of Mo2C needles and a lowering of the carbide lattice parameter that is consistent with the replacement of Mo by Fe in the carbide. The orientation relationship results, including the observed systematic deviation from the exact P/S relationship, are shown to be consistent with the edge-to-edge model. (C) 2002 Kluwer Academic Publishers.

Characterization of redox centers in dimethyl sulfide dehydrogenase from Rhodovulum sulfidophilum

Dimethyl sulfide dehydrogenase from the purple phototrophic bacterium Rhodovulum sulfidophilum catalyzes the oxidation of dimethyl sulfide to dimethyl sulfoxide. Recent DNA sequence analysis of the ddh operon, encoding dimethyl sulfide dehydrogenase (ddhABC), and biochemical analysis (1) have revealed that it is a member of the DMSO reductase family of molybdenum enzymes and is closely related to respiratory nitrate reductase (NarGHI). Variable temperature X-band EPR spectra (120122 K) of purified heterotrimeric dimethyl sulfide dehydrogenase showed resonances arising from multiple redox centers, Mo(V), [3Fe-4S](+), [4Fe-4S](+), and a b-type heme. A pH-dependent EPR study of the Mo(V) center in (H2O)-H-1 and (H2O)-H-2 revealed the presence of three Mo(V) species in equilibrium, Mo(V)-OH2, Mo(v)-anion, and Mo(V)-OH. Above pH 8.2 the dominant species was Mo(V)-OH. The maximum specific activity occurred at pH 9.27. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other molybdenum enzymes of the DMSO reductase family showed that it was most similar to the low-pH nitrite spectrum of Escherichia coli nitrate reductase (NarGHI), consistent with previous sequence analysis of DdhA and NarG. A sequence comparison of DdhB and NarH has predicted the presence of four [Fe-S] clusters in DdhB. A [3Fe-4S](+) cluster was identified in dimethyl sulfide dehydrogenase whose properties resembled those of center 2 of NarH. A [4Fe-4S](+) cluster was also identified with unusual spin Hamiltonian parameters, suggesting that one of the iron atoms may have a fifth non-sulfur ligand. The g matrix for this cluster is very similar to that found for the minor conformation of center 1 in NarH [Guigliarelli, B., Asso, M., More, C., Augher, V., Blasco, F., Pommier, J., Giodano, G., and Bertrand, P. (1992) Eur. J. Biochem. 307,63-68]. Analysis of a ddhC mutant showed that this gene encodes the b-type cytochrome in dimethyl sulfide dehydrogenase. Magnetic circular dichroism studies revealed that the axial ligands to the iron in this cytochrome are a histidine and methionine, consistent with predictions from protein sequence analysis. Redox potentiometry showed that the b-type cytochrome has a high midpoint redox potential (E-o = +315 mV, pH 8).

Crystallization of PNMT, the adrenaline-synthesizing enzyme, is critically dependent on a high protein concentration

Phenylethanolamine N-methyltransferase, PNMT, utilizes the methylating cofactor S-adenosyl-L-methionine to catalyse the synthesis of adrenaline. Human PNMT has been crystallized in complex with an inhibitor and the cofactor product S-adenosyl-L-homocysteine using the hanging-drop technique with PEG 6000 and lithium chloride as precipitant. A critical requirement for crystallization was a high enzyme concentration (>90 mg ml(-1)) and cryocrystallography was used for high-quality data measurement. Diffraction data measured from a cryocooled crystal extend to a resolution of 2.3 Angstrom. Cryocooled crystals belong to space group P4(3)2(1)2 and have unit-cell parameters a = b = 94.3, c = 187.7 Angstrom.

The Phox Homology (PX) domain-dependent, 3-phosphoinositide-mediated association of sorting nexin-1 with an early sorting endosomal compartment is required for its ability to regulate epidermal growth factor receptor degradation

Recent studies have shown that phox homology (PX) domains act as phosphoinositide-binding motifs. The majority of PX domains studied show binding to phosphatidylinositol 3-monophosphate (Ptdlns(3)P), an association that allows the host protein to localize to membranes of the endocytic pathway. One issue, however, is whether PX domains may have alternative phosphoinositide binding specificities that could target their host protein to distinct subcellular compartments or allow their allosteric regulation by phosphoinositides other than PtdIns(3)P. It has been reported that the PX domain of sorting nexin 1 (SNX1) specifically binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3) (Zhong, Q., Lazar, C. S., Tronchere, H., Sato, T., Meerloo, T., Yeo, M., Songyang, Z., Emr, S. D., and Gill, G. N. (2002) Proc. Natl. Acad. Sci. U. S. A. 99,6767-6772). In the present study, we have shown that whereas SNX1 binds PtdIns(3,4,5)P-3 in protein:lipid overlay assays, in liposomes-based assays, binding is observed to PtdIns(3)P and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P-2) but not to PtdIns(3,4,5)P-3. To address the significance of PtdIns(3,4,5)P-3 binding, we examined the subcellular localization of SNX1 under conditions in which plasma membrane PtdIns(3,4,5)P-3 levels were significantly elevated. Under these conditions, we failed to observe association of SNX1 with this membrane. However, consistent with the binding to PtdIns(3)P and PtdIns(3,5)P-2 being of more physiological significance was the observation that the association of SNX1 with an early endosomal compartment was dependent on a 3-phosphoinositide-binding PX domain and the presence of PtdIns(3)P on this compartment. Finally, we somal association of SNX1 is important for its ability to regulate the targeting of internalized epidermal growth factor receptor for lysosomal degradation.

Dynamin-dependent endocytosis is necessary for convergent-extension movements in Xenopus animal cap explants

Cadherin cell-cell adhesion molecules are important determinants of morphogenesis and tissue patterning. C-cadherin plays a key role in the cell-upon-cell movements seen during Xenopus gastrulation. In particular, regulated changes in C-cadherin adhesion critically influence convergence-extension movements, thereby determining organization of the body plan. It is also predicted that remodelling of cadherin adhesive contacts is important for such cell-on-cell movements to occur. The recent demonstration that Epithelial (E-) cadherin is capable of undergoing endocytic trafficking to and from the cell surface presents a potential mechanism for rapid remodelling of such adhesive contacts. To test the potential role for C-cadherin endocytosis during convergence-extension, we expressed in early Xenopus embryos a dominantly-inhibitory mutant of the GTPase, dynamin, a key regulator of clathrin-mediated endocytosis. We report that this dynamin mutant significantly blocked the elongation of animal cap explants in response to activin, accompanied by inhibition of C-cadherin endocytosis. We propose that dynamin-dependent endocytosis of C-cadherin plays an important role in remodelling adhesive contacts during convergence-extension movements in the early Xenopus embryo.

Contribution of cholinergic systems to state-dependent modulation of respiratory control

Respiration is altered during different stages of the sleep-wake cycle. We review the contribution of cholinergic systems to this alteration, with particular reference to the role of muscarinic acetylcholine receptors (MAchRs) during rapid eye movement (REM) sleep. Available evidence demonstrates that MAchRs have potent excitatory effects on medullary respiratory neurones and respiratory motoneurones, and are likely to contribute to changes in central chemosensitive drive to the respiratory control system. These effects are likely to be most prominent during REM sleep, when cholinergic brainstem neurones show peak activity levels. It is possible that MAchR dysfunction is involved in sleep-disordered breathing, Such as obstructive sleep apnea. (C) 2002 Elsevier Science B.V. All rights reserved.

Simulation of the influence of rate- and state-dependent friction on the macroscopic behavior of complex fault zones with the lattice solid model

In order to understand the earthquake nucleation process, we need to understand the effective frictional behavior of faults with complex geometry and fault gouge zones. One important aspect of this is the interaction between the friction law governing the behavior of the fault on the microscopic level and the resulting macroscopic behavior of the fault zone. Numerical simulations offer a possibility to investigate the behavior of faults on many different scales and thus provide a means to gain insight into fault zone dynamics on scales which are not accessible to laboratory experiments. Numerical experiments have been performed to investigate the influence of the geometric configuration of faults with a rate- and state-dependent friction at the particle contacts on the effective frictional behavior of these faults. The numerical experiments are designed to be similar to laboratory experiments by DIETERICH and KILGORE (1994) in which a slide-hold-slide cycle was performed between two blocks of material and the resulting peak friction was plotted vs. holding time. Simulations with a flat fault without a fault gouge have been performed to verify the implementation. These have shown close agreement with comparable laboratory experiments. The simulations performed with a fault containing fault gouge have demonstrated a strong dependence of the critical slip distance D-c on the roughness of the fault surfaces and are in qualitative agreement with laboratory experiments.

SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold

The S-adenosylmethionine-dependent methyltransferase enzymes share little sequence identity, but incorporate a highly conserved structural fold. Surprisingly, residues that bind the common cofactor are poorly conserved, although the binding site is localised to the same region of the fold. The substrate-binding region of the fold varies enormously. Over the past two years, there has been a significant increase in the number of structures that are known to incorporate this fold, including several uncharacterised proteins and two proteins that lack methyltransferase activity.

Multiple maps and activity-dependent representational plasticity in the anterior Wulst of the adult barn owl (Tyto alba)

In the present study we addressed the issue of somatosensory representation and plasticity in a nonmammalian species, the barn owl. Multiunit mapping techniques were used to examine the representation of the specialized receptor surface of the claw in the anterior Wulst. We found dual somatotopic mirror image representations of the skin surface of the contralateral claw. In addition, we examined both representations 2 weeks after denervation of the distal skin surface of a single digit. In both representations, the denervated digital representation became responsive to stimulation of the adjacent, mutually functional, digit. The mutability and multiple representations indicates that the Wulst provides the owl with sensory processing capabilities analogous to those in mammals.

Indirect evidence of density-dependent population regulation in Aponomma hydrosauri (Acari : Ixodidae), an ectoparasite of reptiles

The extent to which density-dependent processes regulate natural populations is the subject of an ongoing debate. We contribute evidence to this debate showing that density-dependent processes influence the population dynamics of the ectoparasite Aponomma hydrosauri (Acari: Ixodidae), a tick species that infests reptiles in Australia. The first piece of evidence comes from an unusually long-term dataset on the distribution of ticks among individual hosts. If density-dependent processes are influencing either host mortality or vital rates of the parasite population, and those distributions can be approximated with negative binomial distributions, then general host-parasite models predict that the aggregation coefficient of the parasite distribution will increase with the average intensity of infections. We fit negative binomial distributions to the frequency distributions of ticks on hosts, and find that the estimated aggregation coefficient k increases with increasing average tick density. This pattern indirectly implies that one or more vital rates of the tick population must be changing with increasing tick density, because mortality rates of the tick's main host, the sleepy lizard, Tiliqua rugosa, are unaffected by changes in tick burdens. Our second piece of evidence is a re-analysis of experimental data on the attachment success of individual ticks to lizard hosts using generalized linear modelling. The probability of successful engorgement decreases with increasing numbers of ticks attached to a host. This is direct evidence of a density-dependent process that could lead to an increase in the aggregation coefficient of tick distributions described earlier. The population-scale increase in the aggregation coefficient is indirect evidence of a density-dependent process or processes sufficiently strong to produce a population-wide pattern, and thus also likely to influence population regulation. The direct observation of a density-dependent process is evidence of at least part of the responsible mechanism.