Structure-Function Analysis of Human TYW2 Enzyme Required for the Biosynthesis of a Highly Modified Wybutosine (yW) Base in Phenylalanine-tRNA

Posttranscriptional modifications are critical for structure and function of tRNAs. Wybutosine (yW) and its derivatives are hyper-modified guanosines found at the position 37 of eukaryotic and archaeal tRNAPhe. TYW2 is an enzyme that catalyzes α-amino-α-carboxypropyl transfer activity at the third step of yW biogenesis. Using complementation of a ΔTYW2 strain, we demonstrate here that human TYW2 (hTYW2) is active in yeast and can synthesize the yW of yeast tRNAPhe. Structure-guided analysis identified several conserved residues in hTYW2 that interact with S-adenosyl-methionine (AdoMet), and mutation studies revealed that K225 and E265 are critical residues for the enzymatic activity. We previously reported that the human TYW2 is overexpressed in breast cancer. However, no difference in the tRNAPhe modification status was observed in either normal mouse tissue or a mouse tumor model that overexpresses Tyw2, indicating that hTYW2 may have a role in tumorigenesis unrelated to yW biogenesis

The Cytoplasmic Location of Chicken Mx Is Not the Determining Factor for Its Lack of Antiviral Activity

Chicken Mx belongs to the Mx family of interferon-induced dynamin-like GTPases, which in some species possess potent antiviral properties. Conflicting data exist for the antiviral capability of chicken Mx. Reports of anti-influenza activity of alleles encoding an Asn631 polymorphism have not been supported by subsequent studies. The normal cytoplasmic localisation of chicken Mx may influence its antiviral capacity. Here we report further studies to determine the antiviral potential of chicken Mx against Newcastle disease virus (NDV), an economically important cytoplasmic RNA virus of chickens, and Thogoto virus, an orthomyxovirus known to be exquisitely sensitive to the cytoplasmic MxA protein from humans. We also report the consequences of re-locating chicken Mx to the nucleus.Chicken Mx was tested in virus infection assays using NDV. Neither the Asn631 nor Ser631 Mx alleles (when transfected into 293T cells) showed inhibition of virus-directed gene expression when the cells were subsequently infected with NDV. Human MxA however did show significant inhibition of NDV-directed gene expression. Chicken Mx failed to inhibit a Thogoto virus (THOV) minireplicon system in which the cytoplasmic human MxA protein showed potent and specific inhibition. Relocalisation of chicken Mx to the nucleus was achieved by inserting the Simian Virus 40 large T antigen nuclear localisation sequence (SV40 NLS) at the N-terminus of chicken Mx. Nuclear re-localised chicken Mx did not inhibit influenza (A/PR/8/34) gene expression during virus infection in cell culture or influenza polymerase activity in A/PR/8/34 or A/Turkey/50-92/91 minireplicon systems.The chicken Mx protein (Asn631) lacks inhibitory effects against THOV and NDV, and is unable to suppress influenza replication when artificially re-localised to the cell nucleus. Thus, the natural cytoplasmic localisation of the chicken Mx protein does not account for its lack of antiviral activity

BRITER: A BMP Responsive Osteoblast Reporter Cell Line

BACKGROUND: BMP signaling pathway is critical for vertebrate development and tissue homeostasis. High-throughput molecular genetic screening may reveal novel players regulating BMP signaling response while chemical genetic screening of BMP signaling modifiers may have clinical significance. It is therefore important to generate a cell-based tool to execute such screens. METHODOLOGY/PRINCIPAL FINDINGS: We have established a BMP responsive reporter cell line by stably integrating a BMP responsive dual luciferase reporter construct in the immortalized calvarial osteoblast cells isolated from tamoxifen inducible Bmp2; Bmp4 double conditional knockout mouse strain. This cell line, named BRITER (BMP Responsive Immortalized Reporter cell line), responds robustly, promptly and specifically to exogenously added BMP2 protein. The sensitivity to added BMP may be further increased by depleting the endogenous BMP2 and BMP4 proteins. CONCLUSION: As the dynamic range of the assay (for BMP responsiveness) is very high for BRITER and as it responds specifically and promptly to exogenously added BMP2 protein, BRITER may be used effectively for chemical or molecular genetic screening for BMP signaling modifiers. Identification of novel molecular players capable of influencing BMP signaling pathway may have clinical significance

The V5 domain of protein kinase C plays a critical role in determining the isoform-specific localization, translocation, and biological function of protein kinase C-delta and -epsilon

The catalytic domain of overexpressed protein kinase C (PKC)-delta mediates phorbol 12-myristate 13-acetate (PMA)-induced differentiation or apoptosis in appropriate model cell lines. To define the portions of the catalytic domain that are critical for these isozyme-specific functions, we constructed reciprocal chimeras, PKC-delta/epsilonV5 and -epsilon/deltaV5, by swapping the V5 domains of PKC-delta and -epsilon. PKC-delta/epsilonV5 failed to mediate PMA-induced differentiation of 32D cells, showing the essential nature of the V5 domain for PKC-delta's functionality. The other chimera, PKC-epsilon/deltaV5, endowed inactive PKC-epsilon with nearly all PKC-delta's apoptotic ability, confirming the importance of PKC-delta in this function. Green fluorescent protein (GFP)-tagged PKC-deltaV5 and -epsilon/deltaV5 in A7r5 cells showed substantial basal nuclear localization, while GFP-tagged PKC-epsilon and -delta/epsilonV5 showed significantly less, indicating that the V5 region of PKC-delta contains determinants critical to its nuclear distribution. PKC-epsilon/deltaV5-GFP showed much slower kinetics of translocation to membranes in response to PMA than parental PKC-epsilon, implicating the PKC-epsilonV5 domain in membrane targeting. Thus, the V5 domain is critical in several of the isozyme-specific functions of PKC-delta and -epsilon

A preservation method that allows recovery of intact RNA from tissues dissected by laser capture microdissection

We report a novel method for preparing samples for laser capture microdissection. The procedure described here permits extraction of intact RNA while preserving morphology, thus being suitable both for identification of specific cells and for analysis of their gene expression. The method is applicable to both mouse embryos and human tumors and may improve the preparation of cDNA libraries from specific cell types without interfering with histological diagnosis. (c)2001 Elsevier Science