Inhibition of nuclear import of LIMK2 in endothelial cells by protein kinase C-dependent phosphorylation at Ser-283

LIM kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Recently, it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of enhanced green fluorescent protein-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, we demonstrate that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predicted two PKC phosphorylation sites in LIMK2 but not in LIMK1. One site at Ser-283 is present between the PDZ and the kinase domain, and the other site at Thr-494 is within the kinase domain. Activation of PKC by phorbol ester treatment of endothelial cells stimulated LIMK2 phosphorylation at Ser-283 and inhibited nuclear import of LIMK2 and the PDZ kinase construct of LIMK2 (amino acids 142-638) but not of LIMK1. The PKC-delta isoform phosphorylated LIMK2 at Ser-283 in vitro. Mutational analysis indicated that LIMK2 phosphorylation at Ser-283 but not Thr-494 was functional. Serum stimulation of endothelial cells also inhibited nuclear import of PDZK-LIMK2 by protein kinase C-dependent phosphorylation of Ser-283. Our study shows that phorbol ester and serum stimulation of endothelial cells inhibit nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-delta-mediated phosphorylation of Ser-283. Since phorbol ester enhanced cyclin D1 expression and subsequent G1-to-S-phase transition of endothelial cells, we suggest that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2.     

De novo methylation of nucleosomal DNA by the mammalian Dnmt1 and Dnmt3A DNA methyltransferases

In the cell, DNA is wrapped on histone octamers, which reduces its accessibility for DNA interacting enzymes. We investigated de novo methylation of nucleosomal DNA in vitro and show that the Dnmt3a and Dnmt1 DNA methyltransferases efficiently methylate nucleosomal DNA without dissociation of the histone octamer from the DNA. In contrast, the prokaryotic SssI DNA methyltransferase and the catalytic domain of Dnmt3a are strongly inhibited by nucleosomes. We also found that full-length Dnmt1 and Dnmt3a bind to nucleosomes much stronger than their isolated catalytic domains, demonstrating that the N-terminal parts of the MTases are required for the interaction with nucleosomes. Variations of the DNA sequence or the histone tails did not significantly influence the methylation activity of Dnmt3a. The observation that mammalian methyltransferases directly modify nucleosomal DNA provides an insight into the mechanisms by which histone tail and DNA methylation patterns can influence each other because the DNA methylation pattern can be established while histones remain associated to the DNA.     

Galactomannan hydrolysis and mannose metabolism in Cellvibrio mixtus

Galactomannan hydrolysis results from the concerted action of microbial endo-mannanases, manosidases and alpha-galactosidases and is a mechanism of intrinsic biological importance. Here we report the identification of a gene cluster in the aerobic soil bacterium Cellvibrio mixtus encoding enzymes involved in the degradation of this polymeric substrate. The family 27 alpha-galactosidase, termed CmAga27A, preferentially hydrolyse galactose containing polysaccharides. In addition, we have characterized an enzyme with epimerase activity, which might be responsible for the conversion of mannose into glucose. The role of the identified enzymes in the hydrolysis of galactomannan by aerobic bacteria is discussed.     

Human kallikrein 10, a predictive marker for breast cancer

Our laboratory is involved in identifying genes that can be used as early diagnostic or prognostic markers in breast cancer. We previously identified a gene (NES1) that is expressed in normal but not in transformed mammary epithelial cells (MECs). NES1 is located on chromosome 19q13.4 within the kallikrein locus and thus was designated as human kallikrein 10 (hK10), although we have been unable to detect any protease activity. Importantly, hK10 expression is decreased in a majority of breast cancer cell lines. Transfection of hK10 into hK10-negative breast cancer cells reduces the tumorigenicity. Using methylation-specific PCR and subsequent sequencing, we demonstrate a strong correlation between hypermethylation of hK10 and loss of mRNA expression. Further analysis showed that essentially 100% of normal breast specimens had hK10 expression, whereas 46% of ductal carcinoma in situ (DCIS) and the majority of infiltrating ductal carcinoma (IDC) samples lacked the hK10 mRNA. Importantly, hK10-negative DCIS diagnosed at the time of biopsy were subsequently diagnosed as IDC at the time of definitive surgery. It has been shown that hK10 protein expression is regulated by steroids. In addition to breast cancers, hK10 is downregulated in cervical cancer, prostate cancer and acute lymphocytic leukemia, whereas it is upregulated in ovarian cancers. These results point to the paradoxical role of hK10 in human cancers and underscore the importance of further studies of this kallikrein.     

Development of Luciferase Expressing Leishmania donovani Axenic Amastigotes as Primary Model for In Vitro Screening of Antileishmanial Compounds

The development of new therapeutic leads against leishmaniasis relies primarily on screening of a large number of compounds on multiplication of clinically irrelevant transgenic promastigotes. The advent of the successful in vitro culture of axenic amastigotes allows the development of transgenic axenic amastigotes as a primary screen which can test compounds in a high throughput mode like promastigotes, still representative of the clinically relevant mammalian amastigotes stage. The present study reports the development of luciferase-tagged axenic amastigotes of Leishmania donovani, the causative agent of Indian Kala-azar, for in vitro drug screening. Luciferase expressing promastigotes were transformed to axenic amastigotes at a low pH and high temperature without the loss of luciferase expression. As compared to transgenic promastigotes, the luciferase expressing axenic amastigotes exhibited more sensitivity to antileishmanial drugs, particularly to pentavalent antimony (~2.8-fold) and also to the test compounds. Hence, the developed luciferase expressing axenic amastigotes make an ideal choice for high throughput drug screening for antileishmanial compounds.     

Ovine middle cerebral artery characterization and quantification of ultrastructure and other features: changes with development

Regulation of tone, blood pressure, and blood flow in the cerebral vasculature is of vital importance, particularly in the developing infant. We tested the hypothesis that, in addition to accretion of smooth muscle cells (SMCs) in cell layers with vessel thickening, significant changes in smooth muscle structure, as well as phenotype, extracellular matrix, and membrane proteins, in the media of cerebral arteries (CAs) during the course of late fetal development account for associated changes in contractility. Using transmission electron, confocal, wide-field epifluorescence, and light microscopy, we examined the structure and ultrastructure of CAs. Also, we utilized wire myography, Western immunoblotting, and real-time quantitative PCR to examine several other features of these arteries. We compared the main branch ovine middle CAs of 95- and 140-gestational day (GD) fetuses with those of adults (n = 5 for each experimental group). We observed a graded increase in phenylephrine- and KCl-induced contractile responses with development. Structurally, lumen diameter, media thickness, and media cross-sectional area increased dramatically from one age group to the next. With maturation, the cross-sectional profiles of CA SMCs changed from flattened bands in the 95-GD fetus to irregular ovoid-shaped fascicles in the 140-GD fetus and adult. We also observed a change in the type of collagen, specific integrin molecules, and several other parameters of SMC morphology with maturation. Ovine CAs at 95 GD appeared morphologically immature and poorly equipped to respond to major hemodynamic adjustments with maturation.