Molecular cloning and characterization of a proliferating cell nuclear antigen gene by chemically induced male sterility in wheat (Triticum aestivum L.).

Although a number of studies have shown that chemical hybridizing agents (CHAs) affect anther growth and regulate cell-cycle progression, little is known about the molecular and cellular mechanisms involved. Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication, and in many other processes in eukaryotic cells. In this study, the open reading frame of TaPCNA, the PCNA in wheat (Triticum aestivum L.), was cloned by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis revealed that this gene was 792-bp long and encoded a protein with 234 amino acids. Alignment of the TaPCNA-predicted sequence revealed a high degree of identity with PCNAs from other plant species. A subcellular localization assay indicated that TaPCNA was localized in the nucleus. The TaPCNA was cloned into the prokaryotic expression plasmid pET32a, and the recombinant plasmid was transformed into BL21 (DE3). TaPCNA expression was induced by 0.5 mM isopropyl-beta-D-thiogalactopyranoside and verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot assays, which indicated that the fusion protein was successfully expressed. The gene involved in the G1-to-S transition, Histone H4, was downregulated by 1376- CIMS, which is a chemically induced male sterility line. However, a semi-quantitative RT-PCR revealed that TaPCNA expression was upregulated in 1376-CIMS. Our results suggest that CHAs (SQ-1) induce DNA damage in wheat anthers. DNA damage results in either the delay or arrest of cell-cycle progression, which affects anther development. This study will help to elucidate the mechanisms of SQ-1-induced male sterility.

Single wire radial junction photovoltaic devices fabricated using aluminum catalyzed silicon nanowires.

Single nanowire radial junction solar cell devices were fabricated using Si nanowires synthesized by Al-catalyzed vapor-liquid-solid growth of the p(+) core (Al auto-doping) and thin film deposition of the n(+)-shell at temperatures below 650 °C. Short circuit current densities of 11.7 mA cm(-2) were measured under 1-sun AM1.5G illumination, showing enhanced optical absorption. The power conversion efficiencies were limited to < 1% by the low open circuit voltage and fill factor of the devices, which was attributed to junction shunt leakage promoted by the high p(+)/n(+) doping. This demonstration of a radial junction device represents an important advance in the use of Al-catalyzed Si nanowire growth for low cost photovoltaics.

Inhibitory action of oxytocin on spontaneous contraction of rat distal colon by nitrergic mechanism: involvement of cyclic GMP and apamin-sensitive K+ channels.

AIM: The mechanisms underlying the inhibitory effects of oxytocin (OT) on colon tone are not totally understood. We explore the mechanisms of OT on spontaneous contractility in rat distal colon and identify the mediators involved in this action. METHODS: In rat distal colon strips, mechanical activity was analysed and the production of nitric oxide (NO) in tissue loaded with the fluorochrome DAF-FM was visualized by confocal microscopy. OT receptor (OTR) expression was determined by Western blotting and immunofluorescence. RESULTS: In rat distal colon, OT produced a concentration-dependent reduction in the spontaneous contraction, which was abolished by the OTR antagonist atosiban, the neural blocker tetrodotoxin and the inhibitor of neuronal nitric oxide synthase (nNOS) NPLA. The inhibitory effects of OT were not affected by propranolol, atropine, the nicotinic cholinoceptor blocker hexamethonium, the vasoactive intestinal peptide receptor antagonist VIPHyb, the P2 purinoceptor antagonist PPADS, the adenosine A1 receptors antagonist DPCPX and the prostacyclin receptor antagonist Ro1138452. The soluble guanylyl cyclase (sGC) inhibitor ODQ and the small conductance Ca2+ -activated K+ (Ca K+ ) channels blocker apamin significantly reduced the relaxation induced by OT, nicotine, sodium nitroprusside and the sGC activator BAY 41-2272. The neural release of NO elicited by OT was prevented by NPLA, tetrodotoxin and atosiban. The presence of the OTR and its co-localization with nNOS was detected by immunohistochemistry and Western blotting experiments. CONCLUSION: These results demonstrate the NO release from enteric neurones induced by activation of OTR mediates distal colon relaxation. sGC and small conductance Ca K+ channels are involved in this relaxation.

Synergistic cytotoxicity of radiation and oncolytic Lister strain vaccinia in (V600D/E)BRAF mutant melanoma depends on JNK and TNF-α signaling.

Melanoma is an aggressive skin cancer that carries an extremely poor prognosis when local invasion, nodal spread or systemic metastasis has occurred. Recent advances in melanoma biology have revealed that RAS-RAF-MEK-ERK signaling has a pivotal role in governing disease progression and treatment resistance. Proof-of-concept clinical studies have shown that direct BRAF inhibition yields impressive responses in advanced disease but these are short-lived as treatment resistance rapidly emerges. Therefore, there is a pressing need to develop new targeted strategies for BRAF mutant melanoma. As such, oncolytic viruses represent a promising cancer-specific approach with significant activity in melanoma. This study investigated interactions between genetically-modified vaccinia virus (GLV-1h68) and radiotherapy in melanoma cell lines with BRAF mutant, Ras mutant or wild-type genotype. Preclinical studies revealed that GLV-1h68 combined with radiotherapy significantly increased cytotoxicity and apoptosis relative to either single agent in (V600D)BRAF/(V600E)BRAF mutant melanoma in vitro and in vivo. The mechanism of enhanced cytotoxicity with GLV-1h68/radiation (RT) was independent of viral replication and due to attenuation of JNK, p38 and ERK MAPK phosphorylation specifically in BRAF mutant cells. Further studies showed that JNK pathway inhibition sensitized BRAF mutant cells to GLV-1h68-mediated cell death, mimicking the effect of RT. GLV-1h68 infection activated MAPK signaling in (V600D)BRAF/(V600E)BRAF mutant cell lines and this was associated with TNF-α secretion which, in turn, provided a prosurvival signal. Combination GLV-1h68/RT (or GLV-1h68/JNK inhibition) caused abrogation of TNF-α secretion. These data provide a strong rationale for combining GLV-1h68 with irradiation in (V600D/E)BRAF mutant tumors.

Use of an oncolytic vaccinia virus for the treatment of canine breast cancer in nude mice: preclinical development of a therapeutic agent.

Mammary cancers together with cancers of the skin account for about 60% of the total cancers occurring in dogs. The veterinary options for therapeutic management of canine mammary cancer are limited and prognosis for such patients is poor. In this study, we analyzed the functionality of the oncolytic vaccinia virus strain GLV-1h68 as a possible therapeutic agent for canine mammary cancer. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the canine mammary adenoma cell line ZMTH3. Furthermore, after systemic administration this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. The efficient tumor colonization process resulted in inhibition of tumor growth and drastic reduction of tumor size. This is the first report demonstrating that vaccinia virus is an effective tool for the therapy of canine mammary cancers, which might next be applied to dogs with breast tumors.

A Renilla luciferase-Aequorea GFP (ruc-gfp) fusion gene construct permits real-time detection of promoter activation by exogenously administered mifepristone in vivo.

In this study, we used a steroid-induced promoter activation system as a molecular switch to study the exogenous activation of transgene expression. This promoter activation system consists of three components: (1) a steroidal inducer drug, mifepristone (RU486), which binds to (2) a chimeric transcription factor complex, consisting of the mutant human progesterone receptor fused to the yeast GAL4 DNA-binding domain and the activation domain of the herpes simplex virus protein VP16, and (3) a synthetic promoter, consisting of a series of GAL4 recognition sequences upstream of the adenovirus major late E1B TATA box, linked to a gene construct (ruc-gfp) encoding a Renilla luciferase- Aequorea green fluorescent protein (GFP) fusion protein. Transcription of the promoter-marker gene cassette is activated by the drug (mifepristone)-bound chimeric transcription factor complex. Monitoring of induced gene expression was carried out using a low-light video camera and a UV microscope to detect luciferase and GFP, respectively. Using this activation system, we observed a 10- to 25-fold activation, depending on the inducer dose, of both luciferase and GFP expression in transiently transfected cells in comparison to cells that were not exposed to mifepristone. We further demonstrated activation of gene expression from the promoter activation system in live animals. The plasmids PAP CMV-GL914VPc'SV, carrying the chimeric transcription factor cassette, and plasmid p17x4-TATA-ruc-gfp, carrying the ruc-gfp reporter gene construct, were co-injected into limb muscles of nude mice. Following DNA injection, mifepristone (50 micro g/kg) was delivered by intraperitoneal injection. Thirty-six hours after DNA and mifepristone injection, significant Renilla luciferase activity was detectable in the limb muscles. The promoter activation system was also demonstrated in limb muscles and livers of nude mice that had received transplants of ex vivo-modified cells, which were transiently transformed with both the chimeric activator plasmid and the ruc-gfp reporter plasmid prior to implantation. Significant Renilla activity and GFP fluorescence were detected externally in limb muscles and in the livers of anesthetized animals that had received an intraperitoneal injection of inducer. This external monitoring method for observing inducible gene expression in live animals will facilitate experimental studies of fundamental questions of biological and therapeutic relevance. It will be especially valuable for the analysis of gene function at specific stages of animal development. The method should also be of general use in gene therapy, since it permits simultaneous monitoring of the expression levels of light-emitting proteins and therapeutic proteins originating from the activation of identical promoters.

Methylation segments are not required for chemotactic signalling by cytoplasmic fragments of Tsr, the methyl-accepting serine chemoreceptor of Escherichia coli.

The serine chemoreceptor Tsr and other methyl-accepting chemotaxis proteins (MCPs) control the swimming behaviour of Escherichia coli by generating signals that influence the direction of flagellar rotation. MCPs produce clockwise (CW) signals by stimulating the autophosphorylation activity of CheA, a cytoplasmic histidine kinase, and counter-clockwise signals by inhibiting CheA. CheW couples CheA to chemoreceptor control by promoting formation of MCP/CheW/CheA ternary complexes. To identify MCP structural determinants essential for CheA stimulation, we inserted fragments of the tsr coding region into an inducible expression vector and used a swimming contest called 'pseudotaxis' to select for transformant cells carrying CW-signalling plasmids. The shortest active fragment we found, Tsr (350-470), stimulated CheA in a CheW-dependent manner, as full-length Tsr molecules do. It spans a highly conserved 'core' (370-420) that probably specifies the CheA and CheW contact sites and other determinants needed for stimulatory control of CheA. Tsr (350-470) also carries portions of the left and right arms flanking the core, which probably play roles in regulating MCP signalling state. However, this Tsr fragment lacks all of the methylation sites characteristic of MCP molecules, indicating that methylation segments are not essential for generating receptor output signals.

Renilla luciferase- Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals.

Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo. Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the "humanized" GFP (gfp) from Aequorea. A plasmid containing the fusion gene construct was successfully transformed into, and expressed in, mammalian cells. The transformed cells exhibited both Renilla luciferase activity in the presence of coelenterazine and GFP fluorescence upon excitation with UV light. Spectrofluorometry of cells containing the Ruc-GFP fusion protein, in the absence of wavelengths capable of exciting GFP fluorescence but in the presence of the luciferase substrate, coelenterazine, showed an emission spectrum with two peaks at 475 nm and 508 nm. These two peaks correspond to the emission maximum of Renilla luciferase at 475 nm and that of GFP at 508 nm. The peak at 508 nm generated in the presence of coelenterazine alone (without UV excitation) is the result of intramolecular energy transfer from Renilla luciferase to Aequorea GFP. Southern analysis of genomic DNA purified from transformed Chinese hamster ovary (CHO) cells and fluorescence in situ hybridization (FISH) to metaphase chromosomes confirmed the integration of the ruc-gfp fusion gene on a single chromosome. The bifunctional Ruc-GFP fusion protein allows the detection of gene expression at the single-cell level based on green fluorescence, and in a group of cells based on luminescence emission. Furthermore, animal experiments revealed that light emission from the Ruc-GFP fusion protein can be detected externally in the organs or tissues of live animals bearing the gene construct.