Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF''s neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF''s effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD.Glial cell line-derived neurotrophic factor (GDNF) is the founding member of the four ligands in the GDNF family, which belong to the transforming growth factor-β superfamily.¹ GDNF was characterized as a potent survival factor for many neurons in culture such as dopaminergic, motor, sympathetic, parasympathetic, sensory and enteric neurons.^{1, 2} In addition, in dopaminergic neuron cultures GDNF stimulates neuronal differentiation, neurite outgrowth, synapse formation and dopamine release.^{1, 2}As degeneration of midbrain dopaminergic neurons in the substantia nigra pars compacta (SNpc) represents a major hallmark of Parkinson disease (PD), the most common neurodegenerative movement disorder, GDNF has raised considerable interest as a therapeutic molecule for the treatment of PD.^{3, 4, 5} PD affects >2% of individuals over the age of 60 years, but no curative treatment is available to date, mainly due to a lack of understanding disease etiology.^{6, 7, 8} Preclinical studies in the established 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) rodent and primate models of PD demonstrated a substantial neuroprotection and regeneration effect by striatal provided GDNF or its close relative neurturin.^{3, 4, 9} However, clinical phase II trials on PD patients using GDNF or neurturin did so far not convincingly recapitulate their beneficial effects on the dopaminergic system in humans most likely due to technical problems and the selection of advanced PD patients.^{10, 11, 12, 13}GDNF signaling is highly complex as this neurotrophic factor can bind to a variety of receptors, thus being able to induce pleiotropic effects. GDNF efficiently binds to the GPI-linked GDNF family receptor α1 (GFRα1).^{1, 2} It has been shown that the GDNF/GFRα1 complex can activate not only the canonical GDNF receptor Ret, a receptor tyrosine kinase which signals through the sarcoma protein (Src)/rat sarcoma (Ras)/mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt, NF-κB (nuclear factor ''kappa-light-chain-enhancer'' of activated B cells), JNK (c-Jun N-terminal kinases) and PLCγ (phospholipase γ) pathway, but also with other signaling inducing receptors.^{1, 2, 4, 5, 13} So far, at least four alternative GDNF receptors have been described which are all expressed in midbrain dopaminergic neurons, NCAM,^{14, 15} the integrins αV and βI,^{14, 16} syndecan 3¹⁷ and N-cadherin.¹⁸ Interestingly, Ret is not essential during pre- and postnatal development of the mouse dopaminergic system,^{19, 20, 21, 22, 23} but specifically required for the maintenance of SNpc dopaminergic neurons and their striatal innervation in aged mice.^{23, 24, 25} In contrast, GDNF seems most likely under physiological conditions to be dispensable during development and maintenance of midbrain dopaminergic neurons in mice, although conflicting results exist.^{26, 27, 28} Thus, Ret might be activated by a GDNF-independent mechanism to stimulate SNpc dopaminergic neuron survival. In addition, the in vivo function of the alternative GDNF receptors in the dopaminergic system under physiological and pathophysiological conditions, like PD, and their dependence on GDNF has not yet been addressed in detail. This raised the important question which GDNF receptor might be required to mediate GDNF''s reported neuroprotective and regenerative effect in the dopaminergic system in PD animal models and potentially in PD patients.^{5, 29}Previously, we showed in dopaminergic neuron-specific Ret knockout mice that Ret receptor loss does not result in a higher vulnerability of midbrain dopaminergic neurons against MPTP but to less resprouting of left over dopaminergic neuron axons in the striatum after MPTP intoxication.³⁰ In adult mice endogenous GDNF levels are rather low.^{26, 31} Therefore, we could not rule out in that study the possibility, that higher levels of GDNF—as also used in the clinical GDNF trials in PD patients—might have neuroprotective and regenerating effects even in the absence of the Ret receptor. Here we addressed now this question by viral overexpression of GDNF in MPTP-treated mice lacking expression of Ret again specifically in dopaminergic neurons.^{23, 30} We found that in the absence of Ret in dopaminergic neurons even a substantial overexpression of GDNF in the striatum does not have a neuroprotective and regenerative effect. Thus, despite the expression of alternative GDNF receptors on midbrain dopaminergic neurons, the presence of the canonical GDNF receptor Ret seems to be mandatory for mediating GDNF''s beneficial survival and axonal resprouting effect in these neurons.     

Ethacrynic acid analogues lacking the α,β-unsaturated carbonyl unit—Potential anti-metastatic drugs

A series of ethacrynic acid analogues, lacking the α,β-unsaturated carbonyl unit, was synthesized and subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, MCF-7/AZ. Several of the analogues were already active in the low micromolar range, whereas ethacrynic acid itself shows no potential to inhibit the migration of these cancer cells. Preliminary studies show that the presence of one or more methoxy groups at the phenyl ring of ethacrynic acid is important in order for the ethacrynic acid analogues to demonstrate an inhibitory effect on the migration.     

PEGylated liposomes of anastrozole for long-term treatment of breast cancer: in vitro and in vivo evaluation

The aim of present study was to develop conventional and PEGylated (long circulating), liposomes containing anastrozole (ANS) for effective treatment of breast cancer. ANS is a third-generation non-steroidal aromatase inhibitor of the triazole class used for the treatment of advanced and late-stage breast cancer in post-menopausal women. Under such disease conditions the median duration of therapy should be prolonged until tumor regression ends (>31 months). Liposomes were prepared by the thin film hydration method by using ANS and various lipids such as soyaphosphatidyl choline, cholesterol and methoxy polyethylene glycol distearoyl ethanolamine in different concentration ratios and evaluated for physical characteristics, in vitro drug release and stability. Optimized formulations of liposome were studied for in vitro cytotoxic activity against the BT-549 and MCF-7 cell lines and in vivo behavior in Wistar rats. Preformulation studies, both Fourier transform infrared study and differential scanning calorimetry analysis showed no interaction between the drug and the excipients used in the formulations. The optimized formulations AL-07 and AL-09 liposomes showed encapsulation efficiencies in the range 65.12?±?1.05% to 69.85?±?3.2% with desired mean particle size distribution of 101.1?±?5.9 and 120.2?±?2.8?nm and zeta potentials of ?43.7?±?4.7 and ?62.9?±?3.5 mV. All the optimized formulations followed Higuchi-matrix release kinetics and when plotted in accordance with the Korsemeyer–Peppas method, the n-value 0.5?n?in vitro cytotoxicity studies (p?(0–∞) values when compared to pure drug (p?相似文献   

Austro-Asiatic tribes of Northeast India provide hitherto missing genetic link between South and Southeast Asia

Northeast India, the only region which currently forms a land bridge between the Indian subcontinent and Southeast Asia, has been proposed as an important corridor for the initial peopling of East Asia. Given that the Austro-Asiatic linguistic family is considered to be the oldest and spoken by certain tribes in India, Northeast India and entire Southeast Asia, we expect that populations of this family from Northeast India should provide the signatures of genetic link between Indian and Southeast Asian populations. In order to test this hypothesis, we analyzed mtDNA and Y-Chromosome SNP and STR data of the eight groups of the Austro-Asiatic Khasi from Northeast India and the neighboring Garo and compared with that of other relevant Asian populations. The results suggest that the Austro-Asiatic Khasi tribes of Northeast India represent a genetic continuity between the populations of South and Southeast Asia, thereby advocating that northeast India could have been a major corridor for the movement of populations from India to East/Southeast Asia.     

Hexameric ring structure of the full-length archaeal MCM protein complex

In eukaryotes, a family of six homologous minichromosome maintenance (MCM) proteins has a key function in ensuring that DNA replication occurs only once before cell division. Whereas all eukaryotes have six paralogues, in some Archaea a single protein forms a homomeric assembly. The complex is likely to function as a helicase during DNA replication. We have used electron microscopy to obtain a three-dimensional reconstruction of the full-length MCM from Methanobacterium thermoautotrophicum. Six monomers are arranged around a sixfold axis, generating a ring-shaped molecule with a large central cavity and lateral holes. The channel running through the molecule can easily accommodate double-stranded DNA. The crystal structure of the amino-terminal fragment of MCM and a model for the AAA+ hexamer have been docked into the map, whereas additional electron density suggests that the carboxy-terminal domain is located at the interface between the two domains. The structure suggests that the MCM complex is likely to act in a different manner to traditional hexameric helicases and is likely to bear more similarity to the SV40 large T antigen or to double-stranded DNA translocases.     

Responses of maize grain yield to changes in acid soil characteristics after soil amendments

An experiment was conducted from 1997 to 2000 on an acid soil in Cameroon to assess the effectiveness of cultivating acid tolerant maize (Zea mays L.) cultivar and the use of organic and inorganic fertilizers as options for the management of soil acidity. The factors investigated were: phosphorus (0 and 60 kg ha^?1), dolomitic lime (0 and 2 t ha^?1), organic manure (no manure, 4 t ha^?1 poultry manure, and 4 t ha^?1 of leaves of Senna spectabilis), and maize cultivars (ATP-SR-Y – an acid soil-tolerant, and Tuxpeño sequia – an acid susceptible). On acid soil, maize grain yield of ATP-SR-Y was 61% higher than the grain yield of Tuxpeño sequia. Continuous maize cultivation on acid soil further increased soil acidity, which was manifested by a decrease in pH (0.23 unit), exchangeable Ca (31%) and Mg (36%) and by an increase in exchangeable Al (20%). Yearly application of 60 kg ha^?1 of P for 3 years increased soil acidity through increases in exchangeable Al (8%) and H (16%) and a decrease in exchangeable Ca (30%), Mg (11%) and pH (0.07 unit). Lime application increased grain yield of the tolerant (82%) and susceptible (208%) cultivars. The grain yield increases were associated with a mean decrease of 43% in exchangeable Al, and 51% in H, a mean increase of 0.27 unit in pH, 5% in CEC, 154% in exchangeable Ca, and 481% in Mg contents of the soil. Poultry manure was more efficient than leaves of Senna producing 38% higher grain yield. This yield was associated with increases in pH, Ca, Mg and P, and a decrease in Al. The highest mean grain yields were obtained with lime added to poultry manure (4.70 t ha^?1) or leaves of Senna (4.72 t ha^?1). Grain yield increase was more related to the decrease in exchangeable Al (r = ?0.86 to ?0.95, P<0.01) and increase in Ca (r = 0.78–0.94, P<0.01), than to pH (r = ?0.57 (non-significant) to ?0.58 (P<0.05)). Exchangeable Al was the main factor determining pH (r = ?0.88 to ?0.92, P<0.01). The yield advantage of the acid tolerant cultivar was evident even after correcting for soil acidity. Acid soil-tolerant cultivars are capable of bringing unproductive acid soils into cultivation on the short run. The integration of soil amendments together with acid soil-tolerant cultivar offers a sustainable and comprehensive strategy for the management of acid soils in the tropics.     

Chitosan and Enteric Polymer Based Once Daily Sustained Release Tablets of Aceclofenac: <Emphasis Type="BoldItalic">In Vitro</Emphasis> and <Emphasis Type="BoldItalic">In Vivo</Emphasis> Studies

The purpose of this study was to develop a once daily sustained release tablet of aceclofenac using chitosan and an enteric coating polymer (hydroxypropyl methylcellulose phthalate or cellulose acetate phthalate). Overall sustained release for 24 h was achieved by preparing a double-layer tablet in which the immediate release layer was formulated for a prompt release of the drug and the sustained release layer was designed to achieve a prolonged release of drug. The preformulation studies like IR spectroscopic and differential scanning calorimetry showed the absence of drug–excipient interactions. The tablets were found within the permissible limits for various physicochemical parameters. Scanning electron microscopy was used to visualize the surface morphology of the tablets and to confirm drug release mechanisms. Good equivalence in the drug release profile was observed when drug release pattern of the tablet containing chitosan and hydroxypropyl methylcellulose phthalate (M-7) was compared with that of marketed tablet. The optimized tablets were stable at accelerated storage conditions for 6 months with respect to drug content and physical appearance. The results of pharmacokinetic studies in human volunteers showed that the optimized tablet (M-7) exhibited no difference in the in vivo drug release in comparison with marketed tablet. No significant difference between the values of pharmacokinetic parameters of M-7 and marketed tablets was observed (p > 0.05; 95% confidence intervals). However the clinical studies in large scale and, long term and extensive stability studies at different conditions are required to confirm these results.     

Role of androgen receptor CAG repeat polymorphism and X-inactivation in the manifestation of recurrent spontaneous abortions in Indian women

The aim of the present study was to investigate the role of CAG repeat polymorphism and X-chromosome Inactivation (XCI) pattern in Recurrent Spontaneous Abortions among Indian women which has not been hitherto explored. 117 RSA cases and 224 Controls were included in the study. Cases were recruited from two different hospitals - Lakshmi Fertility Clinic, Nellore and Fernandez Maternity Hospital, Hyderabad. Controls were roughly matched for age, ethnicity and socioeconomic status. The CAG repeats of the Androgen Receptor gene were genotyped using a PCR-based assay and were analysed using the GeneMapper software to determine the CAG repeat length. XCI analysis was also carried out to assess the inactivation percentages. RSA cases had a significantly greater frequency of allele sizes in the polymorphic range above 19 repeats (p = 0.006), which is the median value of the controls, and in the biallelic mean range above 21 repeats (p = 0.002). We found no evidence of abnormal incidence of skewed X-inactivation. We conclude that longer CAG repeat lengths are associated with increased odds for RSA with statistical power estimated to be ∼90%.     

Preparation of a Matrix Type Multiple-Unit Gastro Retentive Floating Drug Delivery System for Captopril Based on Gas Formation Technique: In Vitro Evaluation

A gastro retentive floating drug delivery system with multiple-unit minitab’s based on gas formation technique was developed in order to prolong the gastric residence time and to increase the overall bioavailability of the drug. The system consists of the drug-containing core units prepared by direct compression process, which are coated with three successive layers of an inner seal coat, effervescent layer (sodium bicarbonate) and an outer gas-entrapped polymeric membrane of an polymethacrylates (Eudragit RL30D, RS30D, and combinations of them). Only the system using Eudragit RL30D and combination of them as a gas-entrapped polymeric membrane could float. The time to float decreased as amount of the effervescent agent increased and coating level of gas-entrapped polymeric membrane decreased. The optimum system floated completely within 3 min and maintained the buoyancy over a period of 12 h. The drug release was controlled and linear with the square root of time. Increasing coating level of gas-entrapped polymeric membrane decreased the drug release. Both the rapid floating and the controlled release properties were achieved in the multiple-unit floating drug delivery system developed in this present study. The analysis of the parameter dissolution data after storage at 40 °C and 75% RH for 3 months showed, no significant change indicating the two dissolution profiles were considered to be similar (f2 value is more than 50).