Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Effect of myostatin depletion on weight gain, hyperglycemia, and hepatic steatosis during five months of high-fat feeding in mice

The marked hypermuscularity in mice with constitutive myostatin deficiency reduces fat accumulation and hyperglycemia induced by high-fat feeding, but it is unclear whether the smaller increase in muscle mass caused by postdevelopmental loss of myostatin activity has beneficial metabolic effects during high-fat feeding. We therefore examined how postdevelopmental myostatin knockout influenced effects of high-fat feeding. Male mice with ubiquitous expression of tamoxifen-inducible Cre recombinase were fed tamoxifen for 2 weeks at 4 months of age. This depleted myostatin in mice with floxed myostatin genes, but not in control mice with normal myostatin genes. Some mice were fed a high-fat diet (60% of energy) for 22 weeks, starting 2 weeks after cessation of tamoxifen feeding. Myostatin depletion increased skeletal muscle mass ～30%. Hypermuscular mice had ～50% less weight gain than control mice over the first 8 weeks of high-fat feeding. During the subsequent 3 months of high-fat feeding, additional weight gain was similar in control and myostatin-deficient mice. After 5 months of high-fat feeding, the mass of epididymal and retroperitoneal fat pads was similar in control and myostatin-deficient mice even though myostatin depletion reduced the weight gain attributable to the high-fat diet (mean weight with high-fat diet minus mean weight with low-fat diet: 19.9 g in control mice, 14.1 g in myostatin-deficient mice). Myostatin depletion did not alter fasting blood glucose levels after 3 or 5 months of high-fat feeding, but reduced glucose levels measured 90 min after intraperitoneal glucose injection. Myostatin depletion also attenuated hepatic steatosis and accumulation of fat in muscle tissue. We conclude that blocking myostatin signaling after maturity can attenuate some of the adverse effects of a high-fat diet.     

Phytoalexin synthesis in soybean cells: elicitor induction of reductase involved in biosynthesis of 6'-deoxychalcone

Chromatofocusing on Mono P proved to be an efficient purification procedure for the NADPH-dependent reductase from soybean (Glycine max L.) cell cultures which acts together with chalcone synthase in the biosynthesis of 2',4',4-trihydroxychalcone (6'-deoxychalcone). By isoelectric focusing the pI of reductase was determined to be 6.3. Addition of pure soybean reductase to cell-free extracts from stimulated cell cultures of parsley and bean (Phaseolus vulgaris) and from young flowers of Dahlia variabilis caused in each case synthesis of 6'-deoxychalcone. When 4-coumaroyl-CoA was replaced by caffeoyl-CoA in the reductase assay, formation of 2',4',3,4-tetrahydrochalcone (butein) was observed. A polyclonal antireductase antiserum was raised in rabbits and proved to be specific in Ouchterlony diffusion experiments, Western blots and immunotitration. The reductase antiserum showed no cross-reactivity with soybean chalcone synthase (CHS). A biotin/[125I]streptavidin system provided a quantitative Western blot for the reductase. Changes in the activities, amounts of protein, and mRNA activities of reductase and CHS were determined after challenge of soybean cell cultures by elicitor (from Phytophthora megasperma f.sp. glycinea or yeast). For both enzymes a pronounced and parallel increase in activity and amounts of protein was observed after elicitor addition with a maximum at about 16 h after challenge. Parallel increases in mRNA activities occurred earlier. The results indicate a parallel induction of de novo synthesis of reductase and CHS which coact in synthesis of 6'-deoxychalcone.     

Insecticidal activity of substituted phenyl N-methylcarbamates

The insecticidal and the acaricidal activities of a number of substituted phenyl N-methylcarbamates have been determined on the housefly (Musca domestica), the black bean aphid (Aphis fabae), the Colorado potato beetle (Leptinotarsa decemlineata), the cabbage worm (Pieris brassicae) and the carmine spider mite (Tetranychus cinnabarinus).It is demonstrated that the thesis of Kolbezen, Metcalf & Fukuto (1954), Metcalf, Fukuto & Winton (1962) and Kohn, Ospenson & Moore (1965), that the meta-isomers of alkylphenyl N-methylcarbamates are the most active, has to be restricted to some insect groups (e.g. flies and caterpillars). In the case of the Colorado potato beetle the o- and m-isomers were equally active; for aphids the o-isomer was the most toxic one.This fact as well as the different responses of the test insects if the compounds are further alkylated indicate that various carbamates exhibit more or less selective activities. Most striking was the high level of activity in the new group of p-dimethylaminomethylphenyl N-methylcarbamates on most of the insects, in combination with a complete lack of toxicity to houseflies.As previously pointed out by Kolbezen et al. (1954) and Metcalf et al. (1962), it was found that lengthening of the N-methyl group or N,N-dialkylation resulted in loss of insecticidal activity.The most active dimethylaminophenyl compounds were those with a p-dimethylamino group in combination with alkyl substituents in the 2,5- and 3,5-positions. Several p-dimethylaminomethylphenyl N-methylcarbamates with two or three alkyl substituents (except the 2,6-combination) proved to be highly active, except on flies, to which they were virtually nontoxic. Greatest broad-spectrum activity was shown by 2,3-dimethyl-4-dimethylaminomethylphenyl N-methylcarbamate. It is demonstrated that by introducing a p-dimethylamino- or a p-dimethylaminomethyl group in alkylphenyl N-methylcarbamates a considerable gain in anticholinesterase and insecticidal activity is obtained.

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Résumé p L'activité insecticide et acaricide de plusieurs N-méthylcarbamates de phényle substitués a été examinée sur la mouche domestique (Musca domestica), le puceron noir (Aphis fabae), le doryphore (Leptinotarsa decemlineata), la chenille de la piéride du chou (Pieris brassicae) et l'araignée rouge des serres (Tetranychus cinnabarinus). Les N-méthylcarbamates de phényle monosubstitués ne présentent qu'une faible activité acaricide. Les substances avec des substitutions en position méta ou ortho montrent une meilleure activité insecticide que celles avec la substitution en position para. Pour les mouches domestiques l'alcoylation en position méta se trouve donner les composés les plus actifs; pour les pucerons l'alcoylation en position ortho était la plus efficace et pour les doryphores les combinaisons o- et m-étaient d'une activité égale. Les isomères ortho des dérivés alcoxylés étaient plus efficaces. En cas de substitution par un groupe diméthylamino il n'y a pas de différence importante quant à l'activité insecticide entre les positions ortho ou méta. En général il en est de même pour les substitutions par diméthylaminométhyle, bien que le m-isomère soit le plus efficace sur les chenilles.L'introduction d'un deuxième groupe alcoyle dans un N-méthylcarbamate de o- ou m-isopropylphényle change le spectre d'action. Un groupe 5-méthyle diminue beaucoup l'activité de l'o-isomère. Un groupe 5-isopropyle diminue l'activité sur les pucerons, mais pas sur les mouches. Quand un groupe 5-méthyle ou 5-isopropyle est introduit dans un N-méthylcarbamate de m-isopropyl-phényle l'activité est généralement augmentée. L'introduction d'un groupe 6-méthyle diminue toutefois l'activité sur les mouches, les pucerons et les chenilles, mais pas sur les Coléoptères et les araignées rouges.Les N-méthylcarbamates de phényle s'avèrent posséder une meilleure activité que les combinaisons N,N-diméthylcarbamates correspondantes. Une prolongation du groupe N-alcoyle diminue également l'activité.Les dérivés de N-méthylcarbamates de 2-diméthylamino-phényle ont une plus grande activité que les isomères méta correspondants. On trouve toutefois la meilleure activité dans les N-méthylcarbamates de 4-diméthylamino-phényle alcoylés. Tout groupe alcoyle, à condition d'être introduit en position 2-, 3-, ou 5-augmente l'activité insecticide. Les composés métasubstitués semblent encore un peu plus actifs que les dérivés d'o-alcoyle. La grande activité sur les pucerons contraste nettement avec la faible activité sur les mouches et les araignées rouges. Le seul dérivé présentant une bonne activité sur les araignées rouges est le N-méthylcarbamate de 3-isopropyl-4-diméthylamino-5-méthylphényle.Les N-méthylcarbamates de p-diméthylaminométhyl-phényle alcoylés présentent également une forte action insecticide, sauf sur les mouches. Le N-méthylcarbamate de 2, 3-diméthyl-4-diméthylaminométhyl-phényle présente la plus grande activité et le plus large spectre d'action.A partir des N-méthylcarbamates d'alcoylphényle l'introduction d'un groupe p-diméthylamino ou p-diméthylamino-méthyle augmente considérablement l'activité anticholinestérasique aussi bien que l'activité insecticide.

     

Neural networks in intestinal immunoregulation

Key physiological functions of the intestine are governed by nerves and neurotransmitters. This complex control relies on two neuronal systems: an extrinsic innervation supplied by the two branches of the autonomic nervous system and an intrinsic innervation provided by the enteric nervous system. As a result of constant exposure to commensal and pathogenic microflora, the intestine developed a tightly regulated immune system. In this review, we cover the current knowledge on the interactions between the gut innervation and the intestinal immune system. The relations between extrinsic and intrinsic neuronal inputs are highlighted with regards to the intestinal immune response. Moreover, we discuss the latest findings on mechanisms underlying inflammatory neural reflexes and examine their relevance in the context of the intestinal inflammation. Finally, we discuss some of the recent data on the identification of the gut microbiota as an emerging player influencing the brain function.     

Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses

Background

A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E₂) are distinct from those in non-E₂-responsive cells.

Methods

FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E₂ proliferative H1793 and non-E₂-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.

Results

LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E₂ or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.

Conclusion

Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.

     

Prevention of murine cryoglobulinemia and associated pathology by monoclonal anti-idiotypic antibody

A murine IgG3 mAb, clone 6-19, derived from non-manipulated autoimmune MRL/MpJ-lpr/lpr mice is a rheumatoid factor specific for IgG2a and is able to generate cryoglobulins via nonspecific IgG3 Fc-Fc interaction. Intraperitoneal passive transfer of ascites containing the 6-19 mAb into BALB/c mice induces, within 18 h, remarkable pathology characterized by skin vasculitis and acute glomerulonephritis associated with cryoglobulinemia. In order to evaluate the possibility of modulating the development of tissue lesions by an anti-Id antibody, we have raised an IgG2b anti-Id mAb specific to the 6-19 mAb. The cryoprecipitation of 6-19 mAb was completely inhibited in the presence of excess amounts of anti-Id mAb in vitro. In vivo, pretreatment of BALB/c mice with anti-6-19 anti-Id mAb inhibited development of skin vasculitis and glomerulonephritis induced by the 6-19 mAb. The cryoglobulin formation was markedly diminished due to enhanced elimination of the 6-19 mAb from the circulation. In contrast, pretreatment with an IgM anti-IgG3 rheumatoid factor mAb neither protected nor aggravated the development of tissue lesions. These results suggest possible implications in the anti-Id treatment of similar vascular diseases in man.     

Lung tissue extracellular matrix-derived hydrogels protect against radiation-induced lung injury by suppressing epithelial–mesenchymal transition

This study aimed to examine whether lung tissue extracellular matrix (ECM) hydrogels have protective effects on radiation-induced lung injury (RILI). The cytocompatibility and histocompatibility were tested for the obtained ECM-derived hydrogel. Sprague–Dawley rats were randomly divided into three groups (n = 18): control group (control); rats receiving irradiation and intratracheal injection of normal saline (IR + NS); and rats receiving irradiation and intratracheal injection of lung ECM-derived hydrogel (IR + ECM). The wet/dry weight ratio was used to evaluate the congestion and edema of the lungs. Histopathological analysis of lung tissues was performed using hemotoxylin and eosin staining and Masson's trichrome staining. Immunohistochemical staining and western blot analyses were carried out to determine the expression of epithelial–mesenchymal transition (EMT)-related proteins in lung tissues (E-cadherin, α-smooth muscle actin [α-SMA], and vimentin). In addition, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6), hydroxyproline, malondialdehyde (MDA), and superoxide dismutase (SOD) levels were also evaluated. The ECM-derived hydrogels had good cytocompatibility and histocompatibility. ECM-derived hydrogel treatment improved lung histopathology injury and pulmonary edema. Higher expression of E-cadherin and lower expression of vimentin and α-SMA were found in the IR + ECM group compared with those in the IR + NS group. Hydroxyproline levels were reduced by ECM-derived hydrogel treatment compared with those in the IR + NS group. Obvious increases of TNF-α, IL-6, and TGF-β1 were identified following irradiation. Marked reductions in MDA content and increases in SOD were induced by ECM-derived hydrogel treatment in rats after radiation. ECM-derived hydrogels were shown to protect against RILI, potentially by reducing EMT, inflammation, and oxidative damage.