Validation of trans-acting elements that promote exon 7 skipping of SMN2 in SMN2-GFP stable cell line

Spinal muscular atrophy is a genetic disease in which the SMN1 gene is deleted. The SMN2 gene exists in all of the patients. Alternative splicing of these two genes are different. More than 90% of exon 7 included form is produced from SMN1 pre-mRNA, whereas only ～20% of exon 7 included form is produced from SMN2 pre-mRNA. Only exon 7 inclusion form produces functional protein. Exon 7 skipped SMN isoform is unstable. Here we constructed a GFP reporter system that recapitulates the alternative splicing of SMN1 and SMN2 pre-mRNA. We designed a system in which GFP protein is expressed only when exon 7 of is included in alternative splicing. The stable cell that expresses SMN1-GFP produces ～4 times more GFP protein than the stable cell line that expresses SMN2-GFP; as demonstrated by microscopy, FACS analysis and immunoblotting. In addition the ratio of exon 7 inclusion and skipping of SMN1-GFP and SMN2-GFP pre-mRNA was similar to endogenous SMN1 and SMN2 pre-mRNA as shown in RT-PCR. Furthermore the knockdown with hnRNP A1 shRNA, a known protein which promotes exon 7 skipping of SMN2, induces exon 7 inclusion of exon 7 in SMN2-GFP pre-mRNA in SMN2-GFP cell line. We conclude that we have established the stable cell lines that recapitulate alternative splicing of the SMN1 and SMN2 genes. The stable cell line can be used to identify the trans-acting elements with siRNA.     

Icaritin shows potent anti-leukemia activity on chronic myeloid leukemia in vitro and in vivo by regulating MAPK/ERK/JNK and JAK2/STAT3 /AKT signalings

Purpose

To explore the effects of Icaritin on chronic myeloid leukemia (CML) cells and underlying mechanisms.

Method

CML cells were incubated with various concentration of Icaritin for 48 hours, the cell proliferation was analyzed by MTT and the apoptosis was assessed with Annexin V and Hoechst 33258 staining. Cell hemoglobinization was determined. Western blotting was used to evaluate the expressions of MAPK/ERK/JNK signal pathway and Jak-2/Phorpho-Stat3/Phorsph-Akt network-related protein. NOD-SCID nude mice were applied to demonstrate the anti-leukemia effect of Icaritin in vivo.

Results

Icaritin potently inhibited proliferation of K562 cells (IC50 was 8 µM) and primary CML cells (IC50 was 13.4 µM for CML-CP and 18 µM for CML-BC), induced CML cells apoptosis and promoted the erythroid differentiation of K562 cells with time-dependent manner. Furthermore, Icaritin was able to suppress the growth of primary CD34+ leukemia cells (CML) and Imatinib-resistant cells, and to induce apoptosis. In mouse leukemia model, Icaritin could prolong lifespan of NOD-SCID nude mice inoculated with K562 cells as effective as Imatinib without suppression of bone marrow. Icaritin could up-regulate phospho-JNK or phospho-C-Jun and down-regulate phospho-ERK, phospho-P-38, Jak-2, phospho-Stat3 and phospho-Akt expression with dose- or time-dependent manner. Icaritin had no influence both on c-Abl and phospho-c-Abl protein expression and mRNA levels of Bcr/Abl.

Conclusion

Icaritin from Chinese herb medicine may be a potential anti-CML agent with low adverse effect. The mechanism of anti-leukemia for Icaritin is involved in the regulation of Bcr/Abl downstream signaling. Icaritin may be useful for an alternative therapeutic choice of Imatinib-resistant forms of CML.     

The in vitro and in vivo effects of anti-galactose antibodies on endothelial cell activation and xenograft rejection

We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the alpha-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal(-/-) mice show a range of affinity for the alpha-Gal epitope, and that affinity was generally increased as the V(H) gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced alpha-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection.     

Chitosan enhanced gene delivery of cationic liposome via non-covalent conjugation

Two new types of stable ternary complexes were formed by mixing chitosan with DOTAP/pDNA lipoplex and DOTAP with chitosan/pDNA polyplex via non-covalent conjugation for the efficient delivery of plasmid DNA. They were characterized by atomic force microscopy, gel retarding, and dynamic light scattering. The DOTAP/CTS/pDNA complexes were in compacted spheroids and irregular lump of larger aggregates in structure, while the short rod- and toroid-like and donut shapes were found in CTS/DOTAP/pDNA complexes. The transfection efficiency of the lipopolyplexes showed higher GFP gene expression than DOTAP/pDNA and CTS/pDNA controls in Hep-2 and Hela cells, and luciferase gene expression 2–3-fold than DOTAP/pDNA control and 70–120-fold than CTS/pDNA control in Hep-2 cells. The intracellular trafficking was examined by confocal laser scanning microscopy. Rapid pDNA delivery to the nucleus enchanced by chitosan was achieved after 4 h transfection.     

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.     

Heteroplasmy levels of mtDNA1555A>G mutation is positively associated with diverse phenotypes and mutation transmission in a Chinese family

The mtDNA 1555A>G mutation was considered to be one of the most common causes of aminoglycoside-induced and non-syndromic hearing loss. However, this mutation was always found in homoplasmy with high phenotypic heterogeneity. Recently this mutation in heteroplasmy has been reported in several studies. In the present study, we have collected a large Chinese family harboring heteroplasmic mtDNA 1555A>G mutation with diverse clinical phenotypes. To investigate the relationship between the mutation load and the severity of hearing loss under Eastern Asian background, we performed clinical, molecular, genetic and phylogenic analysis. This pedigree was characterized by coexistence of eight subjects with homoplasmic mutation and ten subjects with various degrees of heteroplasmy, and the results suggested that there was a strong correlation between the mutation load and the severity/age-onset of hearing loss (r=0.758, p<0.001). We noticed that the mutation level of offspring was associated with their mothers' in this pedigree, which indicated that maybe exist a regular pattern during the process of the heteroplasmic transmission. In addition, analysis of the complete mtDNA genome of this family revealed that it belonged to Eastern Asian haplogroup B4C1. In addition, a rare homoplasmic mtDNA 9128T>C variant was identified, it located at a strictly conserved site of mtDNA ATP6 gene.     

Antidepressant treatment normalizes white matter volume in patients with major depression

Objective

To investigate white matter volume abnormalities in patients with major depression and the effects of antidepressant treatment on white matter volume.

Method

Magnetic resonance imaging (MRI) was performed on 32 treatment-naïve depressed patients, 17 recovered patients who had received antidepressant treatment and subsequently achieved clinical recovery and 34 matched controls.

Results

Relative to the healthy controls, the treatment-naïve depressed patients showed increased white matter volumes in the left dorsolateral prefrontal cortex (DLPFC) and left putamen and reduced white matter volumes in the left cerebellum posterior lobe and left inferior parietal lobule. For the treatment-naïve patients, the length in months of the current depressive episode was positively correlated with the white matter volumes in both the left DLPFC and left putamen. In the recovered patients, the differences in white matter volume were no longer statistically significant relative to healthy controls. No significant difference was found in the total white matter volume among the three groups.

Conclusions

This study demonstrates that there were alterations in the white matter volumes of depressed patients, which might disrupt the neural circuits that are involved in emotional and cognitive function and thus contribute to the pathophysiology of depression. The finding of the significant correlations between refractoriness and the white matter volumes in the left DLPFC and left putamen combined with the finding that antidepressant treatment normalized the white matter volume of recovered patients, suggests that a quantitative, structural MRI measurement could act as a potential biomarker in depression therapy for individual subjects.     

Changes of primary sequence and secondary structure proximal to the 5′ end of the stop codon substantially increases the expression of the variable region of an antibody in <Emphasis Type="Italic">E. coli</Emphasis>

The sequence context at the 5 end of the stop codon may influence the efficiency of termination and translation. To increase the expression of a designed variable region of an antibody (named as VH5) against tumor necrosis factor (TNF), two nucleotides (TC) at 25 and 26 nucleotides (nt) upstream of termination codon were substituted with AG, respectively. The free energy of 70 nt (arbitrarily defined from the 32 nt upstream of termination codon to 38 nt downstream) was changed from –13.5 kcal mol^-1 to –17.3 kcal mol^-1. The expression level was increased from 1 ± 0.3% to 10 ± 1.2% of total cellular protein. Although the precise mechanism of this phenomenon remains to be elucidated, this report provides an alternative means to increase the expression of a foreign gene in E. coli. Revisions requested 18 October 2004; Revisions received 26 November 2004