Tyrosine-specific MAPK phosphatases and the control of ERK signaling in PC12 cells

Background

Spatio-temporal control of extracellular signal-regulated kinase (ERK) activity, a critical determinant of the cell's response to growth factors, requires timely dephosphorylation of its regulatory tyrosine and/or threonine residue by MAPK phosphatases. We studied the physiological role of kinase interaction motif (KIM)-containing protein tyrosine phosphatases (PTPs) in the control of EGF- and NGF-induced ERK activity in neuroendocrine PC12 cells.

Results

We found a single KIM-containing PTP to be endogenously expressed in rat PC12 cells: the transmembrane PTPRR isoform termed PCPTP1. Protein knock-down of PCPTP1, or fourfold overexpression of its mouse orthologue, PTPBR7, left EGF- and NGF-induced ERK1/2 activity in PC12 cells unaltered. Ectopic expression of cytosolic PTPRR isoforms, however, resulted in reduced EGF-induced ERK1/2 activity, an effect that was dependent on the phosphatase activity and the KIM-domain of these PTPs.

Conclusion

The finding that robust changes in tyrosine-specific MAPK phosphatase expression levels have minor effects on temporal ERK1/2 activity control in PC12 cells suggests that dual-specificity MAPK phosphatases may act as major regulators of growth factor-induced ERK1/2 signaling in these cells.     

Pulse radiolysis study of the interaction of retinoids with peroxyl radicals

Vitamin A (retinol) and its derivatives-retinal and retinoic acid-are known for their ability to inhibit lipid peroxidation. Antioxidant actions of retinoids have been attributed to chain-breaking by scavenging of peroxyl radicals. Based on chemical analysis of retinoic acid degradation products formed during microsomal lipid peroxidation, it was previously suggested that retinoids interact with peroxyl radicals forming free carbon-centered radical adducts. However, it can be argued that such a mode of antioxidant action of retinoids is not sufficient to fully explain their effectiveness at inhibiting lipid peroxidation, which in many systems is comparable to, or even exceeds, that of alpha-tocopherol. In order to elucidate the mechanism of interaction of retinoids with peroxyl radicals, (trichloromethyl)peroxyl radical was generated by pulse radiolysis, and its interactions with retinoids solubilized in Triton X-100 micelles were followed by kinetic absorption spectroscopy. All retinoids--retinol, retinal, and retinoic acid--interacted with the peroxyl radical, and at least two transient products were detected. One of these products, absorbing at 590 nm, was identified as retinoid cation radical. Therefore, we postulate that, apart from formation of radical adducts, retinoids may also scavenge peroxyl radicals by electron transfer.     

Modification of proteins by 3-hydroxyanthranilic acid: the role of lysine residues

The mechanism of reaction of proteins with 3-hydroxyanthranilic acid (3OHA) under oxidizing conditions has been examined. A range of proteins were found to tan when exposed to oxidized 3OHA. One exception was lysozyme which tanned only after being denatured by reduction and carboxymethylation. Chemical modification experiments using bovine serum albumin (BSA) suggested that lysine was the primary site of reaction in 3OHA-mediated protein tanning. This reactivity of 3OHA toward lysine was confirmed by autoxidizing 3OHA in the presence of amino acid homopolymers. The rate of modification of both BSA and polylysine was pH dependent. At neutral pH, a component of the coloration of the protein was found to be due to the formation of a lysyl-p-quinone adduct. Other products appear to arise through addition to the 3OHA quinone imine. Poly-(Glu,Lys) was tanned by 3OHA at a greatly reduced rate, suggesting that electrostatic interactions may influence the reaction with lysine residues and may provide an explanation for the lack of tanning of lysozyme. Despite the reaction between 3OHA and lysine, amino acid analysis revealed little quantitative change in the lysine content of proteins even after exposure to 3OHA for a period of 24 h. These results support the proposal that reaction with lysine residues is the major route of protein tanning by 3-hydroxyanthranilic acid.     

Regional Central Nervous System Oligosaccharide Storage in Caprine β-Mannosidosis

Goats affected with beta-mannosidosis, an autosomal recessive disease of glycoprotein metabolism, have deficient activity of the lysosomal enzyme beta-mannosidase along with tissue storage of oligosaccharides, including a trisaccharide [Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc] and a disaccharide [Man(beta 1-4)GlcNAc]. CNS myelin deficiency, with regional variation in severity, is a major pathological characteristic of affected goats. This study was designed to investigate regional CNS differences in oligosaccharide accumulation to assess the extent of correlation between oligosaccharide accumulation and severity of myelin deficits. The concentrations of accumulated disaccharide and trisaccharide and the activity of beta-mannosidase were determined in cerebral hemisphere gray and white matter and in spinal cord from three affected and two control neonatal goats. In affected goats, the content of trisaccharide and disaccharide in spinal cord (moderate myelin deficiency) was similar to or greater than that in cerebral hemispheres (severe myelin deficiency). Thus, greater oligosaccharide accumulation was not associated with more severe myelin deficiency. Regional beta-mannosidase activity levels in control goats were consistent with the affected goat oligosaccharide accumulation pattern. The similarity of trisaccharide and disaccharide content in cerebral hemisphere gray and white matter suggested that lysosomal storage vacuoles, more numerous in gray matter, may not be the only location of stored CNS oligosaccharides.     

Evidence for,and taxonomic value of,an arachidonic acid cascade in the lipomycetaceae

By using specific inhibitors of the lipoxygenase and cyclo-oxygenase pathways, arachidonic acid metabolites with similar sensitivities towards these inhibitors as in humans, were detected inDipodascopsis uninucleata. The taxonomic value of aspirin sensitive arachidonic acid metabolites in the Lipomycetaceae was next assessed. No metabolites of which the production is inhibited by aspirin were detected in strains representing the following species:Lipomyces starkeyi, Lipomyces kononenkoae, Lipomyces tetrasporus, Myxozyma melibiosi, Myxozyma mucilagina, Myxozyma kluyveri, Waltomyces lipofer, Zygozyma oligophaga andZygozyma arxii. The detection of such aspirin sensitive arachidonic acid metabolites in representative strains ofLipomyces anomalus and the genusDipodascopsis, emphasises the isolated position of these taxa in the genusLipomyces and the family Lipomycetaceae, respectively. Finally using long chain fatty acid analyses, electrophoretic karyotyping and other phenotypic characters, a phylogenetic scheme is proposed for some genera in the Lipomycetaceae.     

Evaluation of neem seed powder for Fusarium wilt and Meloidogyne control on tomato

Fusarium wilt and root-knot are important diseases of tomato. The use of chemical is becoming less appealing because of the health implications. Also, the chemicals required are often not within the reach of farmers in most of the developing part of the world. This research is aimed at finding an alternative mode of control. Tomato variety Roma VF inoculated with Meloidogyne and Fusarium were treated with 2 g/kg soil neem seed powder in the screenhouse and 2 Mg ha ^{? 1} in the field. An untreated and Furadan treated plot in the field served as control. Neem seed powder significantly reduced the disease severity of Fusarium and root-knot in both screenhouse and field. Results suggest the possible use of neem seed powder for control of the root-knot nematodes - Fusarium wilt disease complex.     

Testing statistical significance scores of sequence comparison methods with structure similarity

Background  

In the past years the Smith-Waterman sequence comparison algorithm has gained popularity due to improved implementations and rapidly increasing computing power. However, the quality and sensitivity of a database search is not only determined by the algorithm but also by the statistical significance testing for an alignment. The e-value is the most commonly used statistical validation method for sequence database searching. The CluSTr database and the Protein World database have been created using an alternative statistical significance test: a Z-score based on Monte-Carlo statistics. Several papers have described the superiority of the Z-score as compared to the e-value, using simulated data. We were interested if this could be validated when applied to existing, evolutionary related protein sequences.     

Identifying dynamical modules from genetic regulatory systems: applications to the segment polarity network

Background  

It is widely accepted that genetic regulatory systems are 'modular', in that the whole system is made up of smaller 'subsystems' corresponding to specific biological functions. Most attempts to identify modules in genetic regulatory systems have relied on the topology of the underlying network. However, it is the temporal activity (dynamics) of genes and proteins that corresponds to biological functions, and hence it is dynamics that we focus on here for identifying subsystems.     

Pam16 has an essential role in the mitochondrial protein import motor

Mitochondrial preproteins destined for the matrix are translocated by two channel-forming transport machineries, the translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated protein import motor (PAM) contains four essential subunits: the matrix heat shock protein 70 (mtHsp70) and its three cochaperones Mge1, Tim44 and Pam18. Here we report that the PAM contains a fifth essential subunit, Pam16 (encoded by Saccharomyces cerevisiae YJL104W), which is selectively required for preprotein translocation into the matrix, but not for protein insertion into the inner membrane. Pam16 interacts with Pam18 and is needed for the association of Pam18 with the presequence translocase and for formation of a mtHsp70-Tim44 complex. Thus, Pam16 is a newly identified type of motor subunit and is required to promote a functional PAM reaction cycle, thereby driving preprotein import into the matrix.