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.

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.

Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx(2c), either alone (16%) or in combination with stx(1) (74%) or stx(2) (3%) PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.

A review on the role of duckweed in nutrient reclamation and as a source of animal feed

The family of lemnacae colloquially known as duckweed contains the world's smallest species of flowering plants (macrophytes). Aquatic and free-floating, their most striking qualities are a capacity for explosive reproduction and an almost complete lack of fibrous material. They are widely used for reducing chemical loading in facultative sewage lagoons, but their greatest potential lies in their ability to produce large quantities of protein rich biomass, suitable for feeding to a wide range of animals, including fish, poultry and cattle. Despite these qualities there are numerous impediments to these plants being incorporated into western farming systems. Large genetically determined variations in growth in response to nutrients and climate, apparent anti-nutritional factors, concerns about sequestration of heavy metals and possible transference of pathogens raise questions about the safety and usefulness of these plants. A clear understanding of how to address and overcome these impediments needs to be developed before duckweed is widely accepted for nutrient reclamation and as a source of animal feed.

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.

Ion transport induced by proteinase-activated receptors (PAR2) in colon and airways

Protease-activated receptors type 2 (PAR2) are activated by serine proteases like trypsin and mast cell tryptase. The function and physiological significance of PAR2 receptors is poorly understood, but recent studies suggest a role during inflammatory processes in both airways and intestine. PAR2 receptors are also likely to participate in the control of ion transport in these tissues. We demonstrate that stimulation of PAR2 in airways and intestine significantly enhanced ion transport. Trypsin induced CI- secretion in both airways and intestine when added to the basolateral but not to the luminal side of these tissues. In both airways and intestine, stimulation of ion transport was largely dependent on the increase in intracellular Ca2+. Effects of trypsin were largely reduced by basolateral bumetanide and barium and by trypsin inhibitor. Thrombin, an activator of proteinase-activated receptors types 1, 3, and 4 had no effects on equivalent short-circuit current in either airways or intestine. Expression of PAR2 in colon and airways was further confirmed by reverse transcription-polymerase chain reaction. We postulate that these receptors play a significant role in the regulation of electrolyte transport, which might be important during inflammatory diseases of airways and intestine.

Mechanisms for the inhibition of amiloride-sensitive Na+ absorption by extracellular nucleotides in mouse trachea

Purinergic stimulation of airway epithelial cells induces Cl- secretion and modulates Na+ absorption by an unknown mechanism. To gain insight into this mechanism, we used a perfused micro-Ussing chamber to assess transepithelial voltage (V-te) and amiloride-sensitive short-circuit current (Isc-Amil) in mouse trachea. Exposure to apical ATP or UTP (each 100 mumol/l) caused a large initial increase in lumen negative V-te and I-sc corresponding to a transient Cl- secretion, while basolateral application of ATP/UTP induced only a small secretory response. Luminal, but not basolateral, application of nucleotides was followed by a sustained and reversible inhibition of Isc-Amil that was independent of extracellular Ca2+ or activation of protein kinase C and was not induced by carbachol (100 mumol/l) or the Ca2+ ionophore ionomycin (1 mumol/l). Removal of extracellular Cl- or exposure to 200 muM DIDS reduced UTP-mediated inhibition of Isc-Amil Substantially. The phospholipase inhibitor U73122 (10 mumol/l) and pertussis toxin (PTX 200 ng/ml) both attenuated UTP-induced Cl- secretion and inhibition of Isc-Amil. Taken together, these data imply a contribution of Cl- conductance and PTX-sensitive G proteins to nucleotide-dependent inhibition of the amiloride-sensitive Na+ current in the mouse trachea.

Stressful animal housing conditions and their potential effect on sympathetic neurotransmission in mice

Although the sympathetic nervous system (SNS) plays a major role in mediating the peripheral stress response, due consideration is not usually given to the effects of prolonged stress on the SNS. The present study examined changes in neurotransmission in the SNS after exposure of mice (BALB/c) to stressful housing conditions. Focal extracellular recording of excitatory junction currents (EJCs) was used as a relative measure of neurotransmitter release from different regions of large surface areas of the mouse vas deferens. Mice were either group housed (control), isolation housed (social deprivation), group housed in a room containing rats (rat odor stress), or isolation housed in a room containing rats (concurrent stress). Social deprivation and concurrent stressors induced an increase of 30 and 335% in EJC amplitude, respectively. The success rate of recording EJCs from sets of varicosities in the concurrent stressor group was greater compared with all other groups. The present study has shown that some common animal housing conditions act as stressors and induce significant changes in sympathetic neurotransmission.

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.