Regulation of cell motility by tyrosine phosphorylated villin

Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin.     

High-performance liquid chromatographic method for the quantification of unbound evernimicin in human plasma ultrafiltrate

A rapid HPLC method was developed for quantification of unbound evernimicin in human plasma. Protein-free samples prepared by ultrafiltration were injected directly onto a polymeric reversed-phase column and the eluent monitored at 302 nm. Evernimicin that eluted within 3.5 min was well resolved from endogenous components. Linearity was established between peak height and evernimicin concentration from 25 to 2500 ng/ml. Assay precision (C.V.) was within 5% while bias was no greater than 3%. This method has been used for the ex vivo assessment of evernimicin protein binding in human plasma from safety and tolerance as well as liver dysfunction and renal insufficiency studies.     

Alkali‐catalyzed low temperature wet crosslinking of plant proteins using carboxylic acids

We report the development of a new method of alkali‐catalyzed low temperature wet crosslinking of plant proteins to improve their breaking tenacity without using high temperatures or phosphorus‐containing catalysts used in conventional poly(carboxylic acid) crosslinking of cellulose and proteins. Carboxylic acids are preferred over aldehyde‐containing crosslinkers for crosslinking proteins and cellulose because of their low toxicity and cost and ability to improve the desired properties of the materials. However, current knowledge in carboxylic acid crosslinking of proteins and cellulose requires the use of carboxylic acids with at least three carboxylic groups, toxic phosphorous‐containing catalysts and curing at high temperatures (150–185°C). The use of high temperatures and low pH in conventional carboxylic acid crosslinking has been reported to cause substantial strength loss and/or undesired changes in the properties of the crosslinked materials. In this research, gliadin, soyprotein, and zein fibers have been crosslinked with malic acid, citric acid, and butanetetracarboxylic acid to improve the tenacity of the fibers without using high temperatures and phosphorus‐containing catalysts. The new method of wet crosslinking using carboxylic acids containing two or more carboxylic groups will be useful to crosslink proteins for various industrial applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Potential of Mauritius Hemp (Furcraea gigantea Vent.) for the Remediation of Chromium Contaminated Soils

The present study was conducted to evaluate the ability of a high biomass producing, drought tolerant succulent plant Mauritius hemp (Furcraea gigantea Vent.) for its tolerance to different levels of Cr (0, 25, 50, 100 and 200 mg Cr kg soil^?1) and its potential for phytoremediation purposes. Based on the data on inhibition of the growth of plants with Cr, tolerance index and grade of growth inhibition, it was observed that the plant could tolerate up to 50 mg Cr kg ^?1 soil. Absorption of Cr from soil to plant and its translocation into plant tissues were discussed in terms of bio concentration factor (BCF), transfer factor (TF), and translocation efficiency (TE%). Cr was mainly accumulated in the roots and exclusion of Cr was found to be the principal physiological tolerance mechanism followed by a marked increase in proline, ascorbic acid, total free amino acids in the leaf tissue and malic acid in the rhizosphere samples to counter Cr stress. Based on the tissue concentration of Cr (< 300 μg g^?1 in the leaves and TF<1), it was concluded that, Furcraea gigantea could not be considered a hyperaccumulator and therefore unsuitable for phytoextraction of Cr. Nevertheless, Furcraea gigantea could be a suitable candidate for phytostablization of Cr contaminated soils.     

Local but Not Systemic Administration of Uridine Prevents Development of Antigen-Induced Arthritis

Objective

Uridine has earlier been show to down modulate inflammation in models of lung inflammation. The aim of this study was to evaluate the anti-inflammatory effect of uridine in arthritis.

Methods

Arthritis was induced by intra-articular injection of mBSA in the knee of NMRI mice pre-immunized with mBSA. Uridine was either administered locally by direct injection into the knee joint or systemically. Systemic treatment included repeated injections or implantation of a pellet continuously releasing uridine during the entire experimental procedure. Anti-mBSA specific immune responses were determined by ELISA and cell proliferation and serum cytokine levels were determined by Luminex. Immunohistochemistry was used to identify cells, study expression of cytokines and adhesion molecules in the joint.

Results

Local administration of 25–100 mg/kg uridine at the time of arthritis onset clearly prevented development of joint inflammation. In contrast, systemic administration of uridine (max 1.5 mg uridine per day) did not prevent development of arthritis. Protection against arthritis by local administration of uridine did not affect the anti-mBSA specific immune response and did not prevent the rise in serum levels of pro-inflammatory cytokines associated with the triggering of arthritis. In contrast, local uridine treatment efficiently inhibited synovial expression of ICAM-1 and CD18, local cytokine production and recruitment of leukocytes to the synovium.

Conclusion

Local, but not systemic administration of uridine efficiently prevented development of antigen-induced arthritis. The protective effect did not involve alteration of systemic immunity to mBSA but clearly involved inhibition of synovial expression of adhesion molecules, decreased TNF and IL-6 production and prevention of leukocyte extravasation. Further, uridine is a small, inexpensive molecule and may thus be a new therapeutic option to treat joint inflammation in RA.     

Relatedness of Indian Flax Genotypes (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.): An Inter-Simple Sequence Repeat (ISSR) Primer Assay

The objective of this study was to analyze the genetic relationships, using PCR-based ISSR markers, among 70 Indian flax (Linum usitatissimum L.) genotypes actively utilized in flax breeding programs. Twelve ISSR primers were used for the analysis yielding 136 loci, of which 87 were polymorphic. The average number of amplified loci and the average number of polymorphic loci per primer were 11.3 and 7.25, respectively, while the percent loci polymorphism ranged from 11.1 to 81.8 with an average of 63.9 across all the genotypes. The range of polymorphism information content scores was 0.03–0.49, with an average of 0.18. A dendrogram was generated based on the similarity matrix by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), wherein the flax genotypes were grouped in five clusters. The Jaccard’s similarity coefficient among the genotypes ranged from 0.60 to 0.97. When the omega-3 alpha linolenic acid (ALA) contents of the individual genotypes were correlated with the clusters in the dendrogram, the high ALA containing genotypes were grouped in two clusters. This study identified SLS 50, Ayogi, and Sheetal to be the most diverse genotypes and suggested their use in breeding programs and for developing mapping populations.     

PINK1 Is Selectively Stabilized on Impaired Mitochondria to Activate Parkin

Loss-of-function mutations in PINK1 and Parkin cause parkinsonism in humans and mitochondrial dysfunction in model organisms. Parkin is selectively recruited from the cytosol to damaged mitochondria to trigger their autophagy. How Parkin recognizes damaged mitochondria, however, is unknown. Here, we show that expression of PINK1 on individual mitochondria is regulated by voltage-dependent proteolysis to maintain low levels of PINK1 on healthy, polarized mitochondria, while facilitating the rapid accumulation of PINK1 on mitochondria that sustain damage. PINK1 accumulation on mitochondria is both necessary and sufficient for Parkin recruitment to mitochondria, and disease-causing mutations in PINK1 and Parkin disrupt Parkin recruitment and Parkin-induced mitophagy at distinct steps. These findings provide a biochemical explanation for the genetic epistasis between PINK1 and Parkin in Drosophila melanogaster. In addition, they support a novel model for the negative selection of damaged mitochondria, in which PINK1 signals mitochondrial dysfunction to Parkin, and Parkin promotes their elimination.     

Skeletal muscle mitochondrial FAT/CD36 content and palmitate oxidation are not decreased in obese women

A reduction in fatty acid oxidation has been associated with lipid accumulation and insulin resistance in the skeletal muscle of obese individuals. We examined whether this decrease in fatty acid oxidation was attributable to a reduction in muscle mitochondrial content and/or a dysfunction in fatty acid oxidation within mitochondria obtained from skeletal muscle of age-matched, lean [body mass index (BMI) = 23.3 +/- 0.7 kg/m2] and obese women (BMI = 37.6 +/- 2.2 kg/m2). The mitochondrial marker enzymes citrate synthase (-34%), beta-hydroxyacyl-CoA dehydrogenase (-17%), and cytochrome c oxidase (-32%) were reduced (P < 0.05) in obese participants, indicating that mitochondrial content was diminished. Obesity did not alter the ability of isolated mitochondria to oxidize palmitate; however, fatty acid oxidation was reduced at the whole muscle level by 28% (P < 0.05) in the obese. Mitochondrial fatty acid translocase (FAT/CD36) did not differ in lean and obese individuals, but mitochondrial FAT/CD36 was correlated with mitochondrial fatty acid oxidation (r = 0.67, P < 0.05). We conclude that the reduction in fatty acid oxidation in obese individuals is attributable to a decrease in mitochondrial content, not to an intrinsic defect in the mitochondria obtained from skeletal muscle of obese individuals. In addition, it appears that mitochondrial FAT/CD36 may be involved in regulating fatty acid oxidation in human skeletal muscle.