Endothelin-1, Extracellular Volume Overload, and Hemodynamics in Hemodialysis Patients.

INTRODUCTION: Extracellular volume (ECV) overload and endothelial cell dysfunction (ECD) are independent risk factors for mortality in hemodialysis patients. Endothelin-1 (ET-1), an endothelium-derived vasoconstrictive peptide, is associated with poor outcomes in hemodialysis patients and heart failure patients without kidney disease. We hypothesized there would be associations between ET-1 and ECV overload assessed with bioimpedance spectroscopy (BIS) in hypertensive hemodialysis patients. METHODS: We obtained pre-HD plasma ET-1, ECV/weight (using multi-frequency BIS), pre-HD hemodynamic measurements, and ambulatory blood pressure (BP) in a cohort of HD patients. Following appropriate transformations, we conducted correlation and linear regression analyses to identify associations between ET-1 and ECV overload. We further explored associations between ET-1 and total peripheral resistance index (TPRI), cardiac index (CI), and ambulatory BP. RESULTS: Among 66 patients, median ET-1 was 1.93 (1.49-2.56) pg/mL. Median pre-HD ECV/weight, median TPRI, mean CI, and mean systolic ambulatory BP were 0.25 (0.22-0.30) L/kg, 3161 (2711-3642) dynes*sec/cm-5/m2, 2.92 (0.6) L/min/m2, and 143 (14) mmHg, respectively. After reciprocal transformation, ET-1 correlated with reciprocal-transformed ECV/weight (r=0.3, p=.01), log-transformed TPRI (r=-0.3, p=.006), CI (r=0.3, p=.009) and ambulatory BP (r=-0.3, p=.02). Controlling for demographic variables, these associations persisted in linear regression analysis (ß=0.15, p=.002; ß=-0.8, p=.002; ß=0.2, p=.002; ß=-19, p=.03). CONCLUSIONS: In hypertensive HD patients, ECV overload is associated with ET-1. ET-1 is associated with higher TPRI and lower CI before dialysis and higher ambulatory BP. Further research is necessary to determine if ECV reduction lowers ET-1 or if pharmacologic ET-1 antagonism can improve outcomes in HD patients with refractory ECV overload.

UCN2: a new candidate influencing pancreatic ß-cell adaptations in pregnancy.

The corticotropin-releasing hormone (CRH) family of peptides, including urocortin (UCN) 1, 2 and 3, are established hypothalamic neuroendocrine peptides, regulating the physiological and behaviour responses to stress indirectly, via the hypothalamic-pituitary-adrenal (HPA) axis. More recently, these peptides have been implicated in diverse roles in peripheral organs through direct signalling, including in placental and pancreatic islet physiology. CRH has been shown to stimulate insulin release through activation of its cognate receptors, CRH receptor 1 (CRHR1) and 2. However, the physiological significance of this is unknown. We have previously reported that during mouse pregnancy, expression of CRH peptides increase in mouse placenta suggesting that these peptides may play a role in various biological functions associated with pregnancy, particularly the pancreatic islet adaptations that occur in the pregnant state to compensate for the physiological increase in maternal insulin resistance. In the current study, we show that mouse pregnancy is associated with increased circulating levels of UCN2 and that when we pharmacologically block endogenous CRHR signalling in pregnant mice, impairment of glucose tolerance is observed. This effect on glucose tolerance was comparable to that displayed with specific CRHR2 blockade and not with specific CRHR1 blockade. No effects on insulin sensitivity or the proliferative capacity of ß-cells were detected. Thus, CRHR2 signalling appears to be involved in ß-cell adaptive responses to pregnancy in the mouse, with endogenous placental UCN2 being the likely signal mediating this.

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation.

The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.

Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization.

All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N-RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.

SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization.

Feline immunodeficiency virus (FIV) infects many species of cat, and is related to HIV, causing a similar pathology. High-throughput selective 2' hydroxyl acylation analysed by primer extension (SHAPE), a technique that allows structural interrogation at each nucleotide, was used to map the secondary structure of the FIV packaging signal RNA. Previous studies of this RNA showed four conserved stem-loops, extensive long-range interactions (LRIs) and a small, palindromic stem-loop (SL5) within the gag open reading frame (ORF) that may act as a dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Our analyses of wild-type (wt) and mutant RNAs suggest that although the four conserved stem-loops are static structures, the 5' and 3' regions previously shown to form LRI also adopt an alternative, yet similarly conserved conformation, in which the putative DIS is occluded, and which may thus favour translational and splicing functions over encapsidation. SHAPE and in vitro dimerization assays were used to examine SL5 mutants. Dimerization contacts appear to be made between palindromic loop sequences in SL5. As this stem-loop is located within the gag ORF, recognition of a dimeric RNA provides a possible mechanism for the specific packaging of genomic over spliced viral RNAs.

Is there neuroprotection in Parkinson syndrome?

Parkinson's disease (PD) is a neurodegenerative disorder of the aging population with unknown etiopathogenesis. It is assumed that the underlying pathobiochemical processes comprise multifactorial and multigenetic disturbances leading to a progressive and devastating disorder without remission. Subtypes exist suggesting that "PD" is a spectrum disorder with variations in the cascade of pathobiochemical and genetic events. Neuroprotective endogenous processes are lost at the very beginning of PD. Supplementation of substances with neuroprotective and/or neurorescue capacity is eminent for future therapeutic strategies. MAO-B inhibitors, NMDA-receptor antagonists and dopamine receptor agonists fulfill such a criterion in preclinical studies while there is no clear evidence for clinical neuroprotection. However, PET-controlled studies comparing L-DOPA-treated and ropinirol-treated PD patients give evidence for the "concept of neuroprotective treatment strategies".

Parkinson's disease: a major hypokinetic basal ganglia disorder.

Disorders of the basal ganglia can be broadly divided into two: ones that manifest hypokinesia or those that elicit hyperkinesia. Parkinson's disease is the commonest form of hypokinetic disorder. The term Parkinson's disease (PD) usually encompasses idiopathic and Parkinsonian-like syndromes. PD is a chronic and progressive disease, in which the symptoms tend to appear unilateral to begin with. Subsequently subtle dysfunctions on the contralateral side may also be observed. Tremor, akinesia and rigidity are some of the classical features of the disease. Although many hypotheses have been proposed, the cause of PD largely remains obscure. Management of PD includes pharmacological treatment or/and surgical intervention.

Isolated absence of the septum pellucidum.

Absence of the septum pellucidum in the human is a rare congenital anomaly. Previous reports suggest it is almost always associated with other brain anomalies. However, MRI in two patients with absence of the septum pellucidum presented here, indicates that this anomaly may occur without associated anomalies. It may be one manifestation of a spectrum of developmental anomalies. One patient presented with schizophrenic psychosis; developmental disturbances in limbic areas are believed to be associated with schizophrenia. Agenesis of the septum pellucidum may indicate abnormal development of limbic structures and it may be associated with anomalies, such as cytoarchitectural disturbances of cortical layers, as yet undetectable by MRI.

Dopamine D3 receptor gene polymorphism and alcohol dependence: relation to personality rating.

Hereditary dopaminergic mechanisms have been implicated in the aetiology of alcoholism. For this study, the distribution of a dopamine D3 receptor gene polymorphism (Ball) has been investigated in patients suffering from alcohol dependence, and compared with non-dependent controls. The allele A1 occurred significantly more frequently among patients compared to controls. Patients with the genotype A1/A2 showed significantly higher novelty seeking (NS) scores in the tridimensional personality questionnaire (TPQ) than patients with the genotype A1/A1. The distribution of patients with high and low NS scores in heterozygotes (A1/A2) did not follow a random distribution. There were significantly more individuals with higher NS scores, and fewer individuals with lower NS scores than expected. The results of this study support the hypothesis of a genetically determined involvement of the dopaminergic system in alcohol dependence. This is probably related to the modulation of personality traits. The observed effects are relatively small, but statistically significant. Thus, the genetics of the dopaminergic neurotransmitter system alone cannot explain the aetiopathogenesis of alcoholism.

Root resorption of first permanent molar by a supernumerary premolar.

A case is presented of root resorption of the lower right first permanent molar caused by a supernumerary premolar tooth. The first permanent molar was subsequently extracted.

Advanced glycation end products in neurodegeneration: more than early markers of oxidative stress?

Oxidative stress is believed to play a decisive role in the pathogenesis of Parkinson's disease (PD). In addition, Lewy bodies, densely crosslinked intracellular protein deposits formed from cytoskeletal components, accumulate in presymptomatic stages of the disease. Recent findings indicate that "advanced glycation end products" (AGEs) are the major structural crosslinkers that cause the transformation of soluble neurofilament proteins to insoluble Lewy bodies. AGE formation is increased under conditions of oxidative stress, such as early GSH depletion, that are evident in the substantia nigra of PD patients, and is inhibited by radical scavengers and thiol antioxidants. Because AGEs not only are markers of oxidative stress but are also active participants in cell signaling by activation of glial cells to produce superoxide and nitric oxide, they can be considered part of a vicious cycle, which finally leads to neuronal cell death in the substantia nigra in PD.

A novel technique for the isolation of Lewy bodies in brain.

Previously, immunohistochemical methods were primarily used to detect and provide indirect evidence on the composition of Lewy bodies, the pathological hallmark of Parkinson's disease. This was chiefly because there are very few procedures that describe the isolation of these structures. We report here a relatively simple method that we have developed for the exclusive isolation of Lewy bodies from brain tissue. The isolation of the Lewy bodies and subsequent evaluation of their components may furnish an insight into their role in the neurodegenerative mechanism(s) operating in the spectrum of Lewy body disorders.

Comparison of HPLC and enzymatic recycling assays for the measurement of oxidized glutathione in rat brain.

Glutathione (reduced, GSH and oxidized, GSSG) concentrations were analysed in rat cerebellar homogenate using high performance liquid chromatography with ultraviolet detection (HPLC-UV) or enzymatic recycling assays. GSSG levels found using the HPLC-UV assay were 200-fold higher than those obtained with the enzymatic recycling procedure. Reduction of synthetic GSSG by glutathione reductase showed total conversion to GSH as assessed by HPLC-UV analysis. In contrast, only approximately 50% of the HPLC peak for GSSG could be reduced by glutathione reductase. Increasing the period of incubation with glutathione reductase for longer than 15 min did not alter GSSG levels. These results suggest that another substance present in brain tissue is derivatized and eluted at the same time as GSSG using the HPLC-UV assay, thus contributing to the apparently high GSSG levels found employing this technique.

Alterations in glutathione levels in Parkinson's disease and other neurodegenerative disorders affecting basal ganglia.

Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were measured in various brain areas (substantia nigra, putamen, caudate nucleus, globus pallidus, and cerebral cortex) from patients dying with Parkinson's disease, progressive supranuclear palsy, multiple-system atrophy, and Huntington's disease and from control subjects with no neuropathological changes in substantia nigra. GSH levels were reduced in substantia nigra in Parkinson's disease patients (40% compared to control subjects) and GSSG levels were marginally (29%) but insignificantly elevated; there were no changes in other brain areas. The only significant change in multiple-system atrophy was an increase of GSH (196%) coupled with a reduction of GSSG (60%) in the globus pallidus. The only change in progressive supranuclear palsy was a reduced level of GSH in the caudate nucleus (51%). The only change in Huntington's disease was a reduction of GSSG in the caudate nucleus (50%). Despite profound nigral cell loss in the substantia nigra in Parkinson's disease, multiple-system atrophy, and progressive supranuclear palsy, the level of GSH in the substantia nigra was significantly reduced only in Parkinson's disease. This suggests that the change in GSH in Parkinson's disease is not solely due to nigral cell death, or entirely explained by drug therapy, for multiple-system atrophy patients were also treated with levodopa. The altered GSH/GSSG ratio in the substantia nigra in Parkinson's disease is consistent with the concept of oxidative stress as a major component in the pathogenesis of nigral cell death in Parkinson's disease.

Glutathione-related enzymes in brain in Parkinson's disease.

The activities of enzymes related to glutathione synthesis, degradation, and function were analyzed in various brain regions (cerebral cortex, caudate nucleus, putamen, globus pallidus, and substantia nigra) from patients dying with pathologically proven Parkinson's disease (PD) and multiple system atrophy (MSA), and from matched controls with no neurological disorder. The activity of the glutathione degradative enzyme, gamma-glutamyltranspeptidase, was selectively elevated in substantia nigra (SN) in PD. In contrast, the activity of the synthetic enzyme, gamma-glutamylcysteine synthetase, was unaltered in SN and other brain areas in PD. Similarly, glutathione peroxidase and glutathione transferase activities were unaltered in SN or in other brain regions in PD. gamma-Glutamylcysteine synthetase, gamma-glutamyltranspeptidase, glutathione peroxidase, and glutathione transferase activities were normal in SN and most other brain areas in MSA. However, glutathione peroxidase activity was increased in the lateral globus pallidus and caudate nucleus in MSA. The depletion of reduced glutathione (GSH) in the SN in PD, with no change in oxidized glutathione (GSSG), may be due to efflux of GSH mainly out of glia promoted by gamma-glutamyltranspeptidase, perhaps with additional increased conversion of GSH to GSSG (which itself is transported out of cells by gamma-glutamyltranspeptidase), in response to increased hydrogen peroxide formation.

Indices of oxidative stress and mitochondrial function in individuals with incidental Lewy body disease.

Brain tissue from normal individuals with incidental Lewy bodies and cell loss in pigmented substantia nigra neurons (asymptomatic Parkinson's disease) and age-matched control subjects without nigral Lewy bodies was examined biochemically. There was no difference in dopamine levels or dopamine turnover in the caudate and putamen of individuals with incidental Lewy body disease compared to control subjects. There were no differences in levels of iron, copper, manganese, or zinc in the substantia nigra or other brain regions from the individuals with incidental Lewy body disease compared to those from control subjects. Similarly, ferritin levels in the substantia nigra and other brain areas were unaltered. There was no difference in the activity of succinate cytochrome c reductase (complexes II and III) or cytochrome oxidase (complex IV) between incidental Lewy body subjects and control subjects. Rotenone-sensitive NADH coenzyme Q1 reductase activity (complex I) was reduced to levels intermediate between those in control subjects and those in patients with overt Parkinson's disease, but this change did not reach statistical significance. The levels of reduced glutathione in substantia nigra were reduced by 35% in patients with incidental Lewy body disease compared to control subjects. Reduced glutathione levels in other brain regions were unaffected and there were no changes in oxidized glutathione levels in any brain region. Altered iron metabolism is not detectable in the early stages of nigral dopamine cell degeneration. There may be some impairment of mitochondrial complex I activity in the substantia nigra in Parkinson's disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative stress as a cause of nigral cell death in Parkinson's disease and incidental Lewy body disease. The Royal Kings and Queens Parkinson's Disease Research Group.

We examine the evidence for free radical involvement and oxidative stress in the pathological process underlying Parkinson's disease, from postmortem brain tissue. The concept of free radical involvement is supported by enhanced basal lipid peroxidation in substantia nigra in patients with Parkinson's disease, demonstrated by increased levels of malondialdehyde and lipid hydroperoxides. The activity of many of the protective mechanisms against oxidative stress does not seem to be significantly altered in the nigra in Parkinson's disease. Thus, activities of catalase and glutathione peroxidase are more or less unchanged, as are concentrations of vitamin C and vitamin E. The activity of mitochondrial superoxide dismutase and the levels of the antioxidant ion zinc are, however, increased, which may reflect oxidative stress in substantia nigra. Levels of reduced glutathione are decreased in nigra in Parkinson's disease; this decrease does not occur in other brain areas or in other neurodegenerative illnesses affecting this brain region (i.e., multiple system atrophy, progressive supranuclear palsy). Altered glutathione metabolism may prevent inactivation of hydrogen peroxide and enhance formation of toxic hydroxyl radicals. In brain material from patients with incidental Lewy body disease (presymptomatic Parkinson's disease), there is no evidence for alterations in iron metabolism and no significant change in mitochondrial complex I function. The levels of reduced glutathione in substantia nigra, however, are reduced to the same extent as in advanced Parkinson's disease. These data suggest that changes in glutathione function are an early component of the pathological process of Parkinson's disease.(ABSTRACT TRUNCATED AT 250 WORDS)