Molecular and functional characterization of an octopamine receptor from honeybee (Apis mellifera) brain

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera . The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis . Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.

A randomized trial of aggressive lipid reduction for improvement of myocardial ischemia, symptom status, and vascular function in patients with coronary artery disease not amenable to intervention

PURPOSE: To determine the effects of aggressive lipid lowering on markers of ischemia, resistance vessel function, atherosclerotic burden, and Symptom status in patients with symptomatic coronary artery disease. METHODS: Sixty consecutive patients with coronary artery disease that was unsuitable for revascularization were assigned randomly to either usual therapy of lipids for patients with a low-density lipoprotein (LDL) cholesterol target level <116 mg/dL, or to a, more aggressive lipid-lowering strategy involving up to 80 mg/d of atorvastatin, with a target LDL cholesterol level <77 mg/dL. The extent and severity of inducible ischemia (by dobutamine echocardiography), vascular function.(brachial artery reactivity), atheroma burden (carotid intima-media thickness), and symptom status were evaluated blindly at baseline and after 12 weeks of treatment. RESULTS: After 12 weeks of treatment, patients in the aggressive therapy group had a significantly greater decrease in mean (+/- SD) LDL cholesterol level than those in the usual care group (29 +/- 38 mg/dL vs. 7 +/- 24 mg/dL, P = 0.03). Patients in the aggressive therapy group had a reduction in the number of ischemic wall segments (mean between-group difference of 1.3; 95% confidence interval: 0.1 to 2.0; P = 0.04), flow-mediated dilatation (mean between-group difference of 5.9%; 95% confidence interval: 2.5% to 9.4%; P = 0.001), and angina score after 12 weeks. There were no significant changes in atherosclerotic burden in either group. CONCLUSION: Patients with symptomatic coronary artery disease who are treated with aggressive lipid lowering have improvement of symptom status and ischemia that appears to reflect improved vascular function but not atheroma burden. Am J Med. 2003;114:445-453. (C) 2003 by Excerpta Medica Inc.

Vps20p and Vta1p interact with Vps4p and function in multivesicular body sorting and endosomal transport in Saccharomyces cerevisiae

Vps4p (End13p) is an AAA-family ATPase that functions in membrane transport through endosomes, sorting of soluble vacuolar proteins to the vacuole, and multivesicular body (MVB) sorting of membrane proteins to the vacuole lumen. In a yeast two-hybrid screen with Vps4p as bait we isolated VPS20 (YMR077c) and the novel open reading frame YLA181c, for which the name VTA1 has recently been assigned (Saccharomyces Genome Database). Vps4p directly binds Vps20p and Vta1p in vitro and binding is not dependent on ATP-conversely, Vps4p binding to Vps20p is partially sensitive to ATP hydrolysis. Both ATP binding [Vps4p-(K179A)] and ATP hydrolysis [Vps4p-(E233Q)] mutant proteins exhibit enhanced binding to Vps20p and Vta1p in vitro. The Vps4p-Vps20p interaction involves the coiled-coil domain of each protein, whereas the Vps4p-Vta1p interaction involves the (non-coiled-coil) C-terminus of each protein. Deletion of either VPS20 (vps20Delta) or VTA1 (vta1Delta) leads to similar class E Vps(-) phenotypes resembling those of vps4Delta, including carboxypeptidase Y (CPY) secretion, a block in ubiquitin-dependent MVB sorting, and a delay in both post-internalisation endocytic transport and biosynthetic transport to the vacuole. The vacuole resident membrane protein Sna3p (whose MVB sorting is ubiquitin-independent) does not appear to exit the class E compartment or reach the vacuole in cells lacking Vps20p, Vta1p or Vps4p, in contrast to other proteins whose delivery to the vacuole is only delayed. We propose that Vps20p and Vta1p regulate Vps4p function in vivo.

Identification of a novel protein kinase mediating Akt survival signaling to the ATM protein

We identified a novel human AMP-activated protein kinase (AMPK) family member, designated ARK5, encoding 661 amino acids with an estimated molecular mass of 74 kDa. The putative amino acid sequence reveals 47, 45.8, 42.4, and 55% homology to AMPK-alpha1, AMPK-alpha2, MELK and SNARE respectively, suggesting that it is a new member of the AMPK family. It has a putative Akt phosphorylation motif at amino acids 595600, and Ser(600) was found to be phosphorylated by active Akt resulting in the activation of kinase activity toward the SAMS peptide, a consensus AMPK substrate. During nutrient starvation, ARK5 supported the survival of cells in an Akt-dependent manner. In addition, we also demonstrated that ARK5, when activated by Akt, phosphorylated the ATM protein that is mutated in the human genetic disorder ataxia-telangiectasia and also induced the phosphorylation of p53. On the basis of our current findings, we propose that a novel AMPK family member, ARK5, is the tumor cell survival factor activated by Akt and acts as an ATM kinase under the conditions of nutrient starvation.

CD40 and dendritic cell function

CD40 has emerged as a key signaling pathway for the function of B cells, monocytes, and dendritic cells (DC) in the immune system, and plays a major role in inflammatory pathways of nonhemopoletic cells. CD40 is expressed by monocytes and DC and is up-regulated when DC migrate from the periphery to draining lymph nodes (DLN) in response to microbial challenge. CD154 signaling by MHC-restricted, activated CD4* T cells induces differentiation of DC, as defined by an increased surface expression of MHC, costimulatory, and adhesion molecules. Thus, CD40 functions in the adaptive immune response as a trigger for the expression of costimulatory molecules for efficient T-cell activation. CD40 ligation of DC also has the capacity to induce high levels of the cytokine IL-12, which polarizes CD4(+) T cells toward a T helper 1 (Th1) type, enhances proliferation of CD8(+) T cells, and activates NK cells. CD40 may also play an important role in the decision between tolerance and immunity and the generation of regulatory CD4(+) T cells that are thought to maintain peripheral self-tolerance in vivo.

The ultrastructure and function of the silk-producing basitarsus in the Hilarini (Diptera : Ernpididae)

The tribe Hilarini (Diptera: Empididae), commonly known as dance flies, can be recognised by their swollen silk-producing prothoracic basitarsus, a male secondary sexual characteristic. The ultrastructure and function of the silk-producing basitarsus from one undescribed morphospecies of Hilarini, 'Hilarempis 20', is presented. Male H. 20 collect small parcels of diatomaceous algae from the surface of freshwater creeks that they bind with silk produced by the gland in the basitarsus. The gift is then presented to females in a nearby swarm, composed predominately of females. The basitarsus houses approximately 12 pairs of class III dermal glandular units that congregate on the ventral side of the cavity. Each gland cell has a large extracellular lumen where secretion accumulates. The lumen drains to the outside via a conducting canal encompassed by a canal cell and a duct extending through the shaft of a specialised secretory spine. The secretory spines lie in pairs in a ventral groove that runs the length of the basitarsus. A comparison of the basitarsal secretory spines with sensilla on the basitarsi of non gland-bearing legs of males, and with non gland-bearing prothoracic. basitarsi of females, suggests that the glandular units are derived from contact chemosensory sensilla. (C) 2003 Elsevier Ltd. All rights reserved.

Identification of residues which regulate activity of the STE20-related kinase hMINK

Activity of the STE20-related kinase hMINK was investigated. hMINK was expressed widely, though not ubiquitously, in human tissues: highest levels being found in haematopoietic tissues but also in brain, placenta, and lung. Mutagenesis revealed that T-191. and Y-193 in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation. A mutation on T-187 showed reduced enzymatic activity against exogenous substrates but retained autophosphorylationactivity. Phosphorylation was confirmed by the use of a phospho-specific T-187 antibody. hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted. In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway. These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates. Models for receptor-mediated activation of hMINK are discussed. (C) 2002 Elsevier Science (USA). All rights reserved.

Alternatively spliced products of the human kinesin light chain 1 (KNS2) gene

Conventional kinesin is a microtubule-based molecular motor involved in the transport of membranous and non-membranous cargoes. The kinesin holoenzyme exists as a heterotetramer, consisting of two heavy chain and two light chain subunits. It is thought that one function of the light chains is to interact with the cargo. Alternative splicing of kinesin light chain pre-mRNA has been observed in lower organisms, although evidence for alternative splicing of the human gene has not been reported. We have identified 19 variants of the human KNS2 gene (KLC1) that are generated by alternative splicing of downstream exons, but calculate that KNS2 has the potential to produce 285919 spliceforms. Corresponding spliceforms of the mouse KLC1 gene were also identified. The alternative exons are all located 3' of exon 12 and the novel spliceforms produce both alternative carboxy termini and alternative 3' untranslated regions. The observation of multiple light chain isoforms is consistent with their proposed role in specific cargo attachment.

EMA analysis of tongue function in children with dysarthria following traumatic brain injury

Primary objective : To investigate the speed and accuracy of tongue movements exhibited by a sample of children with dysarthria following severe traumatic brain injury (TBI) during speech using electromagnetic articulography (EMA). Methods and procedures : Four children, aged between 12.75-17.17 years with dysarthria following TBI, were assessed using the AG-100 electromagnetic articulography system (Carstens Medizinelektronik). The movement trajectories of receiver coils affixed to each child's tongue were examined during consonant productions, together with a range of quantitative kinematic parameters. The children's results were individually compared against the mean values obtained by a group of eight control children (mean age of 14.67 years, SD 1.60). Main outcomes and results : All four TBI children were perceived to exhibit reduced rates of speech and increased word durations. Objective EMA analysis revealed that two of the TBI children exhibited significantly longer consonant durations compared to the control group, resulting from different underlying mechanisms relating to speed generation capabilities and distances travelled. The other two TBI children did not exhibit increased initial consonant movement durations, suggesting that the vowels and/or final consonants may have been contributing to the increased word durations. Conclusions and clinical implications : The finding of different underlying articulatory kinematic profiles has important implications for the treatment of speech rate disturbances in children with dysarthria following TBI.

Identification and characterization of the hepatic stellate cell transferrin receptor

Activated hepatic stellate cells have been implicated in the fibrogenic process associated with iron overload, both in animal models and in human hemochromatosis. Previous studies have evaluated the role of ferritin/ferritin receptor interactions in the activation of stellate cells and subsequent fibrogenesis; however, the role of transferrin in hepatic stellate cell biology is unknown. This study was designed to identify and characterize the stellate cell transferrin receptor and to evaluate the influence of transferrin on stellate cell activation. Identification and characterization of the stellate cell transferrin receptor was determined by competitive displacement assays. The effect of transferrin on stellate cell activation was assessed using western blot analysis for alpha-smooth muscle actin expression, [H-3]Thymidine incorporation, and real-time RT-PCR for procollagen 1(I) mRNA expression. A specific receptor for rat transferrin was observed on activated but not quiescent stellate cells. Transferrin significantly increased the expression of alpha-smooth muscle actin, but caused a decrease in proliferation. Transferrin induced a significant increase in procollagen alpha1(I) mRNA expression. In conclusion, this study has demonstrated for the first time a specific, high affinity receptor for rat transferrin on activated hepatic stellate cells, which via interaction with transferrin regulates stellate cell activation. This suggests that transferrin may be an important factor in the activation of hepatic stellate cells in conditions of iron overload.

Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms

Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, Candidatus Accumulibacter phosphatis was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions. (C) 2003 Wiley Periodicals, Inc.

Relationship of ventricular longitudinal function to contractile reserve in patients with mitral regurgitation

Background Latent left ventricular (LV) dysfunction in patients with valvular or myocardial disease may be identified by loss of contractile reserve (CR) at exercise echocardiography. Contraction in the LV longitudinal axis may be more sensitive than radial contraction to minor disturbances of LV function. We sought to determine whether tissue Doppler measurement of longitudinal function could be used to identify CR. Methods Exercise echocardiography was performed in 86 patients (20 women, age 53 +/- 18 years), 72 with asymptomatic or minimally symptomatic mitral regurgitation, and 14 normal controls. Pulsed-wave tissue Doppler imaging (DTI) was used to measure maximum annular systolic velocity at rest and stress. Inducible ischemia was excluded by analysis of wall motion by an experienced observer. CR was defined by greater than or equal to5% improvement of stress compared with rest ejection fraction (EF). Exercise capacity was assessed from expired gas analysis. Results CR was present in 34 patients with mitral regurgitation (47%); peak EF in patients with and without CR was 74% +/- 11% versus 54% +/- 15% (P