Differential expression of NPM, GSTA3, and GNMT in mouse liver following long-term in vivo irradiation by means of uranium tailings.

Uranium tailings (UT) are formed as a byproduct of uranium mining and are of potential risk to living organisms. In the present study, we sought to identify potential biomarkers associated with chronic exposure to low dose rate γ radiation originating from UT. We exposed C57BL/6J mice to 30, 100, or 250 µGy/h of gamma radiation originating from UT samples. Nine animals were included in each treatment group. We observed that the liver central vein was significantly enlarged in mice exposed to dose rates of 100 and 250 µGy/h, when compared with nonirradiated controls. Using proteomic techniques, we identified 18 proteins that were differentially expressed (by a factor of at least 2.5-fold) in exposed animals, when compared with controls. We chose glycine N-methyltransferase (GNMT), glutathione S-transferase A3 (GSTA3), and nucleophosmin (NPM) for further investigations. Our data showed that GNMT (at 100 and 250 µGy/h) and NPM (at 250 µGy/h) were up-regulated, and GSTA3 was down-regulated in all of the irradiated groups, indicating that their expression is modulated by chronic gamma radiation exposure. GNMT, GSTA3, and NPM may therefore prove useful as biomarkers of gamma radiation exposure associated with UT. The mechanisms underlying those changes need to be further studied.

Stiff hands and feet, facial deformities.

The appearance of the skin on this woman's face, hands, and feet helped us to recognize an advanced case of an autoimmune disease.

Effects of Saikokaryukotsuboreito on Spermatogenesis and Fertility in Aging Male Mice.

BACKGROUND: Aspermia caused by exogenous testosterone limit its usage in late-onset hypogonadism (LOH) patients desiring fertility. Saikokaryukotsuboreito (SKRBT) is reported to improve serum testosterone and relieve LOH-related symptoms. However, it is unclear whether SKRBT affects fertility. We aimed to examine the effects of SKRBT on spermatogenesis and fertility in aging male mice. METHODS: Thirty aging male mice were randomly assigned to three groups. Mice were orally administered with phosphate-buffer solution or SKRBT (300 mg/kg, daily) or received testosterone by subcutaneous injections (10 mg/kg, every 3 days). Thirty days later, each male mouse was mated with two female mice. All animals were sacrificed at the end of 90 days. Intratesticular testosterone (ITT) levels, quality of sperm, expression of synaptonemal complex protein 3 (SYCP3), and fertility were assayed. RESULTS: In the SKRBT-treated group, ITT, quality of sperm, and expression of SYCP3 were all improved compared with the control group (ITT: 85.50 ± 12.31 ng/g vs. 74.10 ± 11.45 ng/g, P = 0.027; sperm number: [14.94 ± 4.63] × 106 cells/ml vs. [8.79 ± 4.38] × 106 cells/ml, P = 0.002; sperm motility: 43.16 ± 9.93% vs. 33.51 ± 6.98%, P = 0.015; the number of SYCP3-positive cells/tubule: 77.50 ± 11.01 ng/ml vs. 49.30 ± 8.73 ng/ml, P < 0.001; the expression of SYCP3 protein: 1.23 ± 0.09 vs. 0.84 ± 0.10, P < 0.001), but fertility was not significantly changed (P > 0.05, respectively). In the testosterone-treated group, ITT, quality of sperm, and expression of SYCP3 were markedly lower than the control group (ITT: 59.00 ± 8.67, P = 0.005; sperm number: [4.34 ± 2.45] × 106 cells/ml, P = 0.018; sperm motility: 19.53 ± 7.69%, P = 0.001; the number of SYCP3-positive cells/tubule: 30.00 ± 11.28, P < 0.001; the percentage of SYCP3-positive tubules/section 71.98 ± 8.88%, P = 0.001; the expression of SYCP3 protein: 0.71 ± 0.09, P < 0.001), and fertility was also suppressed (P < 0.05, respectively). CONCLUSION: SKRBT had no adverse effect on fertility potential in aging male mice.

Dual-probe electrochemical DNA biosensor based on the "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification for detection of double-strand DNA of PML/RARα related fusion gene.

Taking advantage of "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification, a dual-probe electrochemical DNA (DE-DNA) biosensor was designed to detect double-stranded DNA (dsDNA) of acute promyelocytic leukemia (APL) related gene. Two groups of detection probes were designed, and each group was composed of a biotinylated capture probe and an assisted probe. They were separately complementary with two strands of target dsDNA in order to prevent the reannealing of the two separate strands from target dsDNA. First, thiol functionalized capture probes (C1 and C2) were severally assembled onto two different gold electrodes, followed by hybridizing with target dsDNA (S1a-S1b) and assistant probes to form two Y-junction-structure ternary complexes. Subsequently, restriction sites on the ternary complexes were digested by Rsa I, which can release S1a, S1b and biotins from the electrode surfaces. Meanwhile, the released S1a and S1b can further hybridize with the unhybridized corresponding detection probes and then initiate another new hybridization-cleavage-separation cycle. Finally, the current signals were produced by the enzyme-catalyzed reaction of streptavidin-horse reddish peroxidase (streptavidin-HRP). The distinct difference in current signals between different sequences allowed detection of target dsDNA down to a low detection limit of 47 fM and presented excellent specificity with discriminating only a single-base mismatched dsDNA sequence. Moreover, this biosensor was also used for assay of polymerase chain reaction (PCR) samples with satisfactory results. According to the results, the power of the DE-DNA biosensor as a promising tool for the detection of APL and other diseases.

Transcranial low-level laser therapy enhances learning, memory, and neuroprogenitor cells after traumatic brain injury in mice.

The use of transcranial low-level laser (light) therapy (tLLLT) to treat stroke and traumatic brain injury (TBI) is attracting increasing attention. We previously showed that LLLT using an 810-nm laser 4 h after controlled cortical impact (CCI)-TBI in mice could significantly improve the neurological severity score, decrease lesion volume, and reduce Fluoro-Jade staining for degenerating neurons. We obtained some evidence for neurogenesis in the region of the lesion. We now tested the hypothesis that tLLLT can improve performance on the Morris water maze (MWM, learning, and memory) and increase neurogenesis in the hippocampus and subventricular zone (SVZ) after CCI-TBI in mice. One and (to a greater extent) three daily laser treatments commencing 4-h post-TBI improved neurological performance as measured by wire grip and motion test especially at 3 and 4 weeks post-TBI. Improvements in visible and hidden platform latency and probe tests in MWM were seen at 4 weeks. Caspase-3 expression was lower in the lesion region at 4 days post-TBI. Double-stained BrdU-NeuN (neuroprogenitor cells) was increased in the dentate gyrus and SVZ. Increases in double-cortin (DCX) and TUJ-1 were also seen. Our study results suggest that tLLLT may improve TBI both by reducing cell death in the lesion and by stimulating neurogenesis.

The alternative lengthening of telomere phenotype is significantly associated with loss of ATRX expression in high-grade pediatric and adult astrocytomas: a multi-institutional study of 214 astrocytomas.

Loss-of-function of alpha thalassemia/mental retardation syndrome X-linked (ATRX) protein leads to a phenotype called alternative lengthening of telomeres (ALT) in some tumors. High-grade astrocytomas comprise a heterogeneous group of central nervous system tumors. We examined a large cohort of adult (91) and pediatric (n=88) high-grade astrocytomas as well as lower grade forms (n=35) for immunohistochemical loss of ATRX protein expression and the presence of ALT using telomere-specific fluorescence in situ hybridization, with further correlation to other known genetic alterations. We found that in pediatric high-grade astrocytomas, 29.6% of tumors were positive for ALT and 24.5% were immunonegative for the ATRX protein, these two alterations being highly associated with one another (P<0.0001). In adult high-grade astrocytomas, 26.4% of tumors were similarly positive for ALT, including 80% of ATRX protein immunonegative cases (P<0.0001). Similar frequencies were found in 11 adult low-grade astrocytomas, whereas all 24 pilocytic astrocytomas were negative for ALT. We did not find any significant correlations between isocitrate dehydrogenase status and either ALT positivity or ATRX protein expression in our adult high-grade astrocytomas. In both cohorts, however, the ALT positive high-grade astrocytomas showed more frequent amplification of the platelet-derived growth factor receptor alpha gene (PDGFRA; 45% and 50%, respectively) than the ALT negative counterparts (18% and 26%; P=0.03 for each). In summary, our data show that the ALT and ATRX protein alterations are common in both pediatric and adult high-grade astrocytomas, often with associated PDGFRA gene amplification.

Characterization of a 65 kDa NIF in the nuclear matrix of the monocot Allium cepa that interacts with nuclear spectrin-like proteins.

Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled-coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross-react with anti-intermediate filament and anti-lamin antibodies, form filaments 6-12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin-like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin-like proteins by co-immunoprecipitation and co-localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with Mr and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin-like proteins. Its similarities with some of the proteins described as onion lamin-like proteins suggest that they are highly related or perhaps the same proteins.

Characterization of a rat model of Huntington's disease based on targeted expression of mutant huntingtin in the forebrain using adeno-associated viral vectors.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (htt) gene. Neuropathology is most severe in the striatum and cerebral cortex. As mutant htt is ubiquitously expressed, it has not been possible to establish clear structure-to-function relationships for the clinical aspects. In the present study, we have injected recombinant adeno-associated viral vectors of serotype 5 (rAAV5) expressing an 853-amino-acid fragment of htt with either 79 (mutant) or 18 (wild-type) glutamines (Q) in the dorsal striatum of neonatal rats to achieve expression of htt in the forebrain. Rats were followed for 6 months and compared with control rats. Neuropathological assessment showed long-term expression of the green fluorescent protein (GFP) transgene (used as a marker protein) and accumulation of htt inclusions in the cerebral cortex with the rAAV5-htt-79Q vectors. We estimated that around 10% of NeuN-positive cells in the cerebral cortex and 2% of DARPP-32 neurons in the striatum were targeted with the GFP-expressing vector. Formation of intracellular htt inclusions was not associated with neuronal loss, gliosis or microglia activation and did not lead to altered motor activity or changes in body weight. However, the same mutant htt vector caused orexin loss in the hypothalamus - another area known to be affected in HD. In conclusion, our results demonstrate that widespread forebrain expression of mutant htt can be achieved using rAAV5-vectors and suggest that this technique can be further explored to study region-specific effects of mutant htt or other disease-causing genes in the brain.

Molecular characterization of Wilms' tumor from a resource-constrained region of sub-Saharan Africa.

Sub-Saharan African children have an increased incidence of Wilms' tumor (WT) and experience alarmingly poor outcomes. Although these outcomes are largely due to inadequate therapy, we hypothesized that WT from this region exhibits features of biological aggressiveness that may warrant broader implementation of high-risk therapeutic protocols. We evaluated 15 Kenyan WT (KWT) for features of aggressive disease (blastemal predominance and Ki67/cellular proliferation) and treatment resistance (anaplasia and p53 immunopositivity). To explore the additional biological features of KWT, we determined the mutational status of the CTNNB1/ß-catenin and WT1 genes and performed immunostaining for markers of Wnt pathway activation (ß-catenin) and nephronic progenitor cell self-renewal (WT1, CITED1 and SIX2). We characterized the proteome of KWT using imaging mass spectrometry (IMS). The results were compared to histology- and age-matched North American WT (NAWT) controls. For patients with KWT, blastemal predominance was noted in 53.3% and anaplasia in 13%. We detected increased loss to follow-up (p = 0.028), disease relapse (p = 0.044), mortality (p = 0.001) and nuclear unrest (p = 0.001) in patients with KWT compared to controls. KWT and NAWT showed similar Ki67/cellular proliferation. We detected an increased proportion of epithelial nuclear ß-catenin in KWT (p = 0.013). All 15 KWT specimens were found to harbor wild-type CTNNB1/ß-catenin, and one contained a WT1 nonsense mutation. WT1 was detected by immunostaining in 100% of KWT, CITED1 in 80% and SIX2 in 80%. IMS revealed a molecular signature unique to KWT that was distinct from NAWT. The African WT specimens appear to express markers of adverse clinical behavior and treatment resistance and may require alternative therapies or implementation of high-risk treatment protocols.

Mucoepidermoid carcinoma of the thyroid: a report of three cases and postulated histogenesis.

BACKGROUND: Primary mucoepidermoid carcinoma (MEC) of the thyroid is a rare clinical and pathological entity that accounts for <0.5% of all thyroid malignancies. Although the histogenesis has been controversial, most investigators now favor it as arising from either metaplasia of thyroid follicular epithelium or heterologous de-differentiation from papillary thyroid carcinoma (PTC). We report three cases of thyroid MEC found in continuity with, and clearly arising from de-differentiation of, well-differentiated thyroid carcinomas (WDTCs). PATIENT FINDINGS AND SUMMARY: The cases presented here included two women (aged 22 and 52) and one man (aged 58). One of these cases arose in conjunction with PTC, one with follicular thyroid carcinoma (FTC), and one with Hurthle cell carcinoma (HCC). In all three cases, there was a gradual transition in morphology between the areas of typical WDTC and the areas showing MEC differentiation. In addition, immunohistochemistry demonstrated a gradual loss of thyroid specific markers (thyroid transcription factor-1, thyroglobulin) mirroring the change in morphology. CONCLUSION: We conclude that thyroid MEC can arise from metaplastic de-differentiation of WDTC, including FTC or HCC in addition to PTC. Currently, we recommend that after excision, each of the WDTC and MEC components of these tumors be treated with targeted adjuvant therapies, which may involve radioactive-iodine ablation, thyrotropin suppression, and external beam radiotherapy.

Value of mismatch repair, KRAS, and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer.

PURPOSE: It is uncertain whether modest benefits from adjuvant chemotherapy in stage II colorectal cancer justify the toxicity, cost, and inconvenience. We investigated the usefulness of defective mismatch repair (dMMR), BRAF, and KRAS mutations in predicting tumor recurrence and sensitivity to chemotherapy. PATIENTS AND METHODS: Immunohistochemistry for dMMR and pyrosequencing for KRAS/BRAF were performed for 1,913 patients randomly assigned between fluorouracil and folinic acid chemotherapy and no chemotherapy in the Quick and Simple and Reliable (QUASAR) trial. RESULTS: Twenty-six percent of 695 right-sided colon, 3% of 685 left-sided colon, and 1% of 407 rectal tumors were dMMR. Similarly, 17% of right colon, 2% of left colon, and 2% of rectal tumors were BRAF mutant. KRAS mutant tumors were more evenly distributed: 40% right colon, 28% left colon, and 36% rectal tumors. Recurrence rate for dMMR tumors was half that for MMR-proficient tumors (11% [25 of 218] v 26% [438 of 1,695] recurred; risk ratio [RR], 0.53; 95% CI, 0.40 to 0.70; P < .001). Risk of recurrence was also significantly higher for KRAS mutant than KRAS wild-type tumors (28% [150 of 542] v 21% [219 of 1,041]; RR, 1.40; 95% CI, 1.12 to 1.74; P = .002) but did not differ significantly between BRAF mutant and wild-type tumors (P = .36). No marker predicted benefit from chemotherapy with efficacy not differing significantly by MMR, KRAS, or BRAF status. The prognostic value of MMR and KRAS was similar in the presence and absence of chemotherapy. CONCLUSION: MMR assays identify patients with a low risk of recurrence. KRAS mutational analysis provides useful additional risk stratification to guide use of chemotherapy.

Anti-inflammatory effects of Cynanchum taiwanianum rhizome aqueous extract in IL-1ß-induced NRK-52E cells.

CONTEXT: Cynanchum taiwanianum T. Yamaza (Asclepiadaceae) is a medicinal herb used in folk medicine for the treatment of several inflammation-related diseases such as hepatitis and dermatitis in Taiwan. OBJECTIVE: In the present study, we investigated the anti-inflammatory effect of C. taiwanianum T. Yamaza rhizome aqueous extract (CTAE). MATERIALS AND METHODS: The present study investigated the anti-inflammatory effect of CTAE using IL-1ß-induced NRK-52E cells. Production of NO and PGE(2) by ELISA, the mRNA and protein expression of iNOS and COX-2, phosphorylation of IκBα, and activation of NF-κB by RT-PCR and western blotting were determined. RESULTS: The CTAE significantly (P < 0.05) inhibited NO and PGE(2) production (decreased by 46.1% and 51%, respectively), and also significantly (P < 0.05) attenuated protein and mRNA expression of iNOS and COX-2 (decreased by 90% and 55% for iNOS and by 72% and 74%% for COX-2, respectively) in IL-1ß-induced NRK-52E cells, in a dose-dependent manner, without obvious cytotoxic effects. Furthermore, the CTAE suppressed the NF-κB nuclear translocation, in terms of inhibition of IκBα phosphorylation. DISCUSSION AND CONCLUSION: Our results provided evidence for its folkloric uses and suggest that the anti-inflammatory activities of CTAE may result from the inhibition of inflammatory mediators, such as NO and PGE(2), and an upstream suppression of a NF-κB-dependent mechanism, might be involved.

Differential expression of ruminant ZNF496 variants: Association with quantitative trait locus affecting bovine milk concentration and fertility.

A single nucleotide polymorphism in the intergenic region upstream of the ZNF496 gene on Bos taurus chromosome 7 displayed significant population-wide linkage disequilibrium with milk protein percentage in the Israeli Holstein population. The frequency of the allele associated with increased protein concentration was 10%. This single nucleotide polymorphism was located in the promoter region from which a 10-exon transcript of the bovine and the ovine ZNF496 genes are transcribed. The gene architecture was similar to the mouse ortholog Zkscan17. A 5-exon murine antisense transcript was complementary to the 5' untranslated Zkscan17 region that included a sequence domain conserved between mouse and ruminants, suggesting a regulatory function. In the bovine ZNF496 chromosomal region, segregation of a quantitative trait locus (QTL) for milk protein percentage was confirmed in a daughter design sire family. Concordance was not obtained between QTL status of bulls and any of the polymorphisms in the functional elements of ZNF496. This excludes these variations as the causative polymorphism under the assumption of no epigenetic effect for this locus. However, ZNF496 variants were differentially expressed in bovine ovaries, and only the paternal variant was expressed in liver and kidney in a sheep family with polymorphic ZNF496 sequence. Thus, the search for the mutation underlying the minor QTL allele, which is a top economically favorable allele in Israeli Holstein cattle, may be complicated by the presence of an imprinting center in this QTL confidence interval.

RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma.

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC(50) values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC(50) values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.

Over-expression of PR-10a leads to increased salt and osmotic tolerance in potato cell cultures.

The PR-10a protein (formerly STH-2) is known to be induced by biotic stress in potato. The present study demonstrates that transgenic suspension cells of the potato cultivar Desiree over-expressing the PR-10a protein exhibit significantly increased salt and osmotic tolerance compared to the respective wild type cells. A comparison of the proteome pattern of Solanum tuberosum suspension cultures cv. Desiree before and after the treatment with NaCl or sorbitol under equiosmolar conditions (740mOs/kg) revealed the pathogenesis related protein PR-10a to be one of the predominant differentially expressed proteins in potato cell cultures. The pr-10a mRNA was confirmed to be present by RT-PCR from salt challenged suspension cells and was transcribed into cDNA. For PR-10a over-expression Agrobacterium tumefaciens mediated transformation of the potato cells and a dicistronic vector harboring the cDNA of the pr-10a gene linked to a luciferase gene by an IRES (Internal Ribosome Binding Site) was used. The IRES mediated translation leads to co-expression of PR-10a and luciferase in a fixed ratio. By non-invasive luciferase assay homologous PR-10a over-expressing callus was identified after selection on phosphinothricin supplemented medium. This callus was used for the setup of a transgenic suspension culture. Along with increased salt and osmotic tolerance the transformed culture showed changed proline and glutathione levels under abiotic stress conditions in comparison to the wild type.

Induction of G2/M arrest by pseudolaric acid B is mediated by activation of the ATM signaling pathway.

AIM: The aim of this study was to investigate the mechanism of pseudolaric acid B (PLAB)-induced cell cycle arrest in human melanoma SK-28 cells. METHODS: Cell growth inhibition was detected by MTT assay, the cell cycle was analyzed by flow cytometry, and protein expression was examined by Western blot analysis. RESULTS: PLAB inhibited the growth of human melanoma cells and induced G(2)/M arrest in SK-28 cells, accompanied by an up-regulation of Cdc2 phosphorylation and a subsequent down-regulation of Cdc2 expression. Furthermore, PLAB decreased the expression of Cdc25C phosphatase and increased the expression of Wee1 kinase. Meanwhile, a reduction in Cdc2 activity was partly due to induction of the expression of p21(waf1/cip1) in a p53-dependent manner. In addition, PLAB activated the checkpoint kinase, Chk2, and increased the expression of p53, two major targets of ATM kinase. These effects were inhibited by caffeine, an ATM kinase inhibitor. We also found that PLAB significantly enhanced ATM kinase activity. CONCLUSION: Taken together, these results suggest that PLAB induced G(2)/M arrest in human melanoma cells via a mechanism involving the activation of ATM, and the effect of PLAB on Cdc2 activity was mediated via interactions with the Chk2-Cdc25C and p53 signalling pathways, two distinct downstream pathways of ATM. PLAB may be a promising chemopreventive agent for treating human melanoma.

Tissue-specific expression of the PNZIP promoter is mediated by combinatorial interaction of different cis-elements and a novel transcriptional factor.

Recent studies demonstrated that PNZIP and its homologs encode a special cyclase and play an important role in chlorophyll biosynthesis in higher plants. To investigate the molecular mechanism governing the PNZIP gene, the PNZIP promoter was isolated and analyzed. Deletion analysis indicated that G-box is an important element in the regulation of the reporter gene expression. Further mutation assay demonstrated that G-box and GATACT elements are necessary and sufficient for the high and tissue-specific expression of the GUS gene. Using yeast one-hybrid screening, we have isolated a novel tobacco bZIP protein, NtbZIP, which can specifically recognize the G-box of the PNZIP promoter. The NtbZIP protein shares a limited amino acid homology to Arabidopsis ABI5 and AtAREB1 and very low homology to other bZIP proteins. Northern blot analysis showed that the NtbZIP gene is not induced by exogenous ABA and is expressed in different tobacco organs. Cotransformation assays showed that the NtbZIP protein could activate the transcription of the GUS gene driven by the PNZIP promoter. Transgenic tobaccos analysis demonstrated that constitutively expressing antisense NtbZIP gene resulted in a lower NTZIP synthesis and reduced chlorophyll levels. We suggest that NTZIP is a target gene of NtbZIP, which is involved in the regulation of chlorophyll biosynthesis.

Mipu1, a novel rat zinc-finger protein, inhibits transcriptional activities of AP-1 and SRE in mitogen-activated protein kinase signaling pathway.

Mipu1 is a novel rat gene recently identified in our lab. Mipu1 cDNA contains a 1,824 bp open reading frame (ORF) and encoded a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C(2)H(2) motifs in the C-terminus. Mipu1 protein is located in the nuclei. Fused to Gal-4 DNA-binding domain and cotransfected with pG5-luc, Mipu1 played a transcriptional suppressive effect. Deletion analysis with a series of truncated fusion proteins indicated that the KRAB motif was a basal repression domain. Overexpression of Mipu1 in H9c2 myogenic cells inhibited the transcriptional activities of SRE and AP-1. RNAi of Mipu1 in H9c2 myogenic cells activated the transcriptional activities of SRE and AP-1. These results suggested that Mipu1 protein might act as a transcriptional repressor in mitogen-activated protein kinase (MAPK) signaling pathway to mediate cellular functions.

A small-molecule therapeutic lead for Huntington's disease: preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse.

Huntington's disease (HD) is a progressive neurodegenerative disease caused by a glutamine expansion within huntingtin protein. The exact pathological mechanisms determining disease onset and progression remain unclear. However, aggregates of insoluble mutant huntingtin (mhtt), a hallmark of HD, are readily detected within neurons in HD brain. Although aggregated polyglutamines may not be inherently toxic, they constitute a biomarker for mutant huntingtin useful for developing therapeutics. We previously reported that the small molecule, C2-8, inhibits polyglutamine aggregation in cell culture and brain slices and rescues degeneration of photoreceptors in a Drosophila model of HD. In this study, we assessed the therapeutic potential of C2-8 in the R6/2 mouse model of HD, which has been used to provide proof-of-concept data in considering whether to advance therapies to human HD. We show that, at nontoxic doses, C2-8 penetrates the blood-brain barrier and is present in brain at a high concentration. C2-8-treated mice showed improved motor performance and reduced neuronal atrophy and had smaller huntingtin aggregates. There have been no prior drug-like, non-toxic, brain-penetrable aggregation inhibitors to arise from cell-based high-throughput screens for reducing huntingtin aggregation that is efficacious in preclinical in vivo models. C2-8 provides an essential tool to help elucidate mechanisms of neurodegeneration in HD and a therapeutic lead for further optimization and development.