bluuebunny/crossref_metadata_2025 huggingface dataset:
Endocytic processing of connexin43 gap junctions: a morphological study
Authors: Leithe Edward , Brech Andreas , Rivedal Edgar
Published: December 2005

Gap junctions are plasma membrane areas enriched in channels that provide direct intercellular communication. Gap junctions have a high turnover rate; however, the mechanisms by which gap junctions are degraded are incompletely understood. In the present study, we show that in response to phorbol ester treatment, the gap junction channel protein Cx43 (connexin43) is redistributed from the plasma membrane to intracellular vesicles positive for markers for early and late endosomes and for the endolysosomal protease cathepsin D. Immunoelectron microscopy studies indicate that the double membranes of internalized gap junctions undergo separation and cutting, resulting in multivesicular endosomes enriched in Cx43 protein. Using preloading of BSA–gold conjugates to mark lysosomes, we provide evidence suggesting that the degradation process of the double-membrane structure of annular gap junctions occurs prior to transport of Cx43 to the lysosome. The results further suggest that bafilomycin A1, an inhibitor of vacuolar H+-ATPases, causes accumulation of Cx43 in early endosomes. Taken together, these findings indicate that internalized gap junctions undergo a maturation process from tightly sealed double-membrane vacuoles to connexin-enriched multivesicular endosomes with a single limiting membrane. The results further suggest that along with the processing of the double-membrane structure of annular gap junctions, connexins are trafficked via early and late endosomes, finally resulting in their endolysosomal degradation.
Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants
Authors: Martel Catherine , Macchi Paolo , Furic Luc , Kiebler Michael A. , Desgroseillers Luc
Published: December 2005

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.
Re-evaluation of receptor–ligand interactions of the human neuropeptide Y receptor Y1: a site-directed mutagenesis study
Authors: Sjödin Paula , Holmberg Sara K. S. , Åkerberg Helena , Berglund Magnus M. , Mohell Nina , Larhammar Dan
Published: December 2005

Interactions of the human NPY (neuropeptide Y) receptor Y1 with the two endogenous agonists NPY and peptide YY and two non-peptide antagonists were investigated using site-directed mutagenesis at 17 positions. The present study was triggered by contradictions among previously published reports and conclusions that seemed inconsistent with sequence comparisons across species and receptor subtypes. Our results show that Asp287, at the border between TM (transmembrane) region 6 and EL3 (extracellular loop 3) influences peptide binding, while two aspartic residues in EL2 do not, in agreement with some previous studies but in disagreement with others. A hydrophobic pocket of the Y1 receptor consisting of Tyr100 (TM2), Phe286 (TM6) and His298 (EL3) has been proposed to interact with the amidated C-terminus of NPY, a theory that is unsupported by sequence comparisons between Y1, Y2 and Y5. Nevertheless, our results confirm that these amino acid residues are critical for peptide binding, but probably interact with NPY differently than proposed previously. Studies with the Y1-selective antagonist SR120819A identified a new site of interaction at Asn116 in TM3. Position Phe173 in TM4 is also important for binding of this antagonist. In contrast with previous reports, we found that Phe173 is not crucial for the binding of BIBP3226, another selective Y1 receptor antagonist. Also, we found that position Thr212 (TM5) is important for binding of both antagonists. Our mutagenesis results and our three-dimensional model of the receptor based on the high-resolution structure of bovine rhodopsin suggest new interactions for agonist as well as antagonist binding to the Y1 receptor.
Protein kinase Cϵ interacts with cytochrome c oxidase subunit IV and enhances cytochrome c oxidase activity in neonatal cardiac myocyte preconditioning
Authors: Ogbi Mourad , Johnson John A.
Published: December 2005

We have previously identified a phorbol ester-induced PKCϵ (protein kinase Cϵ) interaction with the (∼18 kDa) COIV [CO (cytochrome c oxidase) subunit IV] in NCMs (neonatal cardiac myocytes). Since PKCϵ has been implicated as a key mediator of cardiac PC (preconditioning), we examined whether hypoxic PC could induce PKCϵ–COIV interactions. Similar to our recent study with phorbol esters [Ogbi, Chew, Pohl, Stuchlik, Ogbi and Johnson (2004) Biochem. J. 382, 923–932], we observed a time-dependent increase in the in vitro phosphorylation of an approx. 18 kDa protein in particulate cell fractions isolated from NCMs subjected to 1–60 min of hypoxia. Introduction of a PKCϵ-selective translocation inhibitor into cells attenuated this in vitro phosphorylation. Furthermore, when mitochondria isolated from NCMs exposed to 30 min of hypoxia were subjected to immunoprecipitation analyses using PKCϵ-selective antisera, we observed an 11.1-fold increase in PKCϵ–COIV co-precipitation. In addition, we observed up to 4-fold increases in CO activity after brief NCM hypoxia exposures that were also attenuated by introducing a PKCϵ-selective translocation inhibitor into the cells. Finally, in Western-blot analyses, we observed a >2-fold PC-induced protection of COIV levels after 9 h index hypoxia. Our studies suggest that a PKCϵ–COIV interaction and an enhancement of CO activity occur in NCM hypoxic PC. We therefore propose novel mechanisms of PKCϵ-mediated PC involving enhanced energetics, decreased mitochondrial reactive oxygen species production and the preservation of COIV levels.
Subcellular localization of rat Abca5, a rat ATP-binding-cassette transporter expressed in Leydig cells, and characterization of its splice variant apparently encoding a half-transporter
Authors: Petry Frauke , Ritz Vera , Meineke Cornelia , Middel Peter , Kietzmann Thomas , Schmitz-Salue Christoph , Hirsch-Ernst Karen I.
Published: December 2005

Several transporters belonging to the ABCA subfamily of ABC (ATP-binding cassette) proteins are involved in lipid trafficking. Human ABCA5 and its rat orthologue, rAbca5, represent recently identified subfamily members whose substrate spectrum remains to be defined. The elucidation of (sub)cellular rAbca5 distribution would be expected to provide a basis for optimization of functional analyses. In the present study, we applied in situ hybridization to examine rAbca5 mRNA distribution within sections of rat testis, a tissue expressing high levels of rAbca5 mRNA. We found rAbca5 mRNA to be predominantly expressed in interstitial Leydig cells, which are major sites of testosterone synthesis. To investigate rAbca5 subcellular localization, we constructed expression vectors yielding rAbca5 fused either to EGFP (enhanced green fluorescent protein) or to a peptide bearing the viral V5 epitope. During rAbca5 cDNA cloning, we discovered a splice variant sequence (rAbca5 V20+16), predicted to give rise to a truncated, half-size transporter, which was highly homologous with a human splice variant described by us previously. Quantitative RT (reverse transcription)–PCR demonstrated that the rAbca5 splice variant was expressed in numerous tissues (including testis, brain and lungs), its cDNA amounting to 2.6–11.2% of total rAbca5 cDNA. Transfection of individual rAbca5-EGFP, rAbca5 splice variant-EGFP or transporter-V5 expression plasmids along with organelle marker plasmids into HEK-293 cells (human embryonic kidney 293 cells) revealed that both rAbca5 and splice variant fusion proteins co-localized with marker protein for the Golgi apparatus. Expression of rAbca5 mRNA in Leydig cells, intracellular localization of rAbca5–EGFP/rAbca5–V5 and involvement of rAbca5-related proteins in lipid transport suggest that rAbca5 may participate in intracellular sterol/steroid trafficking.
Quantitative model of Ras–phosphoinositide 3-kinase signalling cross-talk based on co-operative molecular assembly
Authors: Kaur Harjeet , Park Chang Shin , Lewis Jodee M. , Haugh Jason M.
Published: December 2005

In growth-factor-stimulated signal transduction, cell-surface receptors recruit PI3Ks (phosphoinositide 3-kinases) and Ras-specific GEFs (guanine nucleotide-exchange factors) to the plasma membrane, where they produce 3′-phosphorylated phosphoinositide lipids and Ras-GTP respectively. As a direct example of pathway networking, Ras-GTP also recruits and activates PI3Ks. To refine the mechanism of Ras–PI3K cross-talk and analyse its quantitative implications, we offer a theoretical model describing the assembly of complexes involving receptors, PI3K and Ras-GTP. While the model poses the possibility that a ternary receptor–PI3K–Ras complex forms in two steps, it also encompasses the possibility that receptor–PI3K and Ras–PI3K interactions are competitive. In support of this analysis, experiments with platelet-derived growth factor-stimulated fibroblasts revealed that Ras apparently enhances the affinity of PI3K for receptors; in the context of the model, this suggests that a ternary complex does indeed form, with the second step greatly enhanced through membrane localization and possibly allosteric effects. The apparent contribution of Ras to PI3K activation depends strongly on the quantities and binding affinities of the interacting molecules, which vary across different cell types and stimuli, and thus the model could be used to predict conditions under which PI3K signalling is sensitive to interventions targeting Ras.
TonEBP is inhibited by RNA helicase A via interaction involving the E′F loop
Authors: Colla Emanuela , Lee Sang D. , Sheen Mee R. , Woo Seung K. , Kwon H. Moo
Published: December 2005

TonEBP [TonE (tonicity-responsive enhancer)-binding protein] is a transcriptional activator of the Rel family like NF-κB (nuclear factor κB) and NFAT (nuclear factor of activated T-cells). TonEBP plays a key role in the protection of cells in the kidney medulla from the deleterious effects of hyperosmolality. This is achieved by enhancing expression of HSP70 (heat-shock protein 70) and other genes whose products drive cellular accumulation of organic osmolytes. TonEBP is stimulated by ambient hypertonicity via multiple pathways that regulate nuclear translocation and transactivation. In the present paper, we report that TonEBP is associated in vivo with RHA (RNA helicase A). The N- and C-termini of RHA bound the E′F loop of the DNA-binding domain of TonEBP. The interaction was not affected by DNA binding or dimerization of TonEBP. Overexpression of RHA inhibited the activity of TonEBP; however, catalytic activity of RHA was dispensable for the inhibition. When the ambient tonicity was raised, the TonEBP–RHA interaction decreased, suggesting that dissociation of RHA is a pathway to stimulate TonEBP. We conclude that the E′F loop of TonEBP interacts with RHA like NFAT and NF-κB interact with AP1 (activator protein 1) and the high-mobility group protein HMG-I(Y) respectively. While RHA interacts with and stimulates other transcription factors such as CREB (cAMP-response-element-binding protein), NF-κB and mineralocorticoid receptor, it inhibits TonEBP.
Carbohydrate specificity of an insecticidal lectin isolated from the leaves of Glechoma hederacea (ground ivy) towards mammalian glycoconjugates
Authors: Singh Tanuja , Wu June H. , Peumans Willy J. , Rougé Pierre , Van Damme Els J. M. , Alvarez Richard A. , Blixt Ola , Wu Albert M.
Published: December 2005

Preliminary studies indicated that the potent insecticidal lectin, Gleheda, from the leaves of Glechoma hederacea (ground ivy) preferentially agglutinates human erythrocytes carrying the Tn (GalNAcα1-Ser/Thr) antigen. However, no details have been reported yet with respect to the fine specificity of the lectin. To corroborate the molecular basis of the insecticidal activity and physiological function of Gleheda, it is necessary to identify the recognition factors that are involved in the Gleheda–glycotope interaction. In the present study, the requirement of high-density multivalent carbohydrate structural units for Gleheda binding and a fine-affinity profile were evaluated using ELLSA (enzyme-linked lectinosorbent assay) with our extended glycan/ligand collections, a glycan array and molecular modelling. From the results, we concluded that a high-density of exposed multivalent Tn-containing glycoproteins (natural armadillo and asialo ovine salivary glycoproteins) were the most potent factors for Gleheda binding. They were, on a nanogram basis, 6.5×105, 1.5×104 and 3.1×103 times more active than univalent Gal (galactose), GalNAc (N-acetylgalactosamine) and Tn respectively. Among mono- and oligo-saccharides examined, simple clustered Tn (molecular mass <3000 Da) from ovine salivary glycoprotein was the best, being 37.5 and 1.7×103 times better than GalNAc and Gal respectively. GalNAc glycosides were significantly more active than Gal glycosides, indicating that the N-acetamido group at C-2 plays an important role in Gleheda binding. The results of glycan array support the conclusions drawn with respect to the specificity of Gleheda based on the ELLSA assays. These findings combined with the results of the molecular modelling and docking indicate the occurrence of a primary GalNAcα1-binding site in the Gleheda monomer. However, the extraordinary binding feature of Gleheda for glycoproteins demonstrates the importance of affinity enhancement by high-density multivalent glycotopes in the ligand–lectin interactions in biological processes.
Overproduction of cardiac S-adenosylmethionine decarboxylase in transgenic mice
Authors: Nisenberg Oleg , Pegg Anthony E. , Welsh Patricia A. , Keefer Kerry , Shantz Lisa M.
Published: December 2005

The present study was designed to provide a better understanding of the role played by AdoMetDC (S-adenosylmethionine decarboxylase), the key rate-controlling enzyme in the synthesis of spermidine and spermine, in controlling polyamine levels and the importance of polyamines in cardiac physiology. The αMHC (α-myosin heavy chain) promoter was used to generate transgenic mice with cardiac-specific expression of AdoMetDC. A founder line (αMHC/AdoMetDC) was established with a >100-fold increase in AdoMetDC activity in the heart. Transgene expression was maximal by 1 week of age and remained constant into adulthood. However, the changes in polyamine levels were most pronounced during the first week of age, with a 2-fold decrease in putrescine and spermidine and a 2-fold increase in spermine. At later times, spermine returned to near control levels, whereas putrescine and spermidine levels remained lower, suggesting that compensatory mechanisms exist to limit spermine accumulation. The αMHC/AdoMetDC mice did not display an overt cardiac phenotype, but there was an increased cardiac hypertrophy after β-adrenergic stimulation with isoprenaline (‘isoproterenol’), as well as a small increase in spermine content. Crosses of the αMHC/AdoMetDC with αMHC/ornithine decarboxylase mice that have a >1000-fold increase in cardiac ornithine decarboxylase were lethal in utero, presumably due to increase in spermine to toxic levels. These findings suggest that cardiac spermine levels are highly regulated to avoid polyamine-induced toxicity and that homoeostatic mechanisms can maintain non-toxic levels even when one enzyme of the biosynthetic pathway is greatly elevated but are unable to do so when two biosynthetic enzymes are increased.
The orphan transporter v7-3 (slc6a15) is a Na+-dependent neutral amino acid transporter (B0AT2)
Authors: Bröer Angelika , Tietze Nadine , Kowalczuk Sonja , Chubb Sarah , Munzinger Michael , Bak Lasse K. , Bröer Stefan
Published: December 2005

Transporters of the SLC6 (solute carrier 6) family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. In the present study, we demonstrate that mouse v7-3 (slc6a15) encodes a transporter for neutral amino acids. The transporter is functionally and sequence related to B0AT1 (slc6a19) and was hence named B0AT2. Leucine, isoleucine, valine, proline and methionine were recognized by the transporter, with values of K0.5 (half-saturation constant) ranging from 40 to 200 μM. Alanine, glutamine and phenylalanine were low-affinity substrates of the transporter, with K0.5 values in the millimolar range. Transport of neutral amino acids via B0AT2 was Na+-dependent, Cl−-independent and electrogenic. Superfusion of mouse B0AT2-expressing oocytes with amino acid substrates generated robust inward currents. Na+-activation kinetics of proline transport and uptake under voltage clamp suggested a 1:1 Na+/amino acid co-transport stoichiometry. Susbtrate and co-substrate influenced each other's K0.5 values, suggesting that they share the same binding site. A mouse B0AT2-like transport activity was detected in synaptosomes and cultured neurons. A potential role of B0AT2 in transporting neurotransmitter precursors and neuromodulators is proposed.
Interaction with phospholipids modulates α-synuclein nitration and lipid–protein adduct formation
Authors: Trostchansky Andrés , Lind Summer , Hodara Roberto , Oe Tomoyuki , Blair Ian A. , Ischiropoulos Harry , Rubbo Homero , Souza José M.
Published: December 2005

Intracellular aggregates of α-syn (α-synuclein) represent pathoanatomical hallmarks of neurodegenerative disorders (synucleinopathies). The molecular mechanisms underlying α-syn aggregation into filamentous inclusions may involve oxidation and nitration of the protein. Whereas the effects of oxidants and nitrating species on soluble α-syn have been studied in detail, the effect of these reactive species on α-syn associated with lipids is still unknown. In the present paper, we report that α-syn bound to small unilamellar liposomes composed of phosphatidylcholine/phosphatidic acid is resistant to oxidation and nitration when compared with soluble α-syn. Additionally, increasing concentrations of unsaturated fatty acids diminished the oxidation and nitration of α-syn upon exposure to fluxes of peroxynitrite (8–20 μM·min−1). To investigate the effect of oxidized lipids on α-syn, the protein was incubated with the bifunctional electrophile 4-HNE [4-hydroxy-2(E)-nonenal]. MS analysis showed the formation of three major products corresponding to the native protein and α-syn plus one or two 4-HNE molecules. Trypsin digestion of the modified protein followed by peptide ‘finger-printing’ revealed that 4-HNE modified the peptide E46GVVHGVATVAEK58. Further analysis of the peptides with liquid chromatography–tandem MS identified the modified residue as His50. The data indicate that the association of α-syn with biological membranes protects the protein from oxidation and nitration and thus diminishes the formation of protein molecules capable of forming aggregates. However, products of lipid peroxidation can also modify α-syn, generating novel protein adducts that could serve as biomarkers for documenting oxidative processes in human as well as animal and cellular models of α-syn aggregation and pathology.
KESTREL: a powerful method for identifying the physiological substrates of protein kinases
Authors: Cohen Philip , Knebel Axel
Published: December 2005

The identification of all the substrates of every protein kinase is one of the major challenges of post-genomic research. Here we review a powerful method for tackling this problem that we have developed over the last 5 years. The method has so far been used to identify novel substrates for eight different protein kinases, demonstrating that it is of general utility. Importantly, the method can be used to identify distinct physiological substrates of protein kinases, such as PKB (protein kinase B) and SGK (serum- and glucocorticoid-induced kinase), that are closely related in structure and have similar specificity determinants.
Therapeutic vaccination against HIV: current progress and future possibilities
Authors: Puls Rebekah L. , Emery Sean
Published: December 2005

Although effective in reducing mortality, current antiretroviral therapy for HIV infection involves complex and expensive drug regimens that are toxic and difficult to take. Eradication of HIV reservoirs is not possible with existing therapies. The concept of therapeutic vaccination has been investigated to increase the potency and breadth of anti-HIV immune responses in order to delay or reduce antiretroviral therapy use. A variety of approaches targeted to both cell- and antibody-mediated immunity have been developed, including whole inactivated HIV-1, protein subunits and synthetic peptides, DNA vaccines and a number of viral vectors expressing HIV-1. These investigations have occurred in the absence of a clear understanding of disease pathogenesis or the correlates of protective immunity. At this time, there is no licensed therapeutic vaccine for any viral disease, including HIV; however, this review will consider recent progress in the field and summarize the challenges faced in the development of a therapeutic HIV vaccine.
Reduced glomerular filtration rate in pre-dialysis non-diabetic chronic kidney disease patients is associated with impaired baroreceptor sensitivity and reduced vascular compliance
Authors: Lacy Peter , Carr Susan J. , O'Brien David , Fentum Barbara , Williams Bryan , Paul Sanjoy K. , Robinson Thompson G.
Published: December 2005

Small uncontrolled studies of dialysis-dependent CKD (chronic kidney disease) patients have demonstrated abnormalities of cardiovascular autonomic control and vascular compliance, which may contribute to adverse cardiovascular morbidity in this population. However, there is little information utilizing newer non-invasive techniques in pre-dialysis patients with increasing degrees of uraemia. In the present study, 55 non-dialysis-dependent non-diabetic CKD patients with mean GFR (glomerular filtration rate) of 27 ml·min−1·m−2 were studied. All patients underwent a 10-min period of electrocardiographic and non-invasive blood pressure monitoring. Cardiac BRS (baroreceptor sensitivity) was calculated from the combined α-index. PWV (pulse wave velocity) measurement and determination of arterial wave reflexion by applanation tonometry was performed in all patients. Mean (S.D.) cardiac BRS was 10.8 (7.1) ms/mmHg and mean (S.D.) PWV was 8.6 (1.7) m/s. Reduced GFR was correlated with increased PWV and decreased cardiac BRS. On logistic regression analysis with adjustment for clinical significant risk factors, severely impaired renal function (assessed by GFR <15 ml·min−1·m−2) was associated with increased large artery stiffness [odds ratio for PWV=3.14 (95% confidence intervals, 1.03–9.53); P=0.04] and increased cardiovascular autonomic dysfunction [odds ratio for BRS=0.87 (95% confidence intervals, 0.75–1.80); P=0.06]. In conclusion, non-dialysis dependent non-diabetic CKD patients with decreasing GFR have reduced cardiac BRS and increased large artery stiffness. This may have important prognostic and therapeutic consequences for the management of vascular disease in a pre-dialysis population.
Synthesis rates of total liver protein and albumin are both increased in patients with an acute inflammatory response
Authors: Barle Hans , Hammarqvist Folke , Westman Bo , Klaude Maria , Rooyackers Olav , Garlick Peter J. , Wernerman Jan
Published: December 2005

The general perception that catabolism and inflammation are associated with a high synthesis rate of total liver protein and a low albumin synthesis rate has been challenged in recent years by several studies in man, indicating that the synthesis rate of albumin in response to a catabolic insult is increased rather than decreased. Thus changes in liver protein synthesis rates in conjunction with catabolism and acute inflammation in man need to be characterized better. The aim of the present study was to measure protein synthesis rates of total liver protein and albumin during a state of acute inflammation. Patients (n=10) undergoing acute laparoscopic cholecystectomy due to acute cholecystitis were investigated. FSRs (fractional synthesis rates) of total liver protein (liver biopsy specimens) and albumin (plasma samples) were investigated as early as possible during the surgical procedure, using a flooding dose of L-[2H5]phenylalanine. The results were compared with a reference group of patients without cholecystitis undergoing elective laparoscopic cholecystectomy (n=17). FSR of total liver protein was 60% higher (P<0.001) and the FSR of albumin was 45% higher (P<0.01) in the cholecystitis patients compared with the control group. In conclusion, the synthesis rates of total liver protein and albumin are both increased in patients with an acute general inflammatory reaction undergoing laparoscopic cholecystectomy.
Modifying effects of the R389G β1-adrenoceptor polymorphism on resting heart rate and blood pressure in patients with obstructive sleep apnoea
Authors: Börgel Jan , Schulz Tino , Bartels Nina K. , Epplen Jörg T. , Büchner Nikolaus , Rump Lars Christian , Huesing Anika , Sanner Bernd M. , Mügge Andreas
Published: December 2005

OSA (obstructive sleep apnoea) stimulates sympathetic nervous activity and elevates resting HR (heart rate) and BP (blood pressure). In the present study in a cohort of 309 untreated OSA patients, the resting HR and BP during the daytime were correlated with AHI (apnoea/hypopnea index) and compared with patients with R389R (n=162), R389G (n=125) and G389G (n=22) genotypes of the β1-adrenoreceptor R389G polymorphism. We analysed the impact of the genotype on the decline of HR and BP in a subgroup of 148 patients (R389R, n=86; R389G, n=54; G389G, n=8) during a 6-month follow-up period under CPAP (continuous positive airway pressure) therapy during which cardiovascular medication remained unchanged. In untreated OSA patients, we found an independent relationship between AHI and resting HR (β=0.096, P<0.001), systolic BP (β=0.09, P=0.021) and diastolic BP (β=0.059, P=0.016). The resting HR/BP, however, did not differ among carriers with the R389R, R389G and G389G genotypes. CPAP therapy significantly reduced HR [−2.5 (−1.1 to −4.0) beats/min; values are mean difference (95% confidence intervals)] and diastolic BP [−3.2 (−1.5 to −5.0) mmHg]. The decline in HR was more significantly pronounced in the R389R group compared with the Gly389 carriers [−4.1 (−2.3 to −5.9) beats/min (P<0.001) compared with −0.2 (2.1 to −2.6) beats/min (P=0.854) respectively; Student's t test between groups, P=0.008]. Diastolic BP was decreased significantly (P<0.001) only in Gly389 carriers (R389G or G389G) compared with R389R carriers [−5.0 (−2.3 to −7.6) mmHg compared with −2.0 (0.4 to −4.3) mmHg respectively]. ANOVA revealed a significant difference (P=0.023) in HR reduction between the three genotypes [−4.1 (±8.4) beats/min for R389R, −0.5 (±9.3) beats/min for R389G and +1.9 (±7.2) beats/min for G389G]. In conclusion, although the R389G polymorphism of the β1-adrenoceptor gene did not influence resting HR or BP in untreated OSA patients, it may modify the beneficial effects of CPAP therapy on these parameters.
What role do adrenoreceptor polymorphisms play in modifying cardiovascular responses in obstructive sleep apnoea?
Authors: Mcardle Nigel
Published: December 2005

OSA (obstructive sleep apnoea) is a common condition that is strongly associated with cardiovascular disease. It is remains unclear what role OSA plays in determining cardiovascular risk. The immediate physiological changes that occur during upper airway obstruction are potential contributors to cardiovascular risk in OSA. These changes include increased sympathetic activity, which is responsive to treatment of OSA with CPAP (continuous positive airway pressure). In this issue of Clinical Science, the possible role of a common polymorphism in the β1-adrenoreceptor [R389G (Arg389Gly)] has been investigated by Börgel and co-workers. Measurements of heart rate and blood pressure in untreated OSA patients were not related to the R389G polymorphism. There were changes in heart rate and diastolic blood pressure with CPAP treatment that were related to this polymorphism. Reduction in heart rate with CPAP treatment was associated with the R389R genotype. By contrast, a reduction in diastolic blood pressure was associated with the Gly389 carriers. These findings are intriguing, but difficult to fully explain. Further study is needed to determine if there is an important role of the R389G polymorphism in modifying cardiovascular responses among OSA patients.
Sustainable harvest rates of ironwood, Erythrophleum chlorostachys, in the Northern Territory, Australia
Authors: Cook Garry D. , Williams Richard J. , Banks the late John C. G.
Published: December 2005

Ironwood, Erythrophleum chlorostachys (F.Muell.) Baill., is harvested for timber in the Northern Territory. Available data on ironwood growth rates and stand structures were collated to help inform sustainable management. In 32 plots (0.1 ha each) within 300 km2 of a widespread savanna vegetation type in the Northern Territory, ironwood trees represent ∼11% of the total basal area of all trees and 12% of all individual trees >3 m high. Nevertheless, the density of potentially harvestable trees with a diameter at breast height >35 cm is low at 150 km–2, whereas the density of trees in the preferred size of >40 cm is only 30 km–2. The slow growth rate means that the sustainable harvest is only 1.8 trees km–2 year–1. This represents a maximum estimate that does not account for the high rate of poorly formed boles and the lower density of ironwood throughout most of its range. Limited data on tree growth rates of isolated seedlings and coppice indicate that trees could reach harvestable sizes in decades instead of centuries if competition with other species is reduced; however, there has been no assessment of the relative wood quality of fast- and slow-growing ironwoods.
Luteal stage dependence of pituitary response to gonadotrophin-releasing hormone in cyclic dairy ewes subjected to synchronisation of ovulation
Authors: Amiridis G. S. , Valasi I. , Menegatos I. , Rekkas C. , Goulas P. , Papanikolaou T. , Deligiannis C.
Published: December 2005

Possible hormonal aberrations precluding conception or maintenance of pregnancy in dairy ewes subjected to ovulation synchronisation were investigated in this study. The pituitary response to exogenous gonadotrophin-releasing hormone (GnRH) was tested at different luteal stages in 36 ewes. Oestruses were synchronised by using progestagen-impregnated sponges and the animals were randomly allotted into one of three treatment groups (A, B and C; n = 12 for each). Treatments commenced on Days 4, 9 and 14 of the new cycle (oestrus was defined as Day 0). Ewes were given two GnRH injections, 5 days before and 36 h after a prostaglandin F2α (PGF2α) injection, and the animals were inseminated 12–14 h after the second GnRH injection (modified OVSYNCH). For luteinising hormone (LH) determination blood samples were withdrawn from six ewes of each group at the time of GnRH administration, and 30, 90, 180, 270 and 360 min later. Progesterone was assayed in samples taken every other day starting from oestrus and for 17 days after the second GnRH injection, and in an additional sample collected on the day of insemination. After the first GnRH injection, the LH concentration was higher in Group C than in Groups B and A (mean ± s.d.: 64.8 ± 10.0 ng mL−1, 41.3 ± 3.7 ng mL−1 and 24.6 ± 9.0 ng mL−1, respectively; P < 0.05), whereas after the second GnRH injection a uniform LH release was found in all groups. PGF2α caused a significant decrease in progesterone (P4) concentration in all groups; however, at artificial insemination ewes that conceived had significantly lower P4 concentration in comparison with those that failed to conceive. As early as Day 5, pregnant animals had higher P4 concentrations than non-pregnant animals. Overall, 21 animals conceived (seven, nine and five ewes from Groups A, B and C, respectively). These results indicate that the proposed protocol is equally effective in inducing a preovulatory LH surge at any stage of the luteal phase, and that elevated P4 concentration along with a delayed P4 increase should be considered as a causative factor for inability to conceive.
Mechanism of metal ion activation of the diphtheria toxin repressor DtxR
Authors: D'Aquino J. Alejandro , Tetenbaum-Novatt Jaclyn , White Andre , Berkovitch Fred , Ringe Dagmar
Published: December 2005

The diphtheria toxin repressor (DtxR) is a metal ion-activated transcriptional regulator that has been linked to the virulence of
Corynebacterium diphtheriae
. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear. Calorimetric techniques have demonstrated that although binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 × 10
–7
, binding site 2 (primary) is a low-affinity binding site with a binding constant of 6.3 × 10
–4
. These two binding sites act in an independent fashion, and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A,C102D), reported here, and the previously reported DtxR(H79A) have allowed us to propose a mechanism of metal activation for DtxR.
Modulation of the electronic structure and the Ni–Fe distance in heterobimetallic models for the active site in [NiFe]hydrogenase
Authors: Zhu Wenfeng , Marr Andrew C. , Wang Qiang , Neese Frank , Spencer Douglas J. E. , Blake Alexander J. , Cooke Paul A. , Wilson Claire , Schröder Martin
Published: December 2005

Reaction of the mononuclear Ni(II) thiolate complexes [Ni(L)] [L, L
1
, H
2
L
1
, bis(2-mercaptoethyl)-1,2-dimercaptoethane; L
2
, H
2
L
2
,
N
,
N
′-dimethyl-
N
,
N
′-bis(2-mercaptoethyl)-bis(aminoethyl)sulfide] with [FeCp(CO)
2
I] gives the dithiolate-bridged heterobimetallic species, [Ni(L
1
)FeCp(CO)]PF
6
, 1, and [Ni(L
2
)FeCp]I, 2, respectively. Binding of a Fe(CO)
3
fragment via reaction of square-planar [Ni(pdt)(dppe)] (dppe, 1,2-diphenylphosphinoethane; pdt
2–
, 1,3-propanedithiolate) with Fe
3
(CO)
12
or [Fe(CO)
3
(BDA)] (BDA, benzylidene acetone) affords diamagnetic [(dppe)Ni(μ-pdt)Fe(CO)
3
], 3, in which the Ni(II) center is bound tetrahedrally to two thiolate S-donors and to two P-donors. The complex [(dppe)Ni(μ-pdt)Fe(CO)
3
], 3, reacts in solution via rearrangement to afford [(OC)Ni(μ-dppe)(μ-pdt)Fe(CO)
2
], 4, in which one P-donor of dppe is bound to Ni and the other to Fe, and a CO ligand has transferred from Fe to Ni. Additionally, the syntheses of 3 and 4 afford the side products [(dppe)Ni(CO)
2
] and [(OC)
3
Fe(pdt)Fe(CO)
3
] together with the trinuclear species [(dppe)(CO)Fe(μ-CO)(μ-pdt)Fe(μ-pdt)Fe(CO)
3
], 5. Reaction of [Ni(pdt)(dppe)] with [FeCp(CO)
2
I] in CH
2
Cl
2
affords two products [(dppe)Ni(μ-pdt)FeCp(CO)]PF
6
, 6, and [(dppe)Ni(pdt)(μ-I)Ni(dppe)]PF
6
, 7. The complexes 2, 3, and 4 show Ni–Fe distances of 2.539(4), 2.4666(6), and 2.4777(7) Å, respectively, with relatively acute dihedral angles of 79.5–81.8° for the Ni–S
2
-Fe bridge, thus mimicking the shortened Ni...Fe distance (2.5 Å) and the acute dihedral angle of the Ni–S
2
–Fe moiety observed in certain active forms of [NiFe]hydrogenase. The role of direct Ni–Fe bonding in these complexes is discussed and linked to electronic structure calculations on [(dppe)Ni(pdt)Fe(CO)
3
], 3, which confirm the presence of a bent Ni(d
z
2
)-Fe(d
z
2
) σ-bond in a singlet ground state.
Generating highly ordered DNA nanostrand arrays
Authors: Guan Jingjiao , Lee L. James
Published: December 2005

Highly ordered arrays of stretched DNA molecules were generated over the millimeter scale by using a modified molecular combing method and soft lithography. Topological micropatterning on polydimethyl siloxane stamps was used to mediate the dynamic assembly of DNA molecules into arranged nonostrand arrays. These arrays consisted of either short nanostrands of several micrometers with fixed length and orientation or long nanostrands up to several hundred micrometers in length. The nanostrand arrays were transferred onto flat solid surfaces by contact printing, allowing for the creation of more complex patterns. This technique has potential applications for the construction of next-generation DNA chips and functional circuits of DNA-based 1D nanostructures.
Interrogating multiple aspects of variation in a full resequencing data set to infer human population size changes
Authors: Voight Benjamin F. , Adams Alison M. , Frisse Linda A. , Qian Yudong , Hudson Richard R. , Di Rienzo Anna
Published: December 2005

We present an expanded data set of 50 unlinked autosomal noncoding regions, resequenced in samples of Hausa from Cameroon, Italians, and Chinese. We use these data to make inferences about human demographic history by using a technique that combines multiple aspects of genetic data, including levels of polymorphism, the allele frequency spectrum, and linkage disequilibrium. We explore an extensive range of demographic parameters and demonstrate that our method of combining multiple aspects of the data results in a significant reduction of the compatible parameter space. In agreement with previous reports, we find that the Hausa data are compatible with demographic equilibrium as well as a set of recent population expansion models. In contrast to the Hausa, when multiple aspects of the data are considered jointly, the non-Africans depart from an equilibrium model of constant population size and are compatible with a range of simple bottleneck models, including a 50–90% reduction in effective population size occurring some time after the appearance of modern humans in Africa 160,000–120,000 years ago.
Leucine-rich repeat kinase 2 (LRRK2) interacts with parkin, and mutant LRRK2 induces neuronal degeneration
Authors: Smith Wanli W. , Pei Zhong , Jiang Haibing , Moore Darren J. , Liang Yideng , West Andrew B. , Dawson Valina L. , Dawson Ted M. , Ross Christopher A.
Published: December 2005

Parkinson's disease (PD) is a disorder of movement, cognition, and emotion, and it is characterized pathologically by neuronal degeneration with Lewy bodies, which are cytoplasmic inclusion bodies containing deposits of aggregated proteins. Most PD cases appear to be sporadic, but genetic forms of the disease, caused by mutations in α-synuclein, parkin, and other genes, have helped elucidate pathogenesis. Mutations inleucine-rich repeat kinase 2(LRRK2) cause autosomal-dominant Parkinsonism with clinical features of PD and with pleomorphic pathology including deposits of aggregated protein. To study expression and interactions of LRRK2, we synthesized cDNAs and generated expression constructs coding for human WT and mutant LRRK2 proteins. Expression of full-length LRRK2 in cells in culture suggests that the protein is predominately cytoplasmic, as is endogenous protein by subcellular fractionation. Using coimmunoprecipitation, we find that LRRK2, expressed in cells in culture, interacts with parkin but not with α-synuclein, DJ-1, or tau. A small proportion of the cells overexpressing LRRK2 contain protein aggregates, and this proportion is greatly increased by coexpression of parkin. In addition, parkin increases ubiquitination of aggregated protein. Also, mutant LRRK2 causes neuronal degeneration in both SH-SY5Y cells and primary neurons. This cell model may be useful for studies of PD cellular pathogenesis and therapeutics. These findings suggest a gain-of-function mechanism in the pathogenesis of LRRK2-linked PD and suggest that LRRK2 may be involved in a pathogenic pathway with other PD-related proteins such as parkin, which may help illuminate both familial and sporadic PD.
Augmenting neurotransmitter release by enhancing the apparent Ca
2+
affinity of synaptotagmin 1
Authors: Rhee J.-S. , Li L. Y. , Shin O.-H. , Rah J.-C. , Rizo J. , Südhof T. C. , Rosenmund C.
Published: December 2005

Synaptotagmin 1 likely acts as a Ca
2+
sensor in neurotransmitter release by Ca
2+
-binding to its two C2 domains. This notion was strongly supported by the observation that a mutation in the C2A domain causes parallel decreases in the apparent Ca
2+
affinity of synaptotagmin 1 and in the Ca
2+
sensitivity of release. However, this study was based on a single loss-of-function mutation. We now show that tryptophan substitutions in the synaptotagmin 1 C2 domains act as gain-of-function mutations to increase the apparent Ca
2+
affinity of synaptotagmin 1. The same substitutions, when introduced into synaptotagmin 1 expressed in neurons, enhance the Ca
2+
sensitivity of release. Mutations in the two C2 domains lead to comparable and additive effects in release. Our results thus show that the apparent Ca
2+
sensitivity of release is dictated by the apparent Ca
2+
affinity of synaptotagmin 1 in both directions, and that Ca
2+
binding to both C2 domains contributes to Ca
2+
triggering of release.
Unconscious fear influences emotional awareness of faces and voices
Authors: de Gelder B. , Morris J. S. , Dolan R. J.
Published: December 2005

Nonconscious recognition of facial expressions opens an intriguing possibility that two emotions can be present together in one brain with unconsciously and consciously perceived inputs interacting. We investigated this interaction in three experiments by using a hemianope patient with residual nonconscious vision. During simultaneous presentation of facial expressions to the intact and the blind field, we measured interactions between conscious and nonconsciously recognized images. Fear-specific congruence effects were expressed as enhanced neuronal activity in fusiform gyrus, amygdala, and pulvinar. Nonconscious facial expressions also influenced processing of consciously recognized emotional voices. Emotional congruency between visual and an auditory input enhances activity in amygdala and superior colliculus for blind, relative to intact, field presentation of faces. Our findings indicate that recognition of fear is mandatory and independent of awareness. Most importantly, unconscious fear recognition remains robust even in the light of a concurrent incongruent happy facial expression or an emotional voice of which the observer is aware.
A mouse atlas of gene expression: Large-scale digital gene-expression profiles from precisely defined developing C57BL/6J mouse tissues and cells
Authors: Siddiqui Asim S. , Khattra Jaswinder , Delaney Allen D. , Zhao Yongjun , Astell Caroline , Asano Jennifer , Babakaiff Ryan , Barber Sarah , Beland Jaclyn , Bohacec Slavita , Brown-John Mabel , Chand Steve , Charest David , Charters Anita M. , Cullum Rebecca , Dhalla Noreen , Featherstone Ruth , Gerhard Daniela S. , Hoffman Brad , Holt Robert A. , Hou Juan , Kuo Byron Y.-L. , Lee Lisa L. C. , Lee Stephanie , Leung Derek , Ma Kevin , Matsuo Corey , Mayo Michael , McDonald Helen , Prabhu Anna-liisa , Pandoh Pawan , Riggins Gregory J. , de Algara Teresa Ruiz , Rupert James L. , Smailus Duane , Stott Jeff , Tsai Miranda , Varhol Richard , Vrljicak Pavle , Wong David , Wu Mona K. , Xie Yuan-Yun , Yang George , Zhang Ida , Hirst Martin , Jones Steven J. M. , Helgason Cheryl D. , Simpson Elizabeth M. , Hoodless Pamela A. , Marra Marco A.
Published: December 2005

We analyzed 8.55 million LongSAGE tags generated from 72 libraries. Each LongSAGE library was prepared from a different mouse tissue. Analysis of the data revealed extensive overlap with existing gene data sets and evidence for the existence of ≈24,000 previously undescribed genomic loci. The visual cortex, pancreas, mammary gland, preimplantation embryo, and placenta contain the largest number of differentially expressed transcripts, 25% of which are previously undescribed loci.
Diffusion and scaling during early embryonic pattern formation
Authors: Gregor Thomas , Bialek William , van Steveninck Rob R. de Ruyter , Tank David W. , Wieschaus Eric F.
Published: December 2005

Development of spatial patterns in multicellular organisms depends on gradients in the concentration of signaling molecules that control gene expression. In the
Drosophila
embryo, Bicoid (Bcd) morphogen controls cell fate along 70% of the anteroposterior axis but is translated from mRNA localized at the anterior pole. Gradients of Bcd and other morphogens are thought to arise through diffusion, but this basic assumption has never been rigorously tested in living embryos. Furthermore, because diffusion sets a relationship between length and time scales, it is hard to see how patterns of gene expression established by diffusion would scale proportionately as egg size changes during evolution. Here, we show that the motion of inert molecules through the embryo is well described by the diffusion equation on the relevant length and time scales, and that effective diffusion constants are essentially the same in closely related dipteran species with embryos of very different size. Nonetheless, patterns of gene expression in these different species scale with egg length. We show that this scaling can be traced back to scaling of the Bcd gradient itself. Our results, together with constraints imposed by the time scales of development, suggest that the mechanism for scaling is a species-specific adaptation of the Bcd lifetime.