Evolution of feedback-inhibited beta /alpha barrel isoenzymes by gene duplication and a single mutation

The betaalpha barrel is the common protein fold of numerous enzymes and was proposed recently to be the result of gene duplication and fusion of an ancient half-barrel. The initial enzyme of shikimate biosynthesis possesses the additional feature of feedback regulation. The crystal structure and kinetic studies on chimera and mutant proteins of yeast 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase from Saccharomyces cerevisiae inhibited by phenylalanine (Aro3p) and DAHP synthase S. cerevisiae inhibited by tyrosine (Aro4p) give insight into important regions for regulation in the enzyme: The loop, which is connecting the two half-barrels, and structural elements added to the barrel are prerequisites for regulation and form a cavity on the N-terminal side of the betaalpha barrel. In the cavity of Aro4p at position 226 is a glycine residue, which is highly conserved in all other tyrosine-regulated DAHP synthases as well. Sequence alignments with phenylalanine-regulated DAHP synthases including Aro3p show a highly conserved serine residue at this position. An exchange of glycine to serine and vice versa leads to a complete change in the regulation pattern. Therefore the evolution of these differently feedback-inhibited isoenzymes required gene duplication and a single mutation within the internal extra element. Numerous additional amino acid substitutions present in the contemporary isoenzymes are irrelevant for regulation and occurred independently.     

The genome sequence of Clostridium tetani,the causative agent of tetanus disease

Tetanus disease is one of the most dramatic and globally prevalent diseases of humans and vertebrate animals, and has been reported for over 24 centuries. The manifestation of the disease, spastic paralysis, is caused by the second most poisonous substance known, the tetanus toxin, with a human lethal dose of approximately 1 ng/kg. Fortunately, this disease is successfully controlled through immunization with tetanus toxoid; nevertheless, according to the World Health Organization, an estimated 400,000 cases still occur each year, mainly of neonatal tetanus. The causative agent of tetanus disease is Clostridium tetani, an anaerobic spore-forming bacterium, whose natural habitat is soil, dust, and intestinal tracts of various animals. Here we report the complete genome sequence of toxigenic C. tetani E88, a variant of strain Massachusetts. The genome consists of a 2,799,250-bp chromosome encoding 2,372 ORFs. The tetanus toxin and a collagenase are encoded on a 74,082-bp plasmid, containing 61 ORFs. Additional virulence-related factors could be identified, such as an array of surface-layer and adhesion proteins (35 ORFs), some of them unique to C. tetani. Comparative genomics with the genomes of Clostridium perfringens, the causative agent of gas gangrene, and Clostridium acetobutylicum, a nonpathogenic solvent producer, revealed a remarkable capacity of C. tetani: The organism can rely on an extensive sodium ion bioenergetics. Additional candidate genes involved in the establishment and maintenance of a pathogenic lifestyle of C. tetani are presented.     

Histological classification of interstitial lung diseases

The 2002 ATS/ERS consensus classification of idiopathic interstitial pneumonias standardizes definitions and criteria for classification and diagnosis of idiopathic interstitial pneumonias and replaces previous classifications. Based on clinico-radiologic-pathologic criteria seven entities were defined: idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, cryptogenic organizing pneumonia, acute interstitial pneumonia, respiratory bronchiolitis-associated interstitial lung disease, desquamative interstitial pneumonia and lymphoid interstitial pneumonia. The following paper includes a brief overview of the histopathological diagnosis of these entities as compared to other diffuse interstitial pulmonary diseases and pulmonary manifestations of collagenvascular diseases.     

Persistence of Chlamydia pneumoniae in degenerative aortic valve stenosis indicated by heat shock protein 60 homologues

BACKGROUND AND AIMS OF THE STUDY: Based on the concept of chronic persistent infections with Chlamydia pneumoniae among variable stressors for aortic valve degeneration, the study aim was to assess the presence of chlamydial heat shock protein (cHSP) 60 and its human homologue (hHSP60) in diseased valvular tissue. METHODS: Surgical specimens of high-grade stenosed, native (n = 33) and bioprosthetic (n = 10) aortic valves were examined immunohistochemically for the localization of cHSP60, hHSP60 and macrophages (CD68), supplemented by polymerase chain reaction (PCR) and electron microscopy to prove microbial presence. RESULTS: Degenerated valves showed specific immunostaining of cHSP60 in 27 cases (65%), of hHSP60 in 26 (63%), and of CD68 in 36 (84%). Both HSP60 homologues were predominantly detected in valvular fibrosa, consistently co-localized with macrophages and, quantitatively, showed a strong correlation (r = 0.81, p < 0.001). Presence of C. pneumoniae was demonstrated by PCR in a subset of 11 of 18 valves (61%). Microbial persistence was confirmed by ultrastructural analysis. Degenerated prosthetic valves revealed markedly higher macrophage infiltration and cHSP60 signaling compared with degenerated native valves (each p < 0.05). CONCLUSION: Beyond detection of C. pneumoniae, the present data on co-localization and valvular predilection sites (fibrosa) of both HSP60 homologues indicate the presence of chronic persistent C. pneumoniae infection as well as regional stressor effects, and suggest their involvement in native and prosthetic valve degeneration.     

Influence of residual stenosis after percutaneous coronary intervention with stent implantation on development of restenosis and stent thrombosis

The aim of this study was to assess the effects of residual stenosis after single-stent implantation on the rate of stent thrombosis, as well as restenosis within a 6-month follow-up period. Coronary angiograms of 2,157 patients with 2,523 lesions treated with a single stent were analyzed by quantitative coronary angiography before, immediately after stent implantation, and at a planned 6-month follow-up. Lesions were classified into 4 subgroups according to the degree of residual stenosis after stent implantation: group 1, gross oversizing <-15%; group 2, slight oversizing -15% to <0%; group 3, mild residual 0% to <15%; group 4, moderate residual 15% to <30%. Stent thrombosis rates were not significantly different among the 4 subgroups (group 1: 0 of 60 [0%]; group 2: 2 of 388 [0.5%]; group 3: 8 of 1,370 [0.6%]; group 4: 8 of 705 [1.1%]; p = NS for all). An adequate dosage of ticlopidine (250 mg twice daily) and aspirin (100 mg/day) led to a lower rate of stent thrombosis (6 of 2,189 cases) than inadequate dosages or missing therapy (12 of 343 cases). In 1,882 stenoses with angiographic follow-up (77.7%), gross oversizing of stents lead to a significantly higher increase of percent stenosis (p <0.001) associated with a higher restenosis rate (group 1: 34.7% vs groups 2, 3, and 4: 32.5%, 28.2%, and 29.6%, respectively). A multiple regression analysis was performed. Optimal results with regard to stent thrombosis and restenosis were achieved with mild residual stenoses between 0% and 15% after stent implantation. Oversizing of stents is no longer necessary with an adequate dosage of ticlopidine and aspirin.     

Treatment with the selective muscarinic m1 agonist talsaclidine decreases cerebrospinal fluid levels of A beta 42 in patients with Alzheimer's disease.

The amyloid beta-peptides A beta 40 and A beta 42 are highly amyloidogenic constituents of brain beta-amyloid plaques in Alzheimer's disease (AD). Lowering their formation may be achieved by modulating the activities of proteases that cleave the amyloid precursor protein (A beta PP), including alpha- beta-, and gamma-secretases. Talsaclidine is a functionally selective muscarinic m1 agonist that stimulates non-amyloidogenic alpha-secretase processing in vitro. We compared cerebrospinal fluid (CSF) levels of A beta 40 and A beta 42 measured by ELISA before and at the end of 4 weeks of treatment with talsaclidine. The medication was administered in a double-blind, placebo-controlled, and randomized clinical study to 40 patients with AD. Talsaclidine (n = 34) decreased CSF levels of A beta 42 by a median of 19% (p < 0.001) as compared to baseline. The mean difference between CSF levels of A beta 42 before and after treatment with talsaclidine (n = 34) was -46 +/- 73 (SD) pg/ml as compared to 0 +/- 8 (SD) pg/ml with placebo (n = 6) (p < 0.05). CSF levels of A beta 40 increased during treatment with placebo (n = 6) while they remained stable during treatment with talsaclidine (n = 31) (1.118 +/- 1.710 ng/ml, and -0.170 +/- 0.967 ng/ml, respectively; p < 0.05). These data show that treatment with the m1 agonist talsaclidine reduced A beta peptides, and particularly A beta 42, in AD patients, suggesting it as a potential amyloid lowering therapy of AD.     

Urinary tetrahydroaldosterone as a screening method for primary aldosteronism: a comparative study

BACKGROUND: The major aldosterone metabolite 3 alpha,5 beta tetrahydroaldosterone reflects up to 45% of the aldosterone secretion. Its 24-h urinary excretion is likely to provide an accurate index of the daily aldosterone production and to be an indicator for primary aldosteronism (PA). METHODS: In a prospective study, the validity of tetrahydroaldosterone as a screening test for PA was evaluated in comparison to serum potassium, plasma aldosterone, plasma renin activity, plasma aldosterone/renin activity ratio (PARR), as well as 24-h urinary aldosterone-18-glucuronide and free aldosterone. A total of 111 normotensive individuals, 412 PA patients and 1453 essential hypertensive patients, were studied. The effect of blood sampling technique on potassium level was also investigated. RESULTS: Tetrahydroaldosterone differentiated PA from essential hypertension with a sensitivity of 96% and a specificity of 95%. The sensitivity was 89% for plasma aldosterone, 87% for free aldosterone, 85% for PARR, 71% for aldosterone-18-glucuronide and 51% for renin activity. Specificities varied between 91% and 85%. The combined use of the parameters plasma aldosterone > or =9.0 ng/dL and PARR > or =25 resulted in a sensitivity of 82% and specificity of 95%. Forearm exercise proved to be a source of erroneous elevations of potassium sufficient to obscure the suspicion of PA. CONCLUSION: The data suggest that tetrahydroaldosterone is the most reliable screening test for PA. Tetrahydroaldosterone determination in combination with aldosterone-18-glucuronide and free aldosterone increases diagnostic specificity for PA. Potassium, renin, plasma aldosterone, and basal PARR are inadequate screening procedures because they are subject to high rates of false-positive and false-negative results.     

Inhibition of Calpain Prevents N-Methyl-D-aspartate-Induced Degeneration of the Nucleus Basalis and Associated Behavioral Dysfunction

N-Methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity is thought to underlie a variety of neurological disorders, and inhibition of either the NMDA receptor itself, or molecules of the intracellular cascade, may attenuate neurodegeneration in these diseases. Calpain, a calcium-dependent cysteine protease, has been identified as part of such an NMDA receptor-induced excitotoxic signaling pathway. The present study addressed the question of whether inhibition of calpain can prevent neuronal cell death and associated behavioral deficits in a disease-relevant animal model, which is based on excitotoxic lesions of the cholinergic nucleus basalis magnocellularis of Meynert. Excitotoxic lesions of the nucleus basalis with NMDA induced a markedly impaired performance in the novel object recognition test. Treatment with the calpain inhibitor, N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethlyaminomethylphenyl) ethen-1-yl]benzamide (A-705253), dose-dependently prevented the behavioral deficit. Subsequent analysis of choline acetyltransferase in the cortical mantle of the lesioned animals revealed that application of A-705253 dose-dependently and significantly attenuated cholinergic neurodegeneration. Calpain inhibition also significantly diminished the accompanying gliosis, as determined by immunohistochemical analysis of microglia activation. Finally, inhibition of calpain by A-705253 and the peptidic calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO did not impair long-term potentiation in hippocampal slices, indicating that calpain inhibition interrupts NMDA excitotoxicity pathways without interfering with NMDA receptor-mediated signaling involved in cognition. We conclude that inhibition of calpains may represent a valuable strategy for the prevention of excitotoxicity-induced neuronal decline without interfering with the physiological neuronal functions associated with learning and memory processes. Thus, calpain inhibition may be a promising and novel approach for the treatment of various neurodegenerative disorders.     

Baseline [18F]-FDOPA kinetics are predictive of haloperidol-induced changes in dopamine turnover and cognitive performance: A positron emission tomography study in healthy subjects

The telencephalic dopamine innervations contribute to the modulation of cognitive processing. However, the relationship between cognitive effects of D(2/3)-receptor antagonism and dopamine transmission is not described in healthy subjects. We therefore tested effects of acute haloperidol (5 mg/d over 3 days) on continuous performance task (CPT) performance and 6-[(18)F]-fluoro-l-DOPA (FDOPA) PET parameters. Nine physically and mentally healthy male men performed two FDOPA-PET scans including arterial plasma withdrawal. Over 3 days before the second scan, all subjects were treated with 5 mg/d haloperidol orally. Using our novel steady-state analysis, we calculated the intrinsic rate of the cerebral FDOPA utilization (K), the turnover of [(18)F]fluorodopamine formed in brain (k(loss)) and the storage for FDOPA and its brain metabolites (V(d)). Furthermore, a ds-CPT and EPS-screening was performed before every PET scan. We found that FDOPA kinetics in those normal subjects with relatively high baseline K showed a more pronounced sensitivity to haloperidol treatment, manifesting in reduced storage capacity and elevated turnover of [(18)F]fluorodopamine, whereas subjects with lower K showed the opposite pattern of responses. Furthermore, low baseline K predicted improvements in the CPT task after haloperidol, whereas participants with higher baseline K showed a decline in cognitive performance. We conclude that the initial increase of [(18)F]fluorodopamine turnover after acute haloperidol challenge is associated with an over-stimulation in individuals with initially more pharmacologically responsive dopamine systems, but optimizes cognitive performance in those with lower normal FDOPA utilization at baseline. We hypothesize that these effects may be driven by D(1)-receptor mediated transmission during D(2) blockade.