Characterisation of Human Neutral and Lysosomal alpha-Mannosidases

Neutral alpha-mannosidase and lysosomal MAN2B1 alpha-mannosidase belong to glycoside hydrolase family 38, which contains essential enzymes required for the modification and catabolism of asparagine-linked glycans on proteins. MAN2B1 catalyses lysosomal glycan degradation, while neutral α-mannosidase is most likely involved in the catabolism of cytosolic free oligosaccharides. These mannose containing saccharides are generated during glycosylation or released from misfolded glycoproteins, which are detected by quality control in the endoplasmic reticulum. To characterise the biological function of human neutral α-mannosidase, I cloned the alpha-mannosidase cDNA and recombinantly expressed the enzyme. The purified enzyme trimmed the putative natural substrate Man9GlcNAc to Man5GlcNAc, whereas the reducing end GlcNAc2 limited trimming to Man8GlcNAc2. Neutral α-mannosidase showed highest enzyme activity at neutral pH and was activated by the cations Fe₂+, Co2+ and Mn₂+, Cu2+ in turn had a strong inhibitory effect on alpha-mannosidase activity. Analysis of its intracellular localisation revealed that neutral alpha-mannosidase is cytosolic and colocalises with proteasomes. Further work showed that the overexpression of neutral alpha-mannosidase affected the cytosolic free oligosaccharide content and led to enhanced endoplasmic reticulum associated degradation and underglycosylation of secreted proteins. The second part of the study focused on MAN2B1 and the inherited lysosomal storage disorder α-mannosidosis. In this disorder, deficient MAN2B1 activity is associated with mutations in the MAN2B1 gene. The thesis reports the molecular consequences of 35 alpha-mannosidosis associated mutations, including 29 novel missense mutations. According to experimental analyses, the mutations fall into four groups: Mutations, which prevent transport to lysosomes are accompanied with a lack of proteolytic processing of the enzyme (groups 1 and 3). Although the rest of the mutations (groups 2 and 4) allow transport to lysosomes, the mutated proteins are less efficiently processed to their mature form than is wild type MAN2B1. Analysis of the effect of the mutations on the model structure of human lysosomal alpha-mannosidase provides insights on their structural consequences. Mutations, which affect amino acids important for folding (prolines, glycines, cysteines) or domain interface interactions (arginines), arrest the enzyme in the endoplasmic reticulum. Surface mutations and changes, which do not drastically alter residue volume, are tolerated better. Descriptions of the mutations and clinical data are compiled in an α-mannosidosis database, which will be available for the scientific community. This thesis provides a detailed insight into two ubiquitous human alpha-mannosidases. It demonstrates that neutral alpha-mannosidase is involved in the degradation of cytosolic oligosaccharides and suggests that the regulation of this α-mannosidase is important for maintaining the cellular homeostasis of N-glycosylation and glycan degradation. The study on alpha-mannosidosis associated mutations identifies multiple mechanisms for how these mutations are detrimental for MAN2B1 activity. The α-mannosidosis database will benefit both clinicians and scientific research on lysosomal alpha‑mannosidosis.

Salivary and serum cortisol levels, salivary alpha-amylase and unstimulated whole saliva flow rate in pregnant and non-pregnant

PURPOSE: To compare salivary and serum cortisol levels, salivary alpha-amylase (sAA), and unstimulated whole saliva (UWS) flow rate in pregnant and non-pregnant women. METHOD: A longitudinal study was conducted at a health promotion center of a university hospital. Nine pregnant and 12 non-pregnant women participated in the study. Serum and UWS were collected and analyzed every trimester and twice a month during the menstrual cycle. The salivary and serum cortisol levels were determined by chemiluminescence assay and the sAA was processed in an automated biochemistry analyzer. RESULTS: Significant differences between the pregnant and non-pregnant groups were found in median [interquartile range] levels of serum cortisol (23.8 µL/dL [19.4-29.4] versus 12.3 [9.6-16.8], p<0.001) and sAA (56.7 U/L [30.9-82.2] versus 31.8 [18.1-53.2], p<0.001). Differences in salivary and serum cortisol (µL/dL) and sAA levels in the follicular versus luteal phase were observed (p<0.001). Median UWS flow rates were similar in pregnant (0.26 [0.15-0.30] mL/min) and non-pregnant subjects (0.23 [0.20-0.32] mL/min). Significant correlations were found between salivary and serum cortisol (p=0.02) and between salivary cortisol and sAA (p=0.01). CONCLUSIONS: Serum cortisol and sAA levels are increased during pregnancy. During the luteal phase of the ovarian cycle, salivary cortisol levels increase, whereas serum cortisol and sAA levels decline.

alpha-Smooth muscle actin and proliferating cell nuclear antigen expression in focal segmental glomerulosclerosis: functional and structural parameters of renal disease progression

The aim of the present study was to investigate the expression of alpha-smooth muscle actin (alpha-SM-actin) and proliferating cell nuclear antigen (PCNA) in renal cortex from patients with focal segmental glomerulosclerosis (FSGS) and their correlations with parameters of renal disease progression. We analyzed renal biopsies from 41 patients with idiopathic FSGS and from 14 control individuals. The alpha-SM-actin immunoreaction was evaluated using a score that reflected the changes in the extent and intensity of staining in the glomerular or cortical area. The PCNA reaction was quantified by counting the labeled cells of the glomeruli or renal cortex. The results, reported as median ± percentile (25th; 75th), showed that the alpha-SM-actin scores in the glomeruli and tubulointerstitium from the renal cortex were 2.0 (2.0; 4.0) and 3.0 (3.0; 4.0), respectively, in patients with FSGS, and 0.5 (0.0; 1.0) and 0.0 (0.0; 0.5) in the controls. The number of PCNA-positive cells per glomerulus and graded field of tubulointerstitium from the renal cortex was 0.2 (0.0; 0.4) and 1.1 (0.3; 2.2), respectively, for patients with FSGS, and 0.0 (0.0; 0.5) and 0.0 (0.0; 0.0) for controls. The present data showed an increase of alpha-SM-actin and PCNA expression in glomeruli and renal cortex from FSGS patients. The extent of immunoreaction for alpha-SM-actin in the tubulointerstitial area was correlated with the intensity of proteinuria. However, there was no correlation between the kidney expression of these proteins and the reciprocal of plasma creatinine level or renal fibrosis. These findings suggest that the immunohistochemical alterations may be reversible.

High concentrations of KCl release noradrenaline from noradrenergic neurons in the rat anococcygeus muscle

The aim of the present study was to investigate the effects of high concentrations of KCl in releasing noradrenaline from sympathetic nerves and its actions on postsynaptic alpha-adrenoceptors. We measured the isotonic contractions induced by KCl in the isolated rat anococcygeus muscle under different experimental conditions. The contractile responses induced by KCl were inhibited by alpha-adrenoceptor antagonists in 2.5 mM Ca2+ solution. Prazosin reduced the maximum effect from 100 to 53.9 ± 10.2% (P<0.05) while the pD2 values were not changed. The contractile responses induced by KCl were abolished by prazosin in Ca2+-free solution (P<0.05). Treatment of the rats with reserpine reduced the maximum effect induced by KCl as compared to the contractile responses induced by acetylcholine from 339.5 ± 157.8 to 167.3 ± 65.5% (P<0.05), and increased the pD2 from 1.57 ± 0.01 to 1.65 ± 0.006 (P<0.05), but abolished the inhibitory effect of prazosin (P<0.05). In contrast, L-NAME increased the contractile responses induced by 120 mM KCl by 6.2 ± 2.3% (P<0.05), indicating that KCl could stimulate the neurons that release nitric oxide, an inhibitory component of the contractile response induced by KCl. Our results indicate that high concentrations of KCl induce the release of noradrenaline from noradrenergic neurons, which interacts with alpha1-adrenoceptors in smooth muscle cells, producing a contractile response in 2.5 mM Ca2+ (100%) and in Ca2+-free solution, part of which is due to a direct effect of KCl on the rat anococcygeus muscle.

Effect of salbutamol on innervated and denervated rat soleus muscle

The objective of the present investigation was to perform a 14-day time-course study of treatment with salbutamol, a ß2 adrenoceptor agonist, on rat soleus muscle in order to assess fiber type selectivity in the hypertrophic response and fiber type composition. Male Wistar rats were divided into four groups: control (N = 10), treated with salbutamol (N = 30), denervated (N = 30), and treated with salbutamol after denervation (N = 30). Salbutamol was injected intraperitoneally in the rats of the 2nd and 4th groups at a concentration of 0.3 mg/kg twice a day for 2 weeks. The muscles were denervated using the crush method with pean. The animals were sacrificed 3, 6, 9, 12, and 14 days after treatment. Frozen cross-sections of soleus muscle were stained for myosin ATPase, pH 9.4. Cross-sectional area and percent of muscle fibers were analyzed morphometrically by computerized image analysis. Treatment with salbutamol induced hypertrophy of all fiber types and a higher percentage of type II fibers (21%) in the healthy rat soleus muscle. Denervation caused marked atrophy of all fibers and conversion from type I to type II muscle fibers. Denervated muscles treated with salbutamol showed a significantly larger cross-sectional area of type I muscle fibers, 28.2% compared to the denervated untreated muscle. Moreover, the number of type I fibers was increased. These results indicate that administration of salbutamol is able to induce changes in cross-sectional area and fiber type distribution in the early phase of treatment. Since denervation-induced atrophy and conversion from type I to type II fibers were improved by salbutamol treatment we propose that salbutamol, like other ß2 adrenoceptor agonists, may have a therapeutic potential in improving the condition of skeletal muscle after denervation.

Inducible nitric oxide synthase and tumor necrosis factor-alpha in delayed gastric emptying and gastrointestinal transit induced by lipopolysaccharide in mice

Gastrointestinal motility disturbances during endotoxemia are probably caused by lipopolysaccharide (LPS)-induced factors: candidates include nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-1ß, and interleukin-6. Flow cytometry was used to determine the effects of LPS and these factors on gastric emptying (evaluated indirectly by determining percent gastric retention; %GR) and gastrointestinal transit (GIT) in male BALB/c mice (23-28 g). NO (300 µg/mouse, N = 8) and TNF-alpha (2 µg/mouse, N = 7) increased (P < 0.01) GR and delayed GIT, mimicking the effect of LPS (50 µg/mouse). During early endotoxemia (1.5 h after LPS), inhibition of inducible NO synthase (iNOS) by a selective inhibitor, 1400 W (150 µg/mouse, N = 11), but not antibody neutralization of TNF-alpha (200 µg/mouse, N = 11), reversed the increase of GR (%GR 78.8 ± 3.3 vs 47.2 ± 7.5%) and the delay of GIT (geometric center 3.7 ± 0.4 vs 5.6 ± 0.2). During late endotoxemia (8 h after LPS), both iNOS inhibition (N = 9) and TNF-alpha neutralization (N = 9) reversed the increase of GR (%GR 33.7 ± 2.0 vs 19.1 ± 2.6% (1400 W) and 20.1 ± 2.0% (anti-TNF-alpha)), but only TNF-alpha neutralization reversed the delay of GIT (geometric center 3.9 ± 0.4 vs 5.9 ± 0.2). These findings suggest that iNOS, but not TNF-alpha, is associated with delayed gastric emptying and GIT during early endotoxemia and that during late endotoxemia, both factors are associated with delayed gastric emptying, but only TNF-alpha is associated with delayed GIT.

Effect of aerobic training on EEG alpha asymmetry and depressive symptoms in the elderly: a 1-year follow-up study

The effect of physical exercise on the treatment of depressive elderly adults has not been investigated thus far in terms of changes in cortical hemispheric activity. The objective of the present study was to identify changes in depressive symptoms, quality of life, and cortical asymmetry produced by aerobic activity. Elderly subjects with a diagnosis of major depressive disorder (DSM-IV) were included. Twenty patients (70% females, 71 ± 3 years) were divided into an exercise group (pharmacological treatment plus aerobic training) and a control group (undergoing pharmacological treatment) in a quasi-experimental design. Pharmacological treatment was maintained stable throughout the study (antidepressants and anxiolytics). Subjects were evaluated by depression scales (Beck Depression Inventory, Hamilton Depression Rating Scale, Montgomery-Asberg Depression Rating Scale) and the Short Form Health Survey-36, and electroencephalographic measurements (frontal and parietal alpha asymmetry) before and after 1 year of treatment. After 1 year, the control group showed a decrease in cortical activity on the right hemisphere (increase of alpha power), which was not observed in the exercise group. The exercise group showed a significant decrease of depressive symptoms, which was not observed in the control group. This result was also accompanied by improved treatment response and remission rate after 1 year of aerobic exercise associated with treatment. This study provides support for the effect of aerobic training on alpha activity and on depressive symptoms in elderly patients. Exercise facilitates the treatment of depressive elderly adults, leading to clinical and physical improvement and protecting against a decrease in cortical activity.

Identification of novel retinoid receptors and their roles in vertebrate and invertebrate nervous systems

In vertebrates, signaling by retinoic acid (RA) is known to play an important role in embryonic development, as well as organ homeostasis in the adult. In organisms such as adult axolotls and newts, RA is also important for regeneration of the CNS, limb, tail, and many other organ systems. RA mediates many of its effects in development and regeneration through nuclear receptors, known as retinoic acid receptors (RARs) and retinoid X receptors (RXRs). This study provides evidence for an important role of the RA receptor, RAR~2, in ,( '. regeneration ofthe spinal cord and tail of the adult newt. It has previously been proposed that the ability of the nervous system to regenerate might depend on the presence or absence of this RAR~2 isoform. Here, I show for the very first time, that the regenerating spinal cord of the adult newt expresses this ~2 receptor isoform, and inhibition of retinoid signaling through this specific receptor with a selective antagonist inhibits tail and spinal cord regeneration. This provides the first evidence for a role of this receptor in this process. Another species capable of CNS ~~generation in the adult is the invertebrate, " Lymnaea stagnalis. Although RA has been detected in a small number of invertebrates (including Lymnaea), the existence and functional roles of the retinoid receptors in most invertebrate non-chordates, have not been previously studied. It has been widely believed, however, that invertebrate non-chordates only possess the RXR class of retinoid receptors, but not the RARs. In this study, a full-length RXR cDNA has been cloned, which was the first retinoid receptor to be discovered in Lymnaea. I then went on to clone the very first full-length RAR eDNA from any non-chordate, invertebrate species. The functional role of these receptors was examined, and it was shown that normal molluscan development was altered, to varying degrees, by the presence of various RXR and RAR agonists or antagonists. The resulting disruptions in embryogenesis ranged from eye and shell defects, to complete lysis of the early embryo. These studies strongly suggest an important role for both the RXR and RAR in non-chordate development. The molluscan RXR and RAR were also shown to be expressed in the adult, nonregenerating eNS, as well as in individual motor neurons regenerating in culture. More specifically, their expression displayed a non-nuclear distfibution, suggesting a possible non-genomic role for these 'nuclear' receptors. It was shown that immunoreactivity for the RXR was present in almost all regenerating growth cones, and (together with N. Farrar) it was shown that this RXR played a novel, non-genomic role in mediating growth cone turning toward retinoic acid. Immunoreactivity for the novel invertebrate RAR was also found in the regenerating growth cones, but future work will be required to determine its functional role in nerve cell regeneration. Taken together, these data provide evidence for the importance of these novel '. retinoid receptors in development and regeneration, particularly in the adult nervous system, and the conservation of their effects in mediating RA signaling from invertebrates to vertebrates.

Physiological analysis of central and peripheral insect circadian pacemaker neurons

Alle bisher untersuchten Lebewesen besitzen (circadiane) innere Uhren, die eine endogene Perioden-länge von ungefähr 24 Stunden generieren. Eine innere Uhr kann über Zeitgeber mit der Umwelt synchronisiert werden und ermöglicht dem Organismus, rhythmische Umweltveränderungen vorweg zu nehmen. Neben einem zentralen Schrittmacher, der Physiologie und Verhalten des Organismus steuert, gibt es in unterschiedlichen Organen auch periphere Uhren, die die zeitlichen Abläufe in der spezifischen Funktion dieser Organe steuern. In dieser Arbeit sollten zentrale und periphere Schrittmacherneurone von Insekten physiologisch untersucht und verglichen werden. Die Neurone der akzessorischen Medulla (AME) von Rhyparobia maderae dienten als Modellsystem für zentrale Schrittmacher, während olfaktorische Rezeptorneurone (ORNs) von Manduca sexta als Modellsystem für periphere Schrittmacher dienten. Die zentralen Schrittmacherneurone wurden in extrazellulären Ableitungen an der isolierten AME (Netzwerkebene) und in Patch-Clamp Experimenten an primären AME Zellkulturen (Einzelzellebene) untersucht. Auf Netzwerkebene zeigten sich zwei charakteristische Aktivitätsmuster: regelmäßige Aktivität und Wechsel zwischen hoher und niedriger Aktivität (Oszillationen). Es wurde gezeigt, dass Glutamat ein Neurotransmitter der weitverbreiteten inhibitorischen Synapsen der AME ist, und dass in geringem Maße auch exzitatorische Synapsen vorkommen. Das Neuropeptid pigment-dispersing factor (PDF), das von nur wenigen AME Neuronen exprimiert wird und ein wichtiger Kopplungsfaktor im circadianen System ist, führte zu Hemmungen, Aktivierungen oder Oszillationen. Die Effekte waren transient oder langanhaltend und wurden wahrscheinlich durch den sekundären Botenstoff cAMP vermittelt. Ein Zielmolekül von cAMP war vermutlich exchange protein directly activated by cAMP (EPAC). Auf Einzelzellebene wurde gezeigt, dass die meisten AME Neurone depolarisiert waren und deshalb nicht feuerten. Die Analyse von Strom-Spannungs-Kennlinien und pharmakologische Experimente ergaben, dass unterschiedliche Ionenkanäle vorhanden waren (Ca2+, Cl-, K+, Na+ Kanäle sowie nicht-spezifische Kationenkanäle). Starke, bei hohen Spannungen aktivierende Ca2+ Ströme (ICa) könnten eine wichtige Rolle bei Ca2+-abhängiger Neurotransmitter-Ausschüttung, Oszillationen, und Aktionspotentialen spielen. PDF hemmte unterschiedliche Ströme (ICa, IK und INa) und aktivierte nicht-spezifische Kationenströme (Ih). Es wurde angenommen, dass simultane PDF-abhängige Hyper- und Depolarisationen rhythmische Membranpotential-Oszillationen verursachen. Dieser Mechanismus könnte eine Rolle bei PDF-abhängigen Synchronisationen spielen. Die Analyse peripherer Schrittmacherneurone konzentrierte sich auf die Charakterisierung des olfaktorischen Corezeptors von M. sexta (MsexORCO). In anderen Insekten ist ORCO für die Membran-Insertion von olfaktorischen Rezeptoren (ORs) erforderlich. ORCO bildet Komplexe mit den ORs, die in heterologen Expressionssystemen als Ionenkanäle fungieren und Duft-Antworten vermitteln. Es wurde die Hypothese aufgestellt, dass MsexORCO in pheromonsensitiven ORNs in vivo nicht als Teil eines ionotropen Rezeptors sondern als Schrittmacherkanal fungiert, der unterschwellige Membranpotential-Oszillationen generiert. MsexORCO wurde mit vermeintlichen Pheromonrezeptoren in human embryonic kidney (HEK 293) Zellen coexprimiert. Immuncytochemie und Ca2+ Imaging Experimente zeigten sehr schwache Expressionsraten. Trotzdem war es möglich zu zeigen, dass MsexORCO wahrscheinlich ein spontan-aktiver, Ca2+-permeabler Ionenkanal ist, der durch den ORCO-Agonisten VUAA1 und cyclische Nucleotide aktiviert wird. Außerdem wiesen die Experimente darauf hin, dass MsexOR-1 offensichtlich der Bombykal-Rezeptor ist. Eine weitere Charakterisierung von MsexORCO in primären M. sexta ORN Zellkulturen konnte nicht vollendet werden, weil die ORNs nicht signifikant auf ORCO-Agonisten oder -Antagonisten reagierten.

A dual role for integrin-linked kinase in platelets: regulating integrin function and alpha-granule secretion

Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with beta 1- and beta 3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLC gamma 2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in alpha-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for alpha-granule secretion and therefore may play a central role in the regulation of platelet function. (Blood. 2008; 112: 4523-4531)

The cytoprotective effect of alpha-tocopherol and daidzein against d-galactosamine-induced oxidative damage in the rat liver

We hypothesized that the hepatotoxicity that develops after the induction of oxidative stress (induced by d-galactosamine [GalN]) can be ameliorated by alpha-tocopherol (ATC) and the soy isoflavone daidzein. To test this, we ranked and assigned male Wistar rats into 6 groups, which involved pretreatment (ATC or daidzein) for 1 hour followed by treatment (GalN) for 23 hours. Histopathologic analysis showed that GalN administration induced marked necrosis (P < .001), steatosis (P < .001), both lobular and portal inflammations (P < .001), overall histopathologic score (P < .001), and activation of caspase-3 in the liver (P < .001). Immunohistochemical staining of malondialdehyde-protein adducts, a measure of oxidative stress, was increased in response to GalN (P < .001). Paradoxically, there were increases in total (P < .05) and cytosolic superoxide dismutase (P < .001) activities after GalN administration, indicative of an up-regulation of antioxidant defenses. The concentration of total protein (P < .001), albumin (P < .01), and globulin fractions (P < .001) in the plasma, as well as the activity of aspartate aminotransferase (P < .001), was significantly perturbed after GalN treatment, reflective of overall acute hepatic injury. Administration of daidzein showed a significant amelioration of the Ga1N-induced increase in malondialdehyde-protein adducts (P < .01) and cytosolic superoxide dismutase activities (P < .01) in the liver. However, all other variables were not significantly altered in response to daidzein. In response to ATC pretreatment, the total histopathologic score (P < .05), degree of necrosis (P < .05), and both lobular (P < .05) and portal (P = .05) inflammations were significantly ameliorated. To conclude, both daidzein and ATC protect the liver against oxidative damage possibly via different pathways.

Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alpha A-, alpha beta- and R120G alpha B-Crystallins

alpha B-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alpha B-crystallin in detail, and also that of alpha A-crystallin and the disease-related mutant R120G (alpha B-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alpha B-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. H-1 NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alpha B-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils. (C) 2007 Elsevier Ltd. All rights reserved.

The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis

1,6-alpha-D-Mannosidase from Aspergillits phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 74 kDa by SDS-PAGE and 81 kDa by native-PAGE. The isoelectric point was 4.6. 1,6-alpha-D-Mannosidase had a temperature optimum of 60 degrees C, a pH optimum of 4.0-4.5. a K-m of 14 mM with alpha-D-Manp-(1 -> 6)-D-Manp as substrate. It was strongly inhibited by Mn2+ and did not need Ca2+ or any other metal cofactor of those tested. The enzyme cleaves specifically (1 -> 6)-linked mannobiose and has no activity towards any other linkages, p-nitrophenyl-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-D-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocus sing and size-exclusion chromatography. The apparent molecular weight was 97 kDa by SDS-PAGE and 110 kDa by native-PAGE. The 1,3(1,6)-alpha-D-mannosidase enzyme existed as two charge isomers or isoforms. The isoelectric points of these were 4.3 and 4.8 by isoelectric focussing. It cleaves alpha-D-Manp-(1 -> 3)-D-Manp 10 times faster than alpha-D-Manp-(1 -> 6)-D-Manp, has very low activity towards p-nitrophenyl-alpha-D-mannopyranoside and baker's yeast mannan, and no activity towards alpha-D-Manp-(1 -> 2)-D-Manp. The activity towards (1 -> 3)-linked mannobiose is strongly activated by 1 mM Ca2+ and inhibited by 10 mM EDTA, while (1 -> 6)-activity is unaffected, indicating that the two activities may be associated with different polypeptides. It is also possible that one polypeptide may have two active sites catalysing distinct activities. (c) 2005 Elsevier Ltd. All rights reserved.

Marked influence of the bridging carbonyl ligands on the photo- and electrochemistry of the clusters [Ru-3(CO)(8)(mu-CO)(2)-(alpha-diimine)] (alpha-diimine = 2,2 '-bipyridine, 4,4 '-dimethyl-2,2 '-bipyridine and 2,2 '-bipyrimidine)

Bonding, photochemical and electrochemical properties of the clusters [Ru-3(CO)(8)(mu-CO)(2)(alpha-diimine)] (alpha-diimine=2,2'-bipyridine (1), 4,4'-dimethyl-2,2'-bipyridine (2) and 2,2'-bipyrimidine (3)) are strongly influenced by the presence of bridging carbonyl ligands. Irradiation at 471 nm initially results in the population of a sigma(Ru-3)pi*(alpha-diimine) excited state. From this state, fast decay takes place to the optically hardly directly accessible pi(Ru/mu-CO) pi*(alpha-diimine) lowest excited state. These assignments agree with theoretical (TD-DFT) results, resonance Raman and picosecond time-resolved infrared spectra. The involvement of the bridging carbonyl ligands in the electron transfer increases the energetic barrier for the formation of open-structure photoproducts such as biradicals and zwitterions. Zwitterions were therefore only obtained in strongly coordinating media such as pyridine at 250 K. The bridging carbonyl ligands also stabilize the radical anions produced upon one-electron reduction of the clusters [Ru-3(CO)(8)(mu-CO)(2)(alpha-diimine)] and observed with cyclic voltammetry, EPR and IR spectroelectrochemistry (for alpha-diimine=2,2'-bipyrimidine). In contrast, open-triangle intermediates formed along the reduction path to [Ru(CO)(2)(alpha-diimine)](n) and [Ru-2(CO)(8)](2-) are more reactive than their triosmium analogues.

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.