Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species: comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes

Proteomics is a valuable tool for establishing and comparing the protein content of defined tissues, cell types, or subcellular structures. Its use in non-model species is currently limited because the identification of peptides critically depends on sequence databases. In this study, we explored the potential of a preliminary cDNA database for the non-model species Pisum sativum created by a small number of massively parallel pyrosequencing (MPSS) runs for its use in proteomics and compared it to comprehensive cDNA databases from Medicago truncatula and Arabidopsis thaliana created by Sanger sequencing. Each database was used to identify proteins from a pea leaf chloroplast envelope preparation. It is shown that the pea database identified more proteins with higher accuracy, although the sequence quality was low and the sequence contigs were short compared to databases from model species. Although the number of identified proteins in non-species-specific databases could potentially be increased by lowering the threshold for successful protein identifications, this strategy markedly increases the number of wrongly identified proteins. The identification rate with non-species-specific databases correlated with spectral abundance but not with the predicted membrane helix content, and strong conservation is necessary but not sufficient for protein identification with a non-species-specific database. It is concluded that massively parallel sequencing of cDNAs substantially increases the power of proteomics in non-model species.     

Isosteric ramatroban analogs: selective and potent CRTH-2 antagonists

The chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH-2), also found on eosinophils and basophils, is a prostaglandin D2 receptor involved in the recruitment of these cell types during an inflammatory response. In this report, we describe the synthesis and optimization of a ramatroban isostere that is a selective and potent antagonist of CRTH-2 which may be useful in the treatment of certain diseases.     

Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.     

Role of MAPK/ERK in Neurotrophin-4 Potentiation of Necrotic Neuronal Death

Neurotrophic factors have been proposed for the treatment of a variety of neurological diseases. However, to this point they have failed in clinical trials. One potential problem is that while neurotrophic factors attenuate apoptosis, they have the potential to enhance necrosis. In this study we show that neurotrophin-4 (NT-4) attenuated apoptotic neuronal death while potentiating necrotic neuronal death in cortical cultures. The protective effects of NT-4 were not blocked by the mitogen-activated protein kinase kinase (MEK) inhibitors PD098059 or U0126, while the injury potentiation by NT-4 was blocked by these inhibitors. NT-4 stimulated the phosphorylation of ERK1/2 and this phosphorylation was attenuated by U0126 and PD098059. The results indicate a disassociation between the pathway by which NT-4 potentiates necrosis, and that by which it attenuates apoptosis, and suggest that addition of a MEK inhibitor may enhance the beneficial effects of NT-4 in treating complex injuries such as occur in vivo.     

The alpha-L-iduronidase mutations R89Q and R89W result in an attenuated mucopolysaccharidosis type I clinical presentation

Mucopolysaccharidosis type I (MPS I; McKusick 25280; Hurler syndrome, Hurler-Scheie syndrome and Scheie syndrome) is caused by a deficiency in the lysosomal hydrolase, alpha-L-iduronidase (EC 3.2.1.76). MPS I patients present within a clinical spectrum bounded by the extremes of Hurler and Scheie syndromes. The alpha-L-iduronidase missense mutations R89Q and R89W were investigated and altered an important arginine residue proposed to be a nucleophile activator in the catalytic mechanism of alpha-L-iduronidase. The R89Q alpha-L-iduronidase mutation was shown to result in a reduced level of alpha-L-iduronidase protein (< or =10% of normal control) compared to a normal control level of alpha-L-iduronidase protein that was detected for the R89W alpha-L-iduronidase mutation. When taking into account alpha-L-iduronidase specific activity, the R89W mutation had a greater effect on alpha-L-iduronidase activity than the R89Q mutation. However, overall the R89W mutation produced more residual alpha-L-iduronidase activity than the R89Q mutation. This was consistent with MPS I patients, with an R89W allele, having a less severe clinical presentation compared to MPS I patients with either a double or single allelic R89Q mutation. The effects of the R89Q and R89W mutations on enzyme activity supported the proposed role of R89 as a nucleophile activator in the catalytic mechanism of alpha-L-iduronidase.     

Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate

During embryonic development, organs arise along the gut tube as a series of buds in a stereotyped anterior-posterior (A-P) pattern. Using chick-quail chimeras and in vitro tissue recombination, we studied the interactions governing the induction and maintenance of endodermal organ identify focusing on the pancreas. Though several permissive signals in pancreatic development have been previously identified, here we provide evidence that lateral plate mesoderm sends instructive signals to the endoderm, signals that induce expression of the pancreatic genes Pdx1, p48, Nkx6.1, glucagon, and insulin. Moreover, this instructive signal directs cells to form ectopic insulin-positive islet-like clusters in endoderm that would otherwise form more rostral organs. Once generated, endocrine cells no longer require interaction with mesoderm, but nonendocrine cells continue to require permissive signals from the mesoderm. Stimulation of activin, BMP, or retinoic acid signaling is sufficient to induce Pdx1 expression in endoderm anterior to the pancreas. Lateral plate mesoderm appears to pattern the endoderm in a posterior-dominant fashion as first noted in the patterning of the neural tube at the same embryonic stage. These findings argue for a central role of the mesoderm in coordinating the A-P pattern of all three primary germ layers.     

The development of a method for functional assessment of dentures

Aims: To design and validate a method of assessing complete dentures from a functional standpoint. Subjects: A random sample of 40 complete denture wearers took part in the study. Setting: A university clinical department of prosthetic dentistry. Intervention: We undertook a pilot study to refine the protocol and criteria. All participants and their dentures were examined by two authors independently, with no prior knowledge of the patients'complaints. Design: We defined nine clinical factors of functional quality and applied criteria with binary scoring. We analysed the scores for these factors for inter‐rater reliability. Results: The method proved simple to apply and took less than 5 minutes to complete. The inter‐examiner agreement for all factors was 86% to 100% giving Kappa scores of 0.64 to 1.00 (all Good or Very Good). Conclusions: This study successfully demonstrates that the technique, which we call the Functional Assessment of Dentures (FAD), can give good inter‐examiner reliability. It can therefore be used separately as a routine diagnostic tool and to investigate the relationship between denture qualities and functional ‘outcome’ such as difficulty eating or dietary selection.     

Phylogenetic and taxonomic evaluation of Chalara, Chalaropsis, and Thielaviopsis anamorphs associated with Ceratocystis

Parsimony analysis of sequences of the internal transcribed spacer region of the nuclear rDNA and partial sequences of the large subunit (LSU) place four anamorphic Chalara species as a monophyletic grouping within the teleomorph genus Ceratocystis. Chalara ovoidea, Ch. thielavioides, Ch. populi, and Ch. elegans (synanamorph: Thielaviopsis basicola) form aleurioconidia typical of the anamorph genus Thielaviopsis, to which the species are transferred. Three of these species (T. ovoidea, T. thielavioides, and T. populi) are morphologically similar to each other but are shown to be distinct by rDNA sequences. The anamorphic genera Chalaropsis and Hughesiella are considered synonyms of Thielaviopsis. Thielaviopsis punctulata, which forms aleurioconidia singly, is shown to be the anamorph of Ce. radicicola. The respective anamorphs for Ce. coerulescens, Ce. fagacearum, and Ce. eucalypti, which lack aleurioconidia, are also transferred to the amended genus Thielaviopsis as T. ungeri, T. quercina, and T. eucalypti. Although Ch. australis and Ch. neocaledoniae do not form aleurioconidia, they are placed in Thielaviopsis based on their endoconidial state and clear affinities to Ceratocystis eucalypti. Three apparently asexual Ambrosiella species belong in the Ce. moniliformis clade based on LSU rDNA sequences, but the cultures available are not suitable for detailed morphological study, and these species are not transferred to Thielaviopsis.     

Is there a toxicological advantage for non-hyperbolic kinetics in cytochrome P450 catalysis? Functional allostery from "distributive catalysis"

The cytochrome P450s (CYPs) are the major enzymatic detoxification and drug metabolism system. Recently, it has become clear that several CYP isoforms exhibit positive and negative homotropic cooperativity. However, the toxicological implications of allosteric kinetics have not been considered, nor understood. The allosteric kinetics are particularly enigmatic in several respects. In many cases, CYPs bioactivate substrates to more toxic products, thus making it difficult to rationalize a functional advantage for positive cooperativity. Also, CYPs exhibit cooperativity with many structurally diverse ligands, in marked contrast to the specificity observed with other allosteric systems. Here, kinetic simulations are used to compare the probabilistic time- and concentration-dependent integrated toxicity function during conversion of substrate to product for CYP models exhibiting Michaelis-Menten (non-cooperative) kinetics, positive cooperativity, or negative cooperativity. The results demonstrate that, at low substrate concentrations, the slower substrate turnover afforded by cooperative CYPs compared with Michaelis-Menten enzymes can be a significant toxicological advantage, when toxic thresholds exist. When present, the advantage results from enhanced "distribution" of toxin in two pools, substrate and product, for an extended period, thus minimizing the chance that either exceeds its toxic threshold. At intermediate concentrations, the allosteric kinetics can be a modest advantage or modest disadvantage, depending on the kinetic parameters. However, at high substrate concentrations associated with a high probability of toxicity, fast turnover is desirable, and this advantage is provided also by the cooperative enzymes. For the positive homotropic cooperativity, the allosteric kinetics minimize the probability of toxicity over the widest range of system parameters. Furthermore, this apparent functional cooperativity is achieved without specific molecular recognition that is the hallmark of "traditional" allostery.