Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2'-O-methyl RNA,phosphorothioates and small interfering RNA

Locked nucleic acids (LNAs) and double-stranded small interfering RNAs (siRNAs) are rather new promising antisense molecules for cell culture and in vivo applications. Here, we compare LNA–DNA–LNA gapmer oligonucleotides and siRNAs with a phosphorothioate and a chimeric 2′-O-methyl RNA–DNA gapmer with respect to their capacities to knock down the expression of the vanilloid receptor subtype 1 (VR1). LNA–DNA–LNA gapmers with four or five LNAs on either side and a central stretch of 10 or 8 DNA monomers in the center were found to be active gapmers that inhibit gene expression. A comparative co-transfection study showed that siRNA is the most potent inhibitor of VR1–green fluorescent protein (GFP) expression. A specific inhibition was observed with an estimated IC₅₀ of 0.06 nM. An LNA gapmer was found to be the most efficient single-stranded antisense oligonucleotide, with an IC₅₀ of 0.4 nM being 175-fold lower than that of commonly used phosphorothioates (IC₅₀ ~70 nM). In contrast, the efficiency of a 2′-O-methyl-modified oligonucleotide (IC₅₀ ~220 nM) was 3-fold lower compared with the phosphorothioate. The high potency of siRNAs and chimeric LNA–DNA oligonucleotides make them valuable candidates for cell culture and in vivo applications targeting the VR1 mRNA.     

Ontogenetic changes and environmental effects on ocular transmission in four species of coral reef fishes

Filtration by the humors, cornea and lens limits the spectrum of light available for vision as blocking compounds prevent some wavelengths from reaching photo-sensitive cells of the retina. The visual ecology of fishes is dependent upon factors changing with size and/or habitat. We predicted that ontogeny and habitat depth would affect ocular transmission for four fishes, Mulloidichthys flavolineatus, Parupeneus multifasciatus, Acanthurus triostegas, and Naso lituratus. We measured ocular transmission in specimens from a range of sizes (juvenile-adult) and capture depths (<3-37 m), and used the wavelength (nm) where transmission was reduced 50% as our comparative measure (T(50)). We modeled lens transmission varying pigment concentrations and pathlength, and compared predicted versus measured results. P. multifasciatus, M. flavolineatus, and N. lituratus showed a significant increase in short-wavelength blocking with size. A. triostegas were constant across sizes, and showed a slight but significant effect with depth. Comparisons of predicted versus observed transmission values suggest that pigment concentrations are held constant with age for all species, but species- and family-level differences emerge. The accumulation of blocking compounds in ocular tissues is a contributing means for balancing the costs and benefits of admitting short-wavelength radiation to the retina.     

A critical role of water in the specific cleavage of the anticodon loop of some eukaryotic methionine initiator tRNAs

We have noticed that during a long storage and handling, the plant methionine initiator tRNA is spontaneously hydrolyzed within the anticodon loop at the C34-A35 phosphodiester bond. A literature search indicated that there is also the case for human initiator tRNA^Met but not for yeast tRNA^Met _i or E. coli tRNA^Met _f. All these tRNAs have an identical nucleotide sequence of the anticodon stems and loops with only one difference at position 33 within the loop. It means that cytosine 33 (C33) makes the anticodon loop of plant and human tRNA^Met _i susceptible to the specific cleavage reaction. Using crystallographic data of tRNA^Met _f of E. coli with U33, we modeled the anticodon loop of this tRNA with C33. We found that C33 within the anticodon loop creates a pocket that can accomodate a hydrogen bonded water molecule that acts as a general base and catalyzes a hydrolysis of C-A bond. We conclude that a single nucleotide change in the primary structure of tRNA^Met _i made changes in hydration pattern and readjustment in hydrogen bonding which lead to a cleavage of the phosphodiester bond.     

Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation

Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease. We have ablated the plakophilin 2 gene in mice. The resulting mutant mice exhibit lethal alterations in heart morphogenesis and stability at mid-gestation (E10.5-E11), characterized by reduced trabeculation, disarrayed cytoskeleton, ruptures of cardiac walls, and blood leakage into the pericardiac cavity. In the absence of plakophilin 2, the cytoskeletal linker protein desmoplakin dissociates from the plaques of the adhering junctions that connect the cardiomyocytes and forms granular aggregates in the cytoplasm. By contrast, embryonic epithelia show normal junctions. Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.     

Basic fibroblast growth factor expression following surgical delay of rat transverse rectus abdominis myocutaneous flaps

Partial transverse rectus abdominis myocutaneous (TRAM) flap loss in breast reconstruction can be a devastating complication for both patient and surgeon. Surgical delay of the TRAM flap has been shown to improve flap viability and has been advocated in "high-risk" patients seeking autogenous breast reconstruction. Despite extensive clinical evidence of the effectiveness of surgical delay of TRAM flaps, the mechanisms by which the delay phenomenon occurs remain poorly understood. To examine whether angiogenic growth factors such as basic fibroblast growth factor (bFGF) may play a role in the delay phenomenon, the authors studied the expression of bFGF in rat TRAM flaps subjected to surgical delay. Thirty-five female Sprague-Dawley rats were randomly assigned to one of four TRAM flap groups: no delay (n = 6), 7-day delay (n = 12), 14-day delay (n = 10), or 21-day delay (n = 7). Surgical delay consisted of incising skin around the perimeter of the planned 2.5 x 5.0-cm TRAM flap followed by ablation of both superior epigastric arteries and the left inferior epigastric artery, thus preserving the right inferior epigastric artery (the nondominant blood supply to the rectus abdominis muscle of the rat). TRAM flaps were then elevated after 7, 14, and 21 days of delay by raising zones II, III, and IV off the abdominal wall fascia. Once hemostasis was assured, the flaps were sutured back in place. All flaps were designed with the upper border of the flap 1 cm below the xiphoid tip. Three days after the TRAM procedure, postfluorescein planimetry was used to determine percent area viability of both superficial and deep portions of TRAM flaps. All rats were euthanized and full-thickness TRAM specimens were taken from zones I, II, III, and IV for enzyme-linked immunoabsorbent assay analysis of bFGF levels. Statistical testing was done by t test (percent viability) and two-way analysis of variance (bFGF levels). All delayed flaps had significantly higher bFGF levels when compared with all nondelayed control flaps (p < 0.05). The bFGF levels were not different in the rats that received TRAM flaps 7, 14, or 21 days after delay surgery. There was also no significant difference in bFGF levels among zones I through IV. Control rats had more peripheral zone necrosis compared with all delayed TRAM rats. All delayed flaps had a significantly higher area of flap viability superficially than nondelayed control flaps (p < 0.05). There was no difference in deep flap viability. Surgical delay of rat TRAM flaps is associated with improved flap viability and significantly elevated levels of bFGF over nondelayed TRAM flaps at postoperative day 3 after TRAM surgery. The increases in bFGF noted at this time point suggests that bFGF may play a role in the improved TRAM flap viability observed after delay surgery. Further investigation is needed to evaluate the role bFGF may play in the delay phenomenon.     

beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin

beta-Catenin is an essential molecule in Wnt/wingless signaling, which controls decisive steps in embryogenesis. To study the role of beta-catenin in skin development, we introduced a conditional mutation of the gene in the epidermis and hair follicles using Cre/loxP technology. When beta-catenin is mutated during embryogenesis, formation of placodes that generate hair follicles is blocked. We show that beta-catenin is required genetically downstream of tabby/downless and upstream of bmp and shh in placode formation. If beta-catenin is deleted after hair follicles have formed, hair is completely lost after the first hair cycle. Further analysis demonstrates that beta-catenin is essential for fate decisions of skin stem cells: in the absence of beta-catenin, stem cells fail to differentiate into follicular keratinocytes, but instead adopt an epidermal fate.     

Reversible inhibition of calcineurin by the polyphenolic aldehyde gossypol

The reversible inhibition of calcineurin (CaN), which is the only Ca(2+)/calmodulin-dependent protein Ser/Thr phosphatase, is thought to be a key functional event for most cyclosporin A (CsA)- and tacrolimus (FK506)-mediated biological effects. In addition to CaN inhibition, however, CsA and FK506 have multiple biochemical effects because of their action in a gain-of-function model that requires prior binding to immunophilic proteins. We screened a small molecule library for direct inhibitors of CaN using CaN-mediated dephosphorylation of (33)P-labeled 19-residue phosphopeptide substrate (RII phosphopeptide) as an assay and found the polyphenolic aldehyde gossypol to be a novel CaN inhibitor. Unlike CsA and FK506, gossypol does not require a matchmaker protein for reversible CaN inhibition with an IC(50) value of 15 microm. Gossypolone, a gossypol analog, showed improved inhibition of both RII phosphopeptide and p-nitrophenyl phosphate dephosphorylation with an IC(50) of 9 and 6 microm, respectively. In contrast, apogossypol hexaacetate was inactive. Gossypol acts noncompetitively, interfering with the binding site for the cyclophilin 18.CsA complex in CaN. In contrast to CsA and FK506, gossypol does not inactivate the peptidyl-prolyl-cis/trans-isomerase activity of immunophilins. Similar to CsA and FK506, T cell receptor signaling induced by phorbol 12-myristate 13-acetate/ionomycin is inhibited by gossypol in a dose-dependent manner, demonstrated by the inhibition of nuclear factor of activated T cell (NFAT) c1 translocation from the cytosol into the nucleus and suppression of NFAT-luciferase reporter gene activity.