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During our study on HOXA13, HOXD12, and HOXD13 mRNA expression in human adult and embryonic tissues, we were confronted with the fact that, within our specimen collection, as in other University Departments in Europe, <20% of all samples yielded reliable labeling, while most samples were resistant to hybridization by standard protocols due to over-fixation. Fixation is essential for specimen stability, especially when samples are stored at room temperature and used for histology, and people tend to be more worried about under- than over-fixation. On the other hand fixation inhibits penetration by the probe and may also trap mRNA within ribosomes. Therefore, we developed a nonradioactive in situ hybridization technique, which allows detection of mRNA expressed on low levels from a variety of differentially fixed tissues while maintaining tissue integrity. This was achieved by improving target retrieval and probe detection. In contrast with others, our method allows reliable staining from tissues that are fixed in paraformaldehyde from four hours to over one week, and archived samples that were stored at room temperature for several years (17-19 yr in some cases) and exceeds detection limits of purely fluorescent methods. Our protocol is highly suitable for detecting CDX-2 mRNA in carcinoma specimens, but especially designed to investigate mRNAs in nonpathological adult and embryonic tissues. Due to the use of standardized probes, we do not expect problems in detecting other mRNAs expressed in suitable amounts.

Background: More than 20% of human transcripts have naturally occurring antisense products (or natural antisense transcripts--NATs), some of which may play a key role in a range of human diseases. To date, several databases of in silico defined human sense-antisense (SAS) pairs have appeared, however no study has focused on differential expression of SAS pairs in breast tissue. We therefore investigated the expression levels of sense and antisense transcripts in normal and malignant human breast epithelia using the Affymetrix HG-U133 Plus 2.0 and Almac Diagnostics Breast Cancer DSA microarray technologies as well as massively parallel signature sequencing (MPSS) data., Results: The expression of more than 2500 antisense transcripts were detected in normal breast duct luminal cells and in primary breast tumors substantially enriched for their epithelial cell content by DSA microarray. Expression of 431 NATs were confirmed by either of the other two technologies. A corresponding sense transcript could be identified on DSA for 257 antisense transcripts. Of these SAS pairs, 163 have not been previously reported. A positive correlation of differential expression between normal and malignant breast samples was observed for most SAS pairs. Orientation specific RT-QPCR of selected SAS pairs validated their expression in several breast cancer cell lines and solid breast tumours., Conclusion: Disease-focused and antisense enriched microarray platforms (such as Breast Cancer DSA) confirm the assumption that antisense transcription in the human breast is more prevalent than previously anticipated. Expression of a proportion of these NATs has already been confirmed by other technologies while the true existence of the remaining ones has to be validated. Nevertheless, future studies will reveal whether the relative abundances of antisense and sense transcripts have regulatory influences on the translation of these mRNAs.

Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis, vasculogenesis and vascular permeability. Edema in glioma tumors is considered one of the most pathological characteristics, but the mechanism of regulating vascular permeability is still unclear. In the present study, tumorigenic mice were generated by subcutaneous injection of glioma cell lines, C6-null cells and stable transfected-C6 cells overexpressing mock vector (C6-mock) and antisense VEGF (C6-VEGF(-/-)). Overexpression of antisense VEGF (C6-VEGF(-/-) mice) significantly suppressed tumor growth, decreased angiogenesis and reduced tumoral edema. Further studies by electron microscope revealed that tumor-induced hyperpermeability was mediated by formation of vesiculo-vacuolar organelles (VVO), specifically reducing the number of vesicle and caveolae in VVO, and this effect was blocked, at least partially, by antisense VEGF. These data show a possible mechanism of tumor-induced hyperpermeability and indicate that blockage of VEGF might contribute to therapeutical strategies for tumor edema.

Hepatocellular carcinoma (HCC), one of the most common cancers worldwide, is resistant to anticancer drugs. Hypoxia is a major cause of tumor resistance to chemotherapy, and hypoxia-inducible factor (HIF)-1 is a key transcription factor in hypoxic responses. We have previously demonstrated that gene transfer of an antisense HIF-1alpha expression vector downregulates expression of HIF-1alpha and vascular endothelial growth factor (VEGF), and synergizes with immunotherapy to eradicate lymphomas. The aim of the present study was to determine whether gene transfer of antisense HIF-1alpha could enhance the therapeutic efficacy of doxorubicin to combat HCC. Both antisense HIF-1alpha therapy and doxorubicin suppressed the growth of subcutaneous human HepG2 tumors established in BALB/c nude mice, tumor angiogenesis, and cell proliferation, and induced tumor cell apoptosis. The combination therapy with antisense HIF-1alpha and doxorubicin was more effective in suppressing tumor growth, angiogenesis, and cell proliferation, and inducing cell apoptosis than the respective monotherapies. Gene transfer of antisense HIF-1alpha downregulated the expression of both HIF-1alpha and VEGF, whereas doxorubicin only downregulated VEGF expression. Antisense HIF-1alpha and doxorubicin synergized to downregulate VEGF expression. Both antisense HIF-1alpha and doxorubicin inhibited expression of proliferating cell nuclear antigen, and combined to exert even stronger inhibition of proliferating cell nuclear antigen expression. Antisense HIF-1alpha therapy warrants investigation as a therapeutic strategy to enhance the efficacy of doxorubicin for treating HCC.

Background: As nuclear mediators of wnt/beta-catenin signaling, Lef/Tcf transcription factors play important roles in development and disease. Although it is well established, that the four vertebrate Lef/Tcfs have unique functional properties, most studies unite Lef-1, Tcf-1, Tcf-3 and Tcf-4 and reduce their function to uniformly transduce wnt/beta-catenin signaling for activating wnt target genes. In order to discriminate target genes regulated by XTcf-3 from those regulated by XTcf-4 or Lef/Tcfs in general, we performed a subtractive screen, using neuralized Xenopus animal cap explants., Results: We identified cold-inducible RNA binding protein (CIRP) as novel XTcf-3 specific target gene. Furthermore, we show that knockdown of XTcf-3 by injection of an antisense morpholino oligonucleotide results in a general broadening of the anterior neural tissue. Depletion of XCIRP by antisense morpholino oligonucleotide injection leads to a reduced stability of mRNA and an enlargement of the anterior neural plate similar to the depletion of XTcf-3., Conclusion: Distinct steps in neural development are differentially regulated by individual Lef/Tcfs. For proper development of the anterior brain XTcf-3 and the Tcf-subtype specific target XCIRP appear indispensable. Thus, regulation of anterior neural development, at least in part, depends on mRNA stabilization by the novel XTcf-3 target gene XCIRP.

The genomic architecture of the budding yeast Saccharomyces cerevisiae is typical of other eukaryotes in that genes are spatially organized into discrete and nonoverlapping units. Inherent in this organizational model is the assumption that protein-coding sequences do not overlap completely. Here, we present evidence to the contrary, defining a previously overlooked yeast gene, NAG1 (for nested antisense gene) nested entirely within the coding sequence of the YGR031W open reading frame in an antisense orientation on the opposite strand. NAG1 encodes a 19-kDa protein, detected by Western blotting of hemagglutinin (HA)-tagged Nag1p with anti-HA antibodies and by beta-galactosidase analysis of a NAG1-lacZ fusion. NAG1 is evolutionarily conserved as a unit with YGR031W in bacteria and fungi. Unlike the YGR031WP protein product, however, which localizes to the mitochondria, Nag1p localizes to the cell periphery, exhibiting properties consistent with those of a plasma membrane protein. Phenotypic analysis of a site-directed mutant (nag1-1) disruptive for NAG1 but silent with respect to YGR031W, defines a role for NAG1 in yeast cell wall biogenesis; microarray profiling of nag1-1 indicates decreased expression of genes contributing to cell wall organization, and the nag1-1 mutant is hypersensitive to the cell wall-perturbing agent calcofluor white. Furthermore, production of Nag1p is dependent upon the presence of the cell wall integrity pathway mitogen-activated protein kinase Slt2p and its downstream transcription factor Rlm1p. Thus, NAG1 is important for two reasons. First, it contributes to yeast cell wall biogenesis. Second, its genomic context is novel, raising the possibility that other nested protein-coding genes may exist in eukaryotic genomes.

Purpose: To explore the possibility of the c-erbB2 oncogene antisense probe labeled with superparamagnetic iron oxide (SPIO) nanoparticles as a target contrast agent for magnetic resonance (MR) imaging whose morphology was observed with atomic force microscopy (AFM), and its efficiency was examined by MR imaging., Methods: The c-erbB2 oncogene antisense probe labeled with SPIO was synthesized by a chemical cross-linking approach. Its morphology was observed with AFM., Results: The chemical constitution of c-erbB2 oncogene antisense probes can be observed with AFM. The molecular structure of probes is easily visualized under AFM. Probes with diameters of 25-40 nm are in order, follow uniformity and the arrangement rule, can be separated from each other, and appear as cubes with a rugged surface morphology. Strong, low signals of the probes in transfected cells were observed by MR cellular imaging., Conclusions: AFM is ideal for morphological observation and for analyzing the molecular structure of synthesized c-erbB2 oncogene antisense probes.

Genome-wide DNA rearrangements occur in many eukaryotes but are most exaggerated in ciliates, making them ideal model systems for epigenetic phenomena. During development of the somatic macronucleus, Oxytricha trifallax destroys 95% of its germ line, severely fragmenting its chromosomes, and then unscrambles hundreds of thousands of remaining fragments by permutation or inversion. Here we demonstrate that DNA or RNA templates can orchestrate these genome rearrangements in Oxytricha, supporting an epigenetic model for sequence-dependent comparison between germline and somatic genomes. A complete RNA cache of the maternal somatic genome may be available at a specific stage during development to provide a template for correct and precise DNA rearrangement. We show the existence of maternal RNA templates that could guide DNA assembly, and that disruption of specific RNA molecules disables rearrangement of the corresponding gene. Injection of artificial templates reprogrammes the DNA rearrangement pathway, suggesting that RNA molecules guide genome rearrangement.

Overexpression of the cytokine transforming growth factor-beta 2 (TGF-beta2) is a hallmark of various malignant tumors including pancreatic carcinoma, malignant glioma, metastasizing melanoma, and metastatic colorectal carcinoma. This is due to the pivotal role of TGF-beta2 as it regulates key mechanisms of tumor development, namely immunosuppression, metastasis, angiogenesis, and proliferation. The antisense technology is an innovative technique offering a targeted approach for the treatment of different highly aggressive tumors and other diseases. Antisense oligonucleotides are being developed to inhibit the production of disease-causing proteins at the molecular level. The immunotherapeutic approach with the phosphorothioate oligodeoxynucleotide AP 12009 for the treatment of malignant tumors is based on the specific inhibition of TGF-beta2. After providing preclinical proof of concept, the safety and efficacy of AP 12009 were assessed in clinical phase I/II open-label dose-escalation studies in recurrent or refractory high-grade glioma patients. Median survival time after recurrence exceeded the current literature data for chemotherapy. Currently, phase I/II study in advanced pancreatic carcinoma, metastatic melanoma, and metastatic colorectal carcinoma and a phase IIb study in recurrent or refractory high-grade glioma are ongoing. The preclinical as well as the clinical results implicate targeted TGF-beta2 suppression as a promising therapeutic approach for malignant tumor therapy.

Chromatin allows the eukaryotic cell to package its DNA efficiently. To understand how chromatin structure is controlled across the Saccharomyces cerevisiae genome, we have investigated the role of the ATP-dependent chromatin remodelling complex Isw2 in positioning nucleosomes. We find that Isw2 functions adjacent to promoter regions where it repositions nucleosomes at the interface between genic and intergenic sequences. Nucleosome repositioning by Isw2 is directional and results in increased nucleosome occupancy of the intergenic region. Loss of Isw2 activity leads to inappropriate transcription, resulting in the generation of both coding and noncoding transcripts. Here we show that Isw2 repositions nucleosomes to enforce directionality on transcription by preventing transcription initiation from cryptic sites. Our analyses reveal how chromatin is organized on a global scale and advance our understanding of how transcription is regulated.

Cel1 and Cel2 are members of the tomato (Solanum lycopersicum Mill) endo-beta-1,4-glucanase (EGase) family that may play a role in fruit ripening and organ abscission. This work demonstrates that Cel1 protein is present in other vegetative tissues and accumulates during leaf development. We recently reported the downregulation of both the Cel1 mRNA and protein upon fungal infection, suggesting the involvement of EGases in plant-pathogen interactions. This hypothesis was confirmed by assessing the resistance to Botrytis cinerea infection of transgenic plants expressing both genes in an antisense orientation (Anti-Cel1, Anti-Cel2 and Anti-Cel1-Cel2). The Anti-Cel1-Cel2 plants showed enhanced resistance to this fungal necrotroph. Microscopical analysis of infected leaves revealed that tomato plants accumulated pathogen-inducible callose within the expanding lesion. Anti-Cel1-Cel2 plants presented a faster and enhanced callose accumulation against B. cinerea than wild-type plants. The inhibitor 2-deoxy-d-glucose, a callose synthesis inhibitor, showed a direct relationship between faster callose accumulation and enhanced resistance to B. cinerea. EGase activity appears to negatively modulate callose deposition. The absence of both EGase genes was associated with changes in the expression of the pathogen-related genes PR1 and LoxD. Interestingly, Anti-Cel1-Cel2 plants were more susceptible to Pseudomonas syringae, displaying severe disease symptoms and enhanced bacterial growth relative to wild-type plants. Analysis of the involvement of Cel1 and Cel2 in the susceptibility to B. cinerea in fruits was done with the ripening-impaired mutants Never ripe (Nr) and Ripening inhibitor (rin). The data reported in this work support the idea that enzymes involved in cell wall metabolism play a role in susceptibility to pathogens.

Iron is required for the differentiation of HL-60 cells along the monocyte lineage in vitro, reflecting a requirement for iron in the transcriptional induction of the p21(WAF1/CIP1) gene. To determine if the same requirement holds true for differentiation in other cell lineages and for primary human CD34(+) bone marrow precursor cells, we induced granulocyte differentiation by treating HL-60 cells with dimethyl sulphoxide, and erythroid or megakaryocytic differentiation by treating K562 cells with butyrate or phorbol myristate acetate, respectively. Nitro blue tetrazolium reduction, expression of haem, or expression of CD41 was used to assess granulocytic, erythroid, or megakaryocytic differentiation respectively. Purified CD34(+) cells were cultured with granulocyte/macrophage-colony stimulating factor and stem cell factor to induce myelomonocytic differentiation. Iron deprivation was induced by desferrioxamine. p21(WAF1/CIP1) antisense oligonucleotides were used to inhibit p21 expression. Iron deprivation blocked p21 induction as judged by real-time polymerase chain reaction assays. In addition, both iron deprivation and p21 antisense blocked CD34(+) cell differentiation. These observations were not explained by induction of widespread apoptosis under conditions of iron deprivation. We concluded that both iron and functional p21(WAF1/CIP1) are required for in vitro differentiation of human haematopoietic precursors along multiple cell lineages.

Background: Understanding the functional role(s) of the more than 20,000 proteins of the vertebrate genome is a major next step in the post-genome era. The approximately 4,000 co-translationally translocated (CTT) proteins - representing the vertebrate secretome - are important for such vertebrate-critical processes as organogenesis. However, the role(s) for most of these genes is currently unknown., Results: We identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. We further investigated 150 of these genes (Figure 1) for unique function using morpholino-based analysis in zebrafish embryos. 12% of the CTT protein-deficient embryos resulted in specific developmental defects, a notably higher rate of gene function annotation than the 2%-3% estimate from random gene mutagenesis studies., Conclusion: This initial collection includes novel genes required for the development of vascular, hematopoietic, pigmentation, and craniofacial tissues, as well as lipid metabolism, and organogenesis. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome.

Peptide nucleic acids (PNAs) have shown great promise as potential antisense drugs; however, poor cellular delivery limits their applications. Improved delivery into mammalian cells and enhanced biological activity of PNAs have been achieved by coupling to cell-penetrating peptides (CPPs). Structural requirements for the shuttling ability of these peptides as well as structural properties of the conjugates such as the linker type and peptide position remained controversial, so far. In the present study an 18mer PNA targeted to the cryptic splice site of a mutated beta-globin intron 2, which had been inserted into a luciferase reporter gene coding sequence, was coupled to various peptides. As the peptide lead we used the cell-penetrating alpha-helical amphipathic peptide KLAL KLAL KAL KAAL KLA-NH2 [model amphipathic peptide (MAP)] which was varied with respect to charge and structure-forming properties. Furthermore, the linkage and the localization of the attached peptide (C- vs N-terminal) were modified. Positive charge as well as helicity and amphipathicity of the KLA peptide was all required for efficient dose-dependent correction of aberrant splicing. The highest antisense effect was reached within 4 h without any transfection agent. Stably linked conjugates were also efficient in correction of aberrant splicing, suggesting that a cleavable disulfide bond between CPP and PNA is clearly not essential. Moreover, the placement of the attached peptide turned out to be crucial for attaining antisense activity. Coadministration of endosome disrupting agents such as chloroquine or Ca2+ significantly increased the splicing correction efficiency of some conjugates, indicating the predominant portion to be sequestered in vesicular compartments.

In bacteria, biogenesis of cell wall at the division site requires penicillin-binding protein 3 (PBP3) (or Ftsl). Using pull-down, bacterial two-hybrid, and peptide-based interaction assays, we provide evidence that FtsW of Mycobacterium tuberculosis (FtsWMTB) interacts with PBP3 through two extracytoplasmic loops. Pro306 in the larger loop and Pro386 in the smaller loop of FtsW are crucial for these interactions. Fluorescence microscopy shows that conditional silencing of ftsW in Mycobacterium smegmatis prevents cell septation and positioning of PBP3 at mid-cell. Pull-down assays and conditional depletion of FtsW in M. smegmatis provide evidence that FtsZ, FtsW and PBP3 of mycobacteria are capable of forming a ternary complex, with FtsW acting as a bridging molecule. Bacterial three-hybrid analysis suggests that in M. tuberculosis, the interaction (unique to mycobacteria) of FtsZ with the cytosolic C-tail of FtsW strengthens the interaction of FtsW with PBP3. ftsW of M. smegmatis could be replaced by ftsW of M. tuberculosis. FtsWMTB could support formation of the FtsZ-FtsW-PBP3 ternary complex in M. smegmatis. Our findings raise the possibility that in the genus Mycobacterium binding of FtsZ to the C-tail of FtsW may modulate its interactions with PBP3, thereby potentially regulating septal peptidoglycan biogenesis.

Background: Local structures of target mRNAs play a significant role in determining the efficacies of antisense oligonucleotides (ODNs), but some structure-based target site selection methods are limited by uncertainties in RNA secondary structure prediction. If all the predicted structures of a given mRNA within a certain energy limit could be used simultaneously, target site selection would obviously be improved in both reliability and efficiency. In this study, some key problems in ODN target selection on the basis of multiple predicted target mRNA structures are systematically discussed., Results: Two methods were considered for merging topologically different RNA structures into integrated representations. Several parameters were derived to characterize local target site structures. Statistical analysis on a dataset with 448 ODNs against 28 different mRNAs revealed 9 features quantitatively associated with efficacy. Features of structural consistency seemed to be more highly correlated with efficacy than indices of the proportion of bases in single-stranded or double-stranded regions. The local structures of the target site 5' and 3' termini were also shown to be important in target selection. Neural network efficacy predictors using these features, defined on integrated structures as inputs, performed well in "minus-one-gene" cross-validation experiments., Conclusion: Topologically different target mRNA structures can be merged into integrated representations and then used in computer-aided ODN design. The results of this paper imply that some features characterizing multiple predicted target site structures can be used to predict ODN efficacy.

'Galia' muskmelon (Cucumis melo L. var. reticulatus Ser.) has been recalcitrant to transformation by Agrobacterium tumefaciens. Transformation of the 'Galia' male parental line, 'Krymka', with an ACC oxidase (CMACO-1) gene in antisense orientation is described herein. Explants were transformed using A. tumefaciens strain ABI, which contained a vector pCmACO1-AS plasmid, bearing an antisense gene of CMACO-1 and the CP4 syn gene (glyphosate-tolerance). Both CMACO-1 and CP4 syn genes were assessed by a polymerase chain reaction method. Flow cytometry analysis was performed to determine plant ploidy level of primary transformants. Two completely diploid independent transgenic plants were obtained. Southern blot and segregation analysis in the T1 generation determined that each independent transgenic line had one single insertion of the transgene. These transgenic muskmelon male parental lines have potential for use in the production of 'Galia' F1 hybrids with improved shelf life.

Tamoxifen (TAM) is widely used in the treatment and prevention of breast cancer. There is clear evidence that cytochrome P450 (CYP) 3A enzymes play an important role in TAM metabolism, resulting in metabolites that lead to formation of TAM-DNA adducts. We have investigated the effect of CYP3A2 antisense (AVI-4472) exposure on CYP3A2 transcription, enzyme activity, translation, and TAM-DNA adducts, in livers of rats administered TAM (50 mg/kg body weight [bw]/day) for 7 days. The study design included administration of 0, 0.5, 2.5, or 12.5 mg AVI-4472/kg bw/day for 8 days, beginning 1 day before TAM exposure. The specific activity of CYP3A2 was increased after TAM administration, and decreased significantly (approximately 70%) in the presence of 12.5 mg AVI-4472. CYP3A2 protein levels, determined by immunoblot analysis, showed a similar pattern. Hepatic TAM-DNA adduct levels were measurable in all TAM-exposed groups. However, when rats were co-treated with 2.5 and 12.5 mg AVI-4472/kg bw/day, statistically significant (approximately 50%) reductions in TAM-DNA adduct levels (2.0-2.8 adducts/10(8) nucleotides) were observed compared to rats treated with TAM alone (5.1 adducts/10(8) nucleotides). Rat toxicology U34 arrays (Affymetrix) were used to investigate the modulation of gene expression patterns on co-administration of TAM with AVI-4472. Results indicated that several CYP genes were down regulated although no significant induction of CYP3A2 was observed in the TAM-exposed rats co-treated with AVI-4472. Overall the data suggest the utility of antisense technology in the redirection of TAM metabolism thereby lowering TAM genotoxicity in rat liver.

Several models have been proposed to explain why expression parameters of a gene might be related to the size of the gene's introns. These include the idea that an energetic cost of transcription should favor smaller introns in highly expressed genes (the "economy selection" argument) and that tissue-specific genes reside in genomic locations with complex chromatin level control requiring large amounts of noncoding DNA (the "genomic design" hypothesis). We recently proposed a modification of the economy model arguing that, for some genes, the time that expression takes is more important than the energetic cost, such that some weakly but rapidly expressed genes might also have small introns. We suggested that antisense genes might be such a class and showed that the data appear to be consistent with this. We now reexamine this model to ask (a) whether the effects described were owing solely to the fact that antisense genes are often noncoding RNA and (b) whether we can confidently reject the "genomic design" model as an explanation for the facts. We show that the effects are not specific to noncoding RNAs and that the predictions of the "genomic design" model for the most part are not upheld.

Two transgenic potato lines with antisense AcInv gene and the non-transgenic varieties were used to test the reducing sugar and starch contents. The results indicated that the reducing sugar content increased and total starch content decreased in all samples after tubers were stored at 4 degrees C for 40 d. The reducing sugar content in tubers of transgenic line "Anti-AcInv Atlantic" and "Anti-AcInv Gannongshu No.2" were lower about 23% and 18% than those of Atlantic and Gannongshu No.2 (Table 1). The total starch and the amylopectin were also decreased by 1% and 1.3% in "Anti-AcInv Atlantic" and 1.4% and 1.7% in "Anti-AcInv Gannongshu No.2" respectively (Table 2). The proportions of amylose and amylopectin were lower in the transgenic lines than the non-transgenic varieties (Figs. 1 and 2). It was only 0.29 for "Anti-AcInv Atlantic" and 0.38 for "Anti-AcInv Gannongshu No.2". Meanwhile, there are fewer dark blue starch particles in transgenic tuber, which is less than in tuber of the non-transgenic varieties by paraffin-cut section method (Fig. 3). After the tubers were transferred to the storage temperature 15-17 degrees C for 20 d, the reducing sugar contents in tubers of the two transgenic lines were significantly lower than that of non-transgenic varieties. It was 0.23% for "Anti-AcInv Atlantic" and 0.24% for "Anti-AcInv Gannongshu No.2" (Table 1). It is suggested that the trans-antisense AcInv gene in the transgenic potato down-regulates the AcInv gene expression after the tubers were stored under low temperature.

In this study we present the characterization of a novel transcript, UL81-82ast, UL81-82 antisense transcript, and its protein product. The transcript was initially found in a cDNA library of monocytes from a seropositive donor. mRNA was obtained from monocytes isolated from a healthy donor with a high antibody titer against human cytomegalovirus (HCMV). The mRNAs were cloned into a lambda phage-derived vector to create the cDNA library. Using PCR, UL81-82ast was amplified from the library. The library was tested for the presence of numerous HCMV genes. Neither structural genes nor immediate-early genes were found. UL81-82ast was detected in five bone marrow samples from healthy antibody-positive donors. This same transcript was also found in in vitro-infected human fibroblasts early after infection but disappears at the same time that UL82 transcription begins. Not only was the transcript amplified using reverse transcription-PCR and sequenced but its protein product (UL82as protein) was detected by both Western blot and immunofluorescence. Phylogenetic studies using UL82as protein were conducted, showing a high degree of conservation in clinical isolates, laboratory strains of HCMV, and even in chimpanzee CMV. The transcript could be involved in the posttranscriptional regulation of the UL82 gene, affecting its mRNA stability or translation. Since the UL82 product, pp71, functions as an immediate-early transactivator, its posttranscriptional control could have some effect over latency reactivation and lytic replication.

Macroautophagy is an intracellular degradation system for the majority of proteins and some organelles that is conserved in all eukaryotic species. The precise role of autophagy in mammalian development and potential involvement in disease remain to be discerned. Yeast Atg9p is the first integral membrane protein shown to be essential for the cytoplasm to vacuole targeting (Cvt) pathway and autophagy, whereas its mammalian functional orthologue has yet to be identified. We have identified two human genes homologous to yeast Atg9p and designated these as APG9L1 and APG9L2. We have previously identified APG9L2 as NOS3AS, which participates in the post-transcriptional regulation of the endothelial nitric-oxide synthase (NOS3) gene on chromosome 7 through its antisense overlap. In human adult tissues, APG9L1 was ubiquitously expressed, whereas APG9L2 was highly expressed in placenta (trophoblast cells) and pituitary gland. In transient transfection assays we found that both proteins were primarily localized to the perinuclear region and also scattered throughout the cytosol as dots, a subset of which colocalized with an autophagosome-specific marker LC3 under starvation conditions. Finally, by the small interfering RNA-mediated knockdown of APG9L1 in HeLa cells, we demonstrated that APG9L1 is essential for starvation-induced autophagosome formation. In addition, APG9L2 can functionally complement APG9L1 in this process. These results, taken together with those of phylogenetic and sequence analyses, suggest that both APG9L1 and APG9L2 are functionally orthologous to the yATG9 in autophagosome formation. Moreover, APG9L2 is a vertebrate-specific gene that may have gained critical roles in mammalian-specific developmental events, such as placentation, through rapid evolution.

In extant chordates, the overall patterning along the anteroposterior and dorsoventral axes of the neural tube is remarkably conserved. It has thus been proposed that four domains corresponding to the vertebrate presumptive forebrain, midbrain-hindbrain transition, hindbrain, and spinal cord were already present in the common chordate ancestor. To obtain insights on the evolution of the patterning of the anterior neural tube, we performed a study aimed at characterizing the expression of regulatory genes in the sensory vesicle of Ciona intestinalis, the anteriormost part of the central nervous system (CNS) related to the vertebrate forebrain, at tailbud stages. Selected genes encoded primarily for homologues of transcription factors involved in vertebrate forebrain patterning. Seven of these genes were expressed in the ventral sensory vesicle. A prominent feature of these ascidian genes is their restricted and complementary domains of expression at tailbud stages. These patterning markers thus refine the map of the developing sensory vesicle. Furthermore, they allow us to propose that a large part of the ventral and lateral sensory vesicle consists in a patterning domain corresponding to the vertebrate presumptive hypothalamus.

Antisense agents that inhibit genes at the mRNA level are attractive tools for genome-wide studies and drug target validation. The approach may be particularly well suited to studies of bacteria that are difficult to manipulate with standard genetic tools. Antisense peptide nucleic acids (PNA) with attached carrier peptides can inhibit gene expression in Escherichia coli and Staphylococcus aureus. Here we asked whether peptide-PNAs could mediate antisense effects in Mycobacterium smegmatis. We first targeted the gfp reporter gene and observed dose- and sequence-dependent inhibition at low micromolar concentrations. Sequence alterations within both the PNA and target mRNA sequences eliminated inhibition, strongly supporting an antisense mechanism of inhibition. Also, antisense PNAs with various attached peptides showed improved anti-gfp effects. Two peptide-PNAs targeted to the essential gene inhA were growth inhibitory and caused cell morphology changes that resemble that of InhA-depleted cells. Therefore, antisense peptide-PNAs can efficiently and specifically inhibit both reporter and endogenous essential genes in mycobacteria., (Copyright 2005 S. Karger AG, Basel)

The pea chloroplastic fructose-1,6-bisphosphatase (FBPase) antisense construct reduced the endogenous level of expression of the corresponding Arabidopsis thaliana gene. The reduction of foliar FBPase activity in the transformants T(2) and T(3) generation ranged from 20% to 42%, and correlated with lower levels of FBPase protein. FBPase antisense plants displayed different phenotypes with a clear increase in leaf fresh weight. Measurements of photosynthesis revealed a higher carbon-assimilation rate. Decreased FBPase activity boosted the foliar carbohydrate contents, with a shift in the sucrose:starch ratio, which reached a maximum of 0.99 when the activity loss was 41%. Nitrate reductase activity decreased simultaneously with an increase in glutamine synthetase activity, which could be explained in terms of ammonium assimilation regulation by sugar content. These results suggest the role of FBPase as a key enzyme in CO(2) assimilation, and also in co-ordinating carbon and nitrogen metabolism.

In this research, Rubisco activase gene (rca) was amplified using specific primers and inserted into pGEM T-easy vector, and then cut with EcoRI after confirming by sequencing. The fragment was subcloned into pBluescript KS+, digested with the enzyme BamHI and inserted into the binary expression vector pCAMBIA1301, and the resulting construction with antisense rca was named pCAMR02. The pCAMR02 vector was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation and transformed to embryos of rice (Oryza. Sativa L.ssp.japonica) cultivar Zhonghua11 via Agrobacterium tumefaciens system. Plantlets were regenerated in vitro by resistance selection on medium containing various concentrations of hygromycin. Both GUS histochemical assays and PCR amplification demonstrated that antisense rca was integrated into T0 genomes and inherited to T1. The measurement of phenotypes of transgenic rice plants with antisense rca showed that most of them could hardly survive at ambient CO2 concentration, even could not grow. The antisense plants that survived under natural condition were dwarf and grew slower than the wild-type controls, and their contents of RCA and Rubisco changed significantly. These plants generated in this experiment will be used to study the relationship between RCA and Rubisco and their regulation.

At a time when common regulatory pathways are being identified in several different species and genomics is beginning to allow comparisons of genes, how they are arranged on chromosomes and how they are regulated, zebrafish has emerged as a valuable and complementary vertebrate model. Some of the characteristics that prove of value are described and illustrated. Fluorescent transgenic lines of zebrafish embryos are presented for time-line studies with neurotoxicants. While genetic knockout technology has yet to be developed for the model, the anti-sense, morpholino approach allows for knockdown of expression of genes for the 3 day, embryonic period. This can provide for phenocopies of mutant genes for those genes essential to embryonic development or it can provide for a limited inhibition of gene expression that allows subsequent development of the fish. With the zebrafish genomic sequencing effort, microarray technology is now developing for the model system. These resources and technologies allow one to challenge the system with toxicants, and to view the immediate effects of the toxicants with transgenic embryos that fluoresce in part or all of the nervous system. Behavioral and learning protocols have been developed for the organism so that early exposures can be assayed for effects upon adult fish. Microarray technology should allow for one to identify specific genes and pathways affected by a neurotoxicant. In the future, these approaches should provide a working protocol for exploring molecular mechanisms of neurotoxicants. This type of complementary approach should then allow for more efficient examination and testing of mechanisms in mammalian models.

A transient expression assay system was employed to investigate the possible use of the maize Opaque 2 (O2) and prolamin box binding factor (PBF) proteins as transcriptional activators of rice and wheat storage protein gene promoters. When assayed in developing rice endosperm cells, either O2 or PBF alone could increase transcription from the promoter of the rice glutelin gene, Gt1. However, mutant forms of O2 and PBF that are defective in DNA binding could not. Co-transfection with both transcriptional activators resulted in an additive increase in transactivation of the Gt1 promoter. Co-bombardment of a Gt1::GUS construct with plasmids expressing the DNA binding domains of O2 and PBF in antisense orientation resulted in a decrease of GUS expression below background levels. Similar stimulatory and additive effects of O2 and PBF could be observed on the promoters from other storage protein genes including rice globulin (Glb), prolamins (RP6 and PG5a) and a wheat glutenin (Bx7). However, responsiveness of the promoters from non-storage protein genes like rice actin and CaMV 35S to O2 and PBF was insignificant. Our results indicate that the maize O2 and PBF proteins can act singly or additively as effective stimulators of heterologous storage protein promoters in developing rice endosperm cells. These data support the use of well-characterized transcription factors from maize as an effective means of increasing the expression level of recombinant proteins in developing rice seeds.

Gene function studies in bacteria lag behind progress in genome sequencing, in part because current reverse genetics technology based on genome disruption does not allow subtle control of gene expression for all genes in a range of species. Essential genes and clustered regions are particularly problematic. Antisense technology offers an attractive alternative for microbial genomics. Unfortunately, bacteria lack RNAi mechanisms and conventional oligonucleotides are not taken up efficiently. However, in Escherichia coli, efficient and gene-specific antisense knock down is possible using antisense peptide nucleic acids (PNAs) attached to carrier peptides (KFFKFFKFFK). Carrier peptides can enter a range of microbial species, and in this study we asked whether peptide-PNAs could mediate antisense effects in Staphylococcus aureus. Using low micromolar concentrations we observed dose- and sequence-dependent inhibition of the reporter gene gfp and endogenous gene phoB. Also, antisense peptide-PNAs targeted to the essential genes fmhB, gyrA, and hmrB were growth inhibitory. Control peptide-PNAs were much less effective, and sequence alterations within the PNA and target mRNA sequences reduced or eliminated inhibition. Further development is needed to raise the antibacterial potential of PNAs, but the present results show that the approach can be used to study gene function and requirement in this important pathogen.

We report the design of a unique two-plasmid production system for the first lentiviral vector to be evaluated in humans, VRX496. VRX496 is an optimized VSV-G pseudotyped vector derived from HIV-1 that expresses antisense to the HIV envelope gene. We found that a two-plasmid approach to production resulted in higher vector production titers when compared with a three-plasmid approach, which is particularly important for vector production at the large scale. Therefore, we carefully designed a single packaging construct, VIRPAC, for safety by reducing its homology with VRX496 and by insertion of functionally validated genetic elements designed to reduce the risk of generation of a replication-competent lentivirus (RCL). A native cis-acting ribozyme is used to prevent read through into the envelope gene from the upstream gag-pol genes in the packaging vector, thus preventing RNAs containing gag-pol and env together for comparable safety to a three-plasmid system. We demonstrate that there is no significant in vivo vector mobilization using a primary SCID-hu mouse transplantation model, which correlates with the presence of an anti-HIV payload and suggests that inclusion of antisense may be a useful tool to restrict mobilization in other vector constructs. Gene transfer is achieved using a one-step transduction procedure that is simple and clinically translatable, which reaches stable transduction efficiencies of >99% in CD4+ T lymphocytes within 3 days of culture initiation., (Copyright 2004 John Wiley & Sons, Ltd.)

Glutamate excitotoxicity is strongly implicated as a major contributing factor in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Excitotoxicity results from elevated intracellular calcium ion (Ca(2+)) levels, which in turn recruit cell death signaling pathways. Recent evidence suggests that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit (GluR) stoichiometry is a dominant factor leading to excess Ca(2+) loading in neurodegeneration. In particular, the Ca(2+) permeable glutamate receptor subunit 3 (GluR3) has been implicated in several neurologic conditions such as bipolar disorder and epilepsy. Recent proteomic analysis within our group on the copper zinc superoxide dismutase (SOD1)(G93A) transgenic mouse model of familial ALS (FALS) reveals a potentially deleterious upregulation of GluR3 in spinal cord compared to that in wild-type littermates. Based on this finding we designed a 12mer antisense peptide nucleic acid (PNA) directed against GluR3. This sequence significantly reduced levels of GluR3 protein and protected neuroblastoma x spinal cord (NSC-34) cells against death induced by the AMPA receptor-specific agonist (S)-5-fluorowillardiine. We subsequently treated SOD1(G93A) mice thrice weekly with intraperitoneal injections of the antisense PNA (2.5 mg/kg) commencing at postnatal day 50. Mice treated with the antisense sequence had significantly extended survival compared to mice injected with a nonsense sequence. Western blot analysis, however, did not reveal a significant reduction in GluR3 protein levels in whole extracts of the lumbar spinal cord. These results suggest that interference with the GluR3 component of the AMPA receptor assembly may be a novel strategy for controlling excitotoxic destruction of motor neurons and may lead to new therapeutic opportunities for the treatment of human ALS., (Copyright 2004 Wiley-Liss, Inc.)

Objective: To evaluate the effects of transforming growth factor beta1 (TGFbeta1) autocrine blockage on proliferation activity and drug sensitivity of osteosarcoma. METHODS; Northern blot, MTT determination, and 3H thymidine incorporation were used to investigate the effects of antisense TGF beta1 gene on osteosarcoma., Results: The proliferation of osteosarcoma cells transfected by antisense TGF beta1 gene was suppressed markedly, and adriamycin sensitivity was significantly increased., Conclusion: Blockage of osteosarcoma cells TGF beta1 autocrine loop inhibits cell proliferation and enhances chemotherapy sensitivity.

We present preclinical studies that demonstrate in vitro the feasibility and efficacy of lentivirus-based vector antisense gene therapy for control of HIV replication in primary T lymphocytes isolated from HIV-infected patients discordant for clinical status. VRX496 is a VSV-G-pseudotyped HIV-based vector that encodes an antisense payload against the HIV envelope gene. The antisense payload is under the control of the native LTR promoter, which is highly transactivated by tat upon HIV infection in the cell. Transfer of autologous CD4(+) T lymphocytes genetically modified with VRX496 (VRX496T) into HIV-infected patients is intended to provide a reservoir of cells capable of controlling HIV, potentially delaying AIDS onset. To determine the patient population likely to respond to VRX496 for optimal efficacy, we examined the ability of our research vector, VRX494, to modify and suppress HIV in vitro in lymphocytes isolated from 20 study subjects discordant for CD4 count and viral load. VRX494 is analogous to the clinical vector VRX496, except that it contains GFP as a marker gene instead of the 186-tag marker in the clinical vector. To transfer VRX494 to target cells we developed a novel scalable two-step transduction procedure that has been translated to the clinic in an ongoing clinical trial. This procedure achieved unprecedented transduction efficiencies of 94 +/- 5% in HIV(+) study subject cells. In addition the vector inhibited HIV replication >/=93% in culture regardless of the viral load or CD4 count of the subject or tropism of the virus strain with which they were infected. These findings demonstrate that VRX496T therapy is expected to be beneficial to patients that differ in their status in term of CD4 count and viral load. The methods described represent significant technical advances facilitating execution of lentivirus vector-mediated gene therapy for treatment of HIV and are currently being employed in the first trial evaluating lentivirus vector safety in humans.

The coordinated induced expression of beta-oxidation genes is essential to provide the energy supply for germination and postgerminative development. However, very little is known about other functions of beta-oxidation in nonreserve organs. We have identified a gene-specific pattern of induced beta-oxidation gene expression in wounded leaves of Arabidopsis. Mechanical damage triggered the local and systemic induction of only ACX1 among acyl-coenzyme A oxidase (ACX) genes, and KAT2/PED1 among 3-ketoacyl-coenzyme A thiolase (KAT) genes in Arabidopsis. In turn, wounding induced KAT5/PKT2 only systemically. Although most of the beta-oxidation genes were activated by wound-related factors such as dehydration and abscisic acid, jasmonic acid (JA) induced only ACX1 and KAT5. Reduced expression of ACX1 or KAT2 genes, in transgenic plants expressing their corresponding mRNAs in antisense orientation, correlated with defective wound-activated synthesis of JA and with reduced expression of JA-responsive genes. Induced expression of JA-responsive genes by exogenous application of JA was unaffected in those transgenic plants, suggesting that ACX1 and KAT2 play a major role in driving wound-activated responses by participating in the biosynthesis of JA in wounded Arabidopsis plants.

The design of potent systems for the delivery of charged and noncharged molecules that target genes of interest remains a challenge. We describe a novel technology that combines a new generation of peptide nucleic acids (PNAs), or HypNA-pPNAs, with a new noncovalent peptide-based delivery system, Pep-2, which promotes efficient delivery of PNAs into several cell lines. We have validated the potential of this technology by showing that Pep2-mediated delivery of an antisense HypNA-pPNA chimera directed specifically against cyclin B1 induces rapid and robust downregulation of its protein levels and efficiently blocks cell cycle progression of several cell lines, as well as proliferation of cells derived from a breast cancer. Pep-2-based delivery system was shown to be 100-fold more efficient in delivering HypNA-pPNAs than classical cationic lipid-based methods. Whereas Pep-2 is essential for improving the bioavailability of PNAs and HypNA-pPNAs, the latter contribute significantly to the efficiency and specificity of the biological response. We have found that Pep-2/HypNA-pPNA strategy promotes potent antisense effects, which are approximately 25-fold greater than with classical antisense oligonucleotide directed specifically against the same cyclin B1 target. Taken together, these data demonstrate that peptide-mediated delivery of HypNA-pPNAs constitutes a very promising technology for therapeutic applications.

Background: Human normal cells have active p53 and pRB tumor suppressor pathways and undergo telomere shortening at each cell division. When immortalized, these exhibit functional inactivation of one or both tumor suppressor pathways and activation of telomere-maintaining mechanisms. Regulation of immortalization promoting molecular pathways by other genes is poorly understood and is an essential component of cancer therapeutics. In the present study, we have immortalized human normal cells by functional inactivation of p53 and pRB tumor suppressor proteins and simultaneous activation of telomerase function. We demonstrate that when the expression of mortalin (a member of hsp70 family proteins) was suppressed in such genetically defined immortal cells they underwent a permanent growth arrest., Methods: WI-38 cells were transfected with expression plasmids encoding papilloma virus E6 and E7 proteins and a catalytic subunit of human telomerase enzyme. The derivative cells were compromised for p53 and pRB senescence pathways, showed telomerase activity and were immortalized. These cells were transfected with antisense expression plasmid for mortalin. The derivative clones were analyzed for mortalin expression, proliferation and telomerase activity., Results: WI-38 cells were immortalized with E6, E7 and the catalytic subunit of human telomerase. The derivative-immortalized cells when suppressed for mortalin expression underwent senescence-like growth arrest., Conclusions: The data demonstrate that suppression of mortalin is sufficient to induce growth arrest in human immortalized cells that have compromised p53 and pRB functions and possess telomerase activity., (Copyright 2004 John Wiley & Sons, Ltd.)

c-Met is a receptor tyrosine kinase whose activation by hepatocyte growth factor (HGF) can lead to transformation and tumorigenicity in a variety of tumors. We investigated the effects of suppressing c-Met protein expression in human non-small cell lung tumors. Expression plasmids containing either sense or antisense sequences of the human c-met gene were constructed under control of the U6 snRNA promoter. A U6 control plasmid was also constructed that did not contain any c-met sequence. These constructs have been examined both in vitro and in an in vivo tumor xenograft model. The c-Met protein was downregulated by 50-60% in two lung cancer cell lines that were transiently transfected with the c-Met antisense versus U6 control. Tumor cells treated with the c-Met antisense construct also show decreased phosphorylation of c-Met and MAP kinase when exposed to exogenous HGF. Lung cancer cells were grown as xenografts in mice and treated by intratumoral liposome-mediated transfer of the c-Met sense, antisense or U6 control plasmids. The treatment of lung tumors with c-Met antisense versus U6 control plasmid resulted in the downregulation of the c-Met protein expression, a 50% decrease in tumor growth over a 5-week treatment period and an increased rate of apoptosis. These results suggest that targeting the HGF/c-Met pathway may be an effective novel strategy to treat lung cancer patients.

Brassinosteroids (BRs) are plant steroids essential for normal growth and development. To gain insight into the molecular mechanism by which BRs regulate the growth and development of plants, it is necessary to identify and analyze more genes that are regulated by BRs. A novel brassinolide (BL)-enhanced gene designated OsBLE1, which was originally identified by using rice (Oryza sativa L.) cDNA microarray, was cloned and characterized. Its cDNA is 598 bp long, encoding a predicted polypeptide with 81 amino acid residues. Northern blots analysis revealed that OsBLE1 expression began to increase at 6 h and reached its maximum at 12 h after BL treatment. OsBLE1 expression was most responsive to BL in lamina joint in rice seedlings; besides, IAA and GA3 also enhanced its expression. OsBLE1 expressed mainly in active tissues such as vascular bundles and root primordial. Transgenic rice expressing antisense OsBLE1 exhibits various degrees of repressed growth. Results suggest that OsBLE1 might be involved in BL-regulated growth processes in rice seedlings.

To study the mechanism of action of BAG-1 in drug-induced apoptosis, we constructed an antisense BAG-1 vector and established a stably transfected cell line from BAG-1-over-expressing HeLa cells. Reduced BAG-1 protein was confirmed by Western blot. Treatment of the antisense BAG-1-transfected cells with the anti-cancer drugs staurosporine, paclitaxel, all-trans retinoic acid (ATRA), and N-(4-hydroxyphenyl) retinamide (4-HPR) resulted in significantly enhanced apoptosis and reduced cell viability relative to vector-transfected cells. While the expression of p53 was increased, the level of Bcl-2 and Bax was decreased. Cells underexpressing BAG-1 had reduced cytosolic cytochrome c level. Treatment with staurosporine and paclitaxel resulted in increased cytochrome c release from mitochondria, whereas there was no change induced by treatment with ATRA and 4-HPR. Our experiments suggest that BAG-1 inhibits anti-cancer drug-induced apoptosis through apoptosis regulation pathways that may involve the mitochondrial Bcl-2/Bax ratio, p53, and differential anti-cancer drug-mediated cytochrome c release.

Although most of the components of the cell cycle machinery are conserved in all eukaryotes, plants differ strikingly from animals by the absence of a homolog of E-type cyclin, an important regulator involved in G1/S-checkpoint control in animals. By contrast, plants contain a complex range of A-type cyclins, with no fewer than 10 members in Arabidopsis. We previously identified the tobacco A-type cyclin Nicta;CYCA3;2 as an early G1/S-activated gene. Here, we show that antisense expression of Nicta;CYCA3;2 in tobacco plants induces defects in embryo formation and impairs callus formation from leaf explants. The green fluorescent protein (GFP)-Nicta;CYCA3;2 fusion protein was localized in the nucleoplasm. Transgenic tobacco plants overproducing GFP-Nicta;CYCA3;2 could not be regenerated from leaf disc transformation, whereas some transgenic Arabidopsis plants were obtained by the floral-dip transformation method. Arabidopsis plants that overproduce GFP-Nicta;CYCA3;2 showed reduced cell differentiation and endoreplication and a dramatically modified morphology. Calli regenerated from leaf explants of these transgenic Arabidopsis plants were defective in shoot and root regeneration. We propose that Nicta;CYCA3;2 has important functions, analogous to those of cyclin E in animals, in the control of plant cell division and differentiation.

Oxygenation of pheophorbide a is a key step in chlorophyll breakdown. Several biochemical studies have implicated that this step was catalyzed by an iron-containing and ferredoxin-dependent monooxygenase, pheophorbide a oxygenase (PaO). It has been proposed that inhibition of its activity arrests the chlorophyll breakdown and leads to the "stay-green" phenotype. We searched the Arabidopsis genome for a possible PaO-encoding gene and hypothesized that it has homology to known iron-containing Rieske-type monooxygenase sequences. We identified three such open reading frames, Tic55, ACD1 and ACD1-like. We produced transgenic Arabidopsis plants which expressed antisense RNA as a method to inhibit the expression of these genes. The appearance of these antisense plants were indistinguishable from that of the wild type under illumination. However, after they were kept under darkness for 5 d and again illuminated, the leaves of the antisense ACD1 plants (AsACD1) were bleached. Leaves of AsACD1 accumulated 387 nmol (g FW)(-1) pheophorbide a which corresponded to 60% of chlorophyll a degraded. The rate of decrease in chlorophyll a was not influenced in senesced AsACD1 leaves. These results demonstrated that ACD1 is involved in PaO activity, and its inhibition led to photooxidative destruction of the cell instead of the "stay-green" phenotype.

The formation of laminae within the retina requires the coordinate regulation of cell differentiation and migration. The cell adhesion molecule and member of the immunoglobulin superfamily, receptor protein tyrosine phosphatase Mu, PTPmu, is expressed in precursor and early, differentiated cells of the prelaminated retina, and later becomes restricted to the inner plexiform, ganglion cell, and optic fiber layers. Since the timing of PTPmu expression correlates with the peak period of retinal lamination, we examined whether this RPTP could be regulating cell adhesion and migration within the retina, and thus influencing retinal development. Chick retinal organ cultures were infected with herpes simplex viruses encoding either an antisense sequence to PTPmu, wild-type PTPmu, or a catalytically inactive mutant form of PTPmu, and homophilic adhesion was blocked by using a function-blocking antibody. All conditions that perturbed PTPmu dramatically disrupted retinal histogenesis. Our findings demonstrate that catalytic activity and adhesion mediated by PTPmu regulate lamination of the retina, emphasizing the importance of adhesion and signaling via receptor protein tyrosine phosphatases in the developing nervous system. To our knowledge, this is the first demonstration that an Ig superfamily RPTP regulates the lamination of any neural tissue.

Objective: To observe the sensitivity change of SMMC-7721 cells transfected with antisense DNMT1 gene fragment to tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its mechanism., Methods: Cell survival rate was measured by trypan blue, apoptosis rate by TUNEL method and the expression of bcl-2, bax and bad by flow cytometry., Results: Cell survival rate of SMMC-7721 cells transfected with antisense DNMT1 gene fragment was markedly lower than that transfected with sense DNMT1 gene fragment or empty vector (P < 0.05 and 0.01), but the apoptosis rate was on the contrary (P < 0.05 or 0.01). The expression of bax and bad (especially the former), but not bcl-2 of SMMC-7721 cells transfected with antisense DNMT1 gene fragment was markedly higher than those of SMMC-7721 cells transfected with sense DNMT1 gene fragment or empty vector., Conclusion: The sensitivity of SMMC-7721 cells to TRAIL can be enhanced by the transfection of antisense DNMT1 gene fragment, which may be related to the increase of bax and bad expression.

Background: Fibrosis characteristically occurs in the advanced stages of chronic inflammatory diseases, occasionally as the primary lesion, and frequently determines the disease prognosis. Fibrotic lesions consist mostly of collagen, and therefore it may be possible to prevent or treat fibrosis by inhibiting collagen production. Of the currently available therapeutic approaches, however, none is sufficiently effective and specific for inhibition of collagen. Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been reported to play a pivotal role in secretion of procollagen molecules. Therefore, we have tried to suppress its function to inhibit these various types of collagen., Methods: We have developed a novel type of ribozyme by ligating a hammerhead sequence to a tRNA(Val) promoter to facilitate displacing the ribozyme from nucleus to cytoplasm and to constitutive transport element, a binding motif of helicase which unwinds mRNA to render the target sequence on the mRNA accessible to the ribozyme., Results: The ribozyme thus constructed showed strong activity to cleave HSP47 mRNA and suppress the secretion of type I procollagen in the human primary fibroblast., Conclusion: We suggest applicability of this ribozyme as a new modality for antifibrosis therapy., (Copyright 2003 John Wiley & Sons, Ltd.)

Wolfram syndrome is a rare condition in which the pancreatic beta-cells of patients are selectively deleted during the early years of life by a non-autoimmune-mediated mechanism. The condition is associated with mutations in the gene encoding wolframin, suggesting that this protein exerts a critical, but currently unknown, function in beta-cells. We have used an antisense strategy to modulate the expression of wolframin in insulin-secreting BRIN-BD11 cells to study its function. Stably transfected clones were established expressing full-length human wolframin antisense transcripts. These cells exhibited a dramatic reduction in cell proliferation rate and changes in morphology, although insulin secretion was not modified. The results imply that wolframin expression is required to sustain normal rates of beta-cell proliferation.

The AtMYB103 gene is a member of the R2R3 MYB gene family in Arabidopsis thaliana. Using the GUS reporter gene, AtMYB103 expression was found to be restricted to the tapetum of developing anthers. Employing RT-PCR and in situ hybridisation, we now show that AtMYB103 is also expressed in trichomes. GUS expression in trichomes was obtained by incorporating the coding and 3'-untranslated regions of AtMYB103 into the promoter-GUS constructs. Sense and antisense technologies were used to downregulate AtMYB103 expression. In transgenic lines with reduced AtMYB103 transcript levels, pollen, tapetum and trichome development were altered. The majority of the pollen grains were distorted in shape and had reduced or no cytoplasmic content. Tapetal degeneration occurred early, and large opaque bodies appeared in the tapetal cytoplasm. In transgenic plants, trichomes on cauline and rosette leaves produced additional branches. These overbranched trichomes contained more nuclear DNA than the wild-type trichomes. The results indicate that AtMYB103 is required for tapetal development and microsporogenesis, and negatively regulates trichome endoreduplication linked to the trichome branching.

Arabidopsis plants were transformed with acyl carrier protein (ACP)-4 in antisense conformation driven by the cauliflower mosaic virus 35S promoter. It was hypothesized that reduction of ACP4 in leaf tissue would result in a reduction in lipid biosynthesis and, in addition, affect fatty acid composition and leaf physiology. Several transgenic lines have been generated with reduced ACP4 protein in leaf tissue. Dramatic reductions in ACP4 resulted in a reduction of leaf lipid content (22%-60%) based on fresh leaf weight and a bleached appearance and reduced photosynthetic efficiency. In addition, a decrease in 16:3 as a percentage of the total fatty acid composition was noted. There were no changes in leaf lipid class distribution; however, there was a decrease in the relative amount of 16:3 in monogalactosyldiacylglycerol. These results suggest that ACP4 plays a major role in the biosynthesis of fatty acids for chloroplast membrane development. Alterations in the ACP isoform profile of Arabidopsis leaf also appear to alter the flow of fatty acids between the prokaryotic and eukaryotic pathways for assembly of galactolipids. However, it has not yet been determined if the changes in fatty acid composition are due to changes in the profile of ACP isoforms, or if they are actually a reaction to a reduction in fatty acid precursors.

We have previously isolated a CCAAT-binding factor B subunit gene ( BnCBF-B) from Brassica napus that is widely expressed in different plant tissues and whose role is still unknown. To investigate the importance of this transcription factor subunit in plant reproductive tissues, we targeted antisense BnCBF-B transcripts to the tapetum of transgenic B. napus plants. Of the 24 independent transformants, 13 yielded reduced quantities of viable pollen, of which five were unable to produce the elongated siliques indicative of normal seed set. The decrease in pollen viability probably resulted from the precocious degeneration of the tapetal cell layer observed in these plants. Surprisingly, the male-sterile phenotype was also accompanied by a decrease in female fertility, which could be due to the expression of the antisense BnCBF-B transcripts in the female reproductive structures of the transgenic plants. These results suggest that the BnCBF-B gene plays a critical non-redundant role in plant reproductive tissues.