The regiospecific one-pot phosphorylation of either the 5'- or 2'-hydroxyl in 3'-deoxycytidines without protection: critical role of the base

We report methodology which enables direct phosphorylation of 3'-deoxycytidine exclusively either at the 5'-hydroxyl or the 2'-hydroxyl. Protection of the base is not required. Standard phosphoramidochloridates in combination with pyridine and tert-butyl magnesium chloride is employed, in which the ratio of nucleoside to Grignard reagent is crucial. These findings, which appear to be general for 3'-deoxycytidines, are not applicable to 3'-deoxyuridine or 3'-deoxyguanosine.     

Accurate determination of ataxin-2 polyglutamine expansion in patients with intermediate-range repeats

Spinocerebellar ataxia, type 2 (SCA2), results from an expansion of a stretch of polyglutamine repeats within the coding sequence of the ataxin-2 gene (ATX2), localized to chromosome 12q23-24. Recent studies have widened the clinical phenotype, notably for individuals with repeats of intermediate size, from 32 to 35 glutamine residues. This narrow range necessitates precise determination of repeat size. Diagnostic laboratories most often perform direct genotyping of ATX2 from polymerase chain-amplified patient DNA with subsequent sizing utilizing slab gel polyacrylamide gel electrophoresis (PAGE) or capillary electrophoresis. Using cloning and sequencing methods, we have constructed a ladder of ATX2 alleles of known size and sequence composition. This freely available size ladder will facilitate future quantification of expansions of the ATX2 locus.     

Human and murine glycerol kinase: influence of exon 18 alternative splicing on function

Glycerol kinase (GK) is a key enzyme in glycerol metabolism with two alternatively spliced forms-one with an 87bp insertion corresponding to exon 18 (GK+EX18), and one lacking exon 18 (GK-EX18). We report the expression of GK+/-EX18 in various tissues and cell lines, as well as their enzymatic characteristics and subcellular localization. RT-PCR revealed differential expression in tissues and cell lines. Northern blot analysis revealed that both forms of the murine ortholog, Gyk, were highly expressed in murine heart and increased during embryonic development. K(m) values for glycerol for GK+/-EX18 were not significantly different, although GK-EX18 had a higher V(max) for glycerol. GK-EX18 had a lower K(m) and V(max) for ATP than GK+EX18. Immunofluorescence experiments showed that GK+EX18 co-localized to the mitochondria and the perinuclear region while GK-EX18 had a diffuse expression pattern. These data suggest specific and divergent roles for GK+EX18 and GK-EX18 in cellular metabolism and development.     

Constitutive activation of GSK3 down-regulates glycogen synthase abundance and glycogen deposition in rat skeletal muscle cells

The effects of inhibition or constitutive activation of glycogen synthase kinase-3 (GSK3) on glycogen synthase (GS) activity, abundance, and glycogen deposition in L6 rat skeletal muscle cells were investigated. GS protein expression increased approximately 5-fold during differentiation of L6 cells (comparing cells at the end of day 5 with those at the beginning of day 3). However, exposure of undifferentiated myoblasts (day 3) to 50 microM SB-415286, a GSK3 inhibitor, led to a significant elevation in GS protein that was not accompanied by changes in the abundance of GLUT4, another late differentiation marker. In contrast, stable expression of a constitutively active form of GSK3beta (GSK3S9A) led to a significant reduction (approximately 80%) in GS protein that was antagonized by SB-415286. Inhibition of GSK3 or expression of the constitutively active GSK3S9A did not result in any detectable changes in GS mRNA abundance. However, the increase in GS protein in undifferentiated myoblasts or that seen following incubation of cells expressing GSK3S9A with GSK3 inhibitors was blocked by cycloheximide suggesting that GSK3 influences GS abundance possibly via control of mRNA translation. Consistent with the reduction in GS protein, cells expressing GSK3S9A were severely glycogen depleted as judged using a specific glycogen-staining antibody. Inhibiting GSK3 in wild-type or GSK3S9A-expressing cells using SB-415286 resulted in an attendant activation of GS, but not that of glucose transport. However, GS activation alone was insufficient for stimulating glycogen deposition. Only when muscle cells were incubated simultaneously with insulin and SB-415286 or with lithium (which stimulates GS and glucose transport) was an increase in glycogen accretion observed. Our findings suggest that GSK3 activity is an important determinant of GS protein expression and that while glycogen deposition in muscle cells is inherently dependent upon the activity/expression of GS, glucose transport is a key rate-determining step in this process.     

In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag

The introduction of green fluorescent protein and its variants (GFPs) has allowed protein analysis at the level of the cell. Now, chemical methods are needed to label proteins in vivo with a wider variety of functionalities so that mechanistic questions about protein function in the complex cellular environment can be addressed. Here we demonstrate that trimethoprim derivatives can be used to selectively tag Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins in wild-type mammalian cells with minimal background and fast kinetics.     

α-Tocopheryl succinate sensitizes established tumors to vaccination with nonmatured dendritic cells

Purpose: Dendritic cells (DCs) are considered potential candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC-based vaccines have exhibited minimal effectiveness against established tumors in mice and human cancer patients. The use of appropriate adjuvants can enhance the efficacy of DC-based cancer vaccines in treating established tumors. Methods: In this study we have employed -tocopheryl succinate (-TOS), a nontoxic esterified analogue of vitamin E, as an adjuvant to enhance the effectiveness of DC vaccines in treating established murine Lewis lung (3LL) carcinomas. Results: We demonstrate that locally or systemically administered -TOS in combination with nonmatured DCs injected intratumorally (i.t.) or subcutaneously (s.c.) significantly inhibits the growth of preestablished 10-day tumors (mean tumor volume of 77.5 ± 17.8 mm³ on day 30 post–tumor injection) as compared to -TOS alone (mean tumor volume of 471 ± 68 mm³ on day 30 post–tumor injection). Additionally, the adjuvant effect of -TOS was superior to that of cyclophosphamide (CTX). The mean tumor volume on day 28 post–tumor injection in mice treated with CTX+DCs was 611 ± 94 mm³ as compared to 105 ± 36 mm³ in mice treated with -TOS+DCs. Analysis of purified T lymphocytes from mice treated with -TOS+DC revealed significantly increased secretion of IFN- as compared to T cells from the various control groups. Conclusion: This study demonstrates the potential usefulness of -tocopheryl succinate, an agent nontoxic to normal cell types, as an adjuvant to augment the effectiveness of DC-based vaccines in treating established tumors.Abbreviations AO acridine orange - CTX cyclophosphamide - DC dendritic cell - dUTP deoxyuridine triphosphate - FACS fluorescence-activated cell sorter - FBS fetal bovine serum - FITC fluorescein isothiocyanate - GM-CSF granulocyte-macrophage colony-stimulating factor - IFN- interferon-gamma - IL-4 interleukin-4 - NaS sodium succinate - OCT optimal cutting temperature - PBS phosphate-buffered saline - PI propidium iodide - Tdt terminal deoxynucleotidyl transferase - TNF- tumor necrosis factor alpha - -TOS -tocopheryl succinateSupported by grants 1 RO1 CA94111-02 from the NIH and DAMD 17010126 from the DOD.     

Stage-dependent modulation of limb regeneration by caffeic acid phenethyl ester (CAPE)--immunocytochemical evidence of a CAPE-evoked delay in mesenchyme formation and limb regeneration

Caffeic acid phenethyl ester (CAPE), a natural compound of bee propolis, selectively inhibits proliferation of transformed cells in several cancer models in vitro. To examine in vivo CAPE function, we used the newt regeneration blastema as a model system wherein the processes of de-differentiation and subsequent proliferation of undifferentiated cells mimic changes associated with oncogenic transformation and tumorigenesis. We have shown that a single dose of CAPE significantly increased cell proliferation at the stages of blastema growth and re-differentiation. At the de-differentiation stage, CAPE significantly stimulated proliferation of wound epidermis keratinocytes, but decreased proliferation in the blastema mesenchyme. Immunohistochemistry with a mesenchymal cell marker, vimentin, revealed a highly significant reduction of vimentin staining in the mesenchyme of CAPE-treated regenerates (p<0.001). These results, together with morphological observations indicate that, at the de-differentiation stage, CAPE stimulated wound re-epithelization, increased keratinocyte proliferation and increased thickness of the wound epidermis. However, CAPE inhibited mesenchyme formation and proliferation. The functional consequence of the CAPE inhibitory action was a delay in limb regeneration.     

The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae

Attack by the host powdery mildew Erysiphe cichoracearum usually results in successful penetration and rapid proliferation of the fungus on Arabidopsis. By contrast, the nonhost barley powdery mildew Blumeria graminis f. sp. hordei (Bgh) typically fails to penetrate Arabidopsis epidermal cells. In both instances the plant secretes cell wall appositions or papillae beneath the penetration peg of the fungus. Genetic screens for mutations that result in increased penetration of Bgh on Arabidopsis have recently identified the PEN1 syntaxin. Here we examine the role of PEN1 and of its closest homologue, SYP122, identified as a syntaxin whose expression is responsive to infection. pen1 syp122 double mutants are both dwarfed and necrotic, suggesting that the two syntaxins have overlapping functions. Although syp122-1 and the cell wall mur mutants have considerably more pronounced primary cell wall defects than pen1 mutants, these have relatively subtle or no effects on penetration resistance. Upon fungal attack, PEN1 appears to be actively recruited to papillae, and there is a 2-h delay in papillae formation in the pen1-1 mutant. We conclude that SYP122 may have a general function in secretion, including a role in cell wall deposition. By contrast, PEN1 appears to have a basal function in secretion and a specialized defense-related function, being required for the polarized secretion events that give rise to papilla formation.