Caspase-independent pathways of hair cell death induced by kanamycin in vivo

Cochlear and vestibular sensory cells undergo apoptosis when exposed to aminoglycoside antibiotics in organ culture, but mechanisms of chronic drug-induced hair cell loss in vivo are unclear. We investigated cell death pathways in a mouse model of progressive kanamycin-induced hair cell loss. Hair cell nuclei showed both apoptotic- and necrotic-like appearances but markers for classic apoptotic pathways (cytochrome c, caspase-9, caspase-3, JNK, TUNEL) were absent. In contrast, drug treatment caused EndoG translocation, activation of mu-calpain, and both the synthesis and activation of cathepsin D. Poly (ADP-ribose) polymerase 1 (PARP1) was decreased, but a caspase-derived 89 kDa PARP1 fragment was not present. The mRNA level of PARP1 remained unchanged. Thus, chronic administration of aminoglycosides causes multiple forms of cell death, without a major contribution by classic apoptosis. These results provide a better understanding of the toxic effects of aminoglycosides and are relevant to design protection from aminoglycoside-induced hearing loss.     

Major histocompatibility complex class IIB allele polymorphism and its association with resistance/susceptibility to Vibrio anguillarum in Japanese flounder (Paralichthys olivaceus)

The full length of major histocompatibility complex (MHC) class IIB cDNA was cloned from a Chinese population of Paralichthys olivaceus by homology cloning and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The MHC IIB genomic sequence is 1,864 bp long and consists of 34-bp 5′UTR, 741-bp open reading frame, 407-bp 3′UTR, 96-bp intron1, 392-bp intron2, 85-bp intron3, and 109-bp intron4. Phylogenetic analysis showed that the putative MHC class IIB amino acid of the Chinese P. olivaceus shared 28.3% to 85.4% identity with that of the reported MHC class IIB in other species. A significant association between MHC IIB polymorphism and disease resistance/susceptibility was found in Chinese P. olivaceus. Thirteen different MHC IIB alleles were identified among 411 clones from 84 individuals. Among the 280 (268) nucleotides, 32 (11.4%) nucleotide positions were variable. Most alleles such as alleles a, b, c, d, e, f, j, k, i, m were commonly found in both resistant and susceptible stock. Via χ² test, allele d was significantly more prevalent in individuals from susceptible stock than from resistant stock, and their percentages were 23.80% and 7.14%, respectively. In addition, allele g occurred in 9 and allele h in 4 of 42 resistant individuals that were not present in the susceptible stock; their percentages were 21.4% and 9.52%, respectively. Although allele l was found only in 8 individuals from the susceptible stock, its percentage is 19.05%.     

NYD-SP15: a novel gene potentially involved in regulating testicular development and spermatogenesis

By hybridizing human adult testis cDNA microarrays with human adult and embryo testis cDNA probes, we identified a novel human testis gene, NYD-SP15. NYD-SP15 expression was 3.26-fold higher in adult than in fetal testis; however, there was almost no NYD-SP15 expression in the sperm. NYD-SP15 comprises 3364 base pairs, including a 1545 bp open reading frame encoding a 514 amino acid protein possessing 89% sequence identity with the mouse testis homologous protein. NYD-SP15 is located on human chromosome 13q14.2. The deduced structure of the protein contains two dCMP_cyt_deam domains, indicating a potential functional role for zinc ion binding. The gene is expressed variably in a wide range of tissues, with high expression levels in the testis. Sequence analysis revealed that NYD-SP15 is not a highly conserved protein, with its distribution in high-level species such as vertebrates including Homo, Mus, Rattus, and Canis. The results of semiquantitative polymerase chain reaction in mouse testis representing different developmental stages indicate that NYD-SP15 expression was developmentally regulated. These results suggest the putative NYD-SP15 protein may play an important role in testicular development and spermatogenesis and may be an important factor governing male infertility.     

NYD-SP15: A Novel Gene Potentially Involved in Regulating Testicular Development and Spermatogenesis

By hybridizing human adult testis cDNA microarrays with human adult and embryo testis cDNA probes, we identified a novel human testis gene, NYD-SP15. NYD-SP15 expression was 3.26-fold higher in adult than in fetal testis; however, there was almost no NYD-SP15 expression in the sperm. NYD-SP15 comprises 3364 base pairs, including a 1545 bp open reading frame encoding a 514 amino acid protein possessing 89% sequence identity with the mouse testis homologous protein. NYD-SP15 is located on human chromosome 13q14.2. The deduced structure of the protein contains two dCMP_cyt_deam domains, indicating a potential functional role for zinc ion binding. The gene is expressed variably in a wide range of tissues, with high expression levels in the testis. Sequence analysis revealed that NYD-SP15 is not a highly conserved protein, with its distribution in high-level species such as vertebrates including Homo, Mus, Rattus, and Canis. The results of semiquantitative polymerase chain reaction in mouse testis representing different developmental stages indicate that NYD-SP15 expression was developmentally regulated. These results suggest the putative NYD-SP15 protein may play an important role in testicular development and spermatogenesis and may be an important factor governing male infertility. These authors contributed equally to this work     

The statistics of identifying differentially expressed genes in Expresso and TM4: a comparison

Background  

Analysis of DNA microarray data takes as input spot intensity measurements from scanner software and returns differential expression of genes between two conditions, together with a statistical significance assessment. This process typically consists of two steps: data normalization and identification of differentially expressed genes through statistical analysis. The Expresso microarray experiment management system implements these steps with a two-stage, log-linear ANOVA mixed model technique, tailored to individual experimental designs. The complement of tools in TM4, on the other hand, is based on a number of preset design choices that limit its flexibility. In the TM4 microarray analysis suite, normalization, filter, and analysis methods form an analysis pipeline. TM4 computes integrated intensity values (IIV) from the average intensities and spot pixel counts returned by the scanner software as input to its normalization steps. By contrast, Expresso can use either IIV data or median intensity values (MIV). Here, we compare Expresso and TM4 analysis of two experiments and assess the results against qRT-PCR data.     

A convenient cell fusion assay for the study of SARS-CoV entry and inhibition

SARS-CoV spike (S) protein-mediated cell fusion is important for the viral entry mechanism and identification of SARS-CoV entry inhibitors. In order to avoid the high risks involved in handling SARS-CoV and to facilitate the study of viral fusion mechanism, we established the cell lines: SR-COS7 cells that stably express both SARS-CoV S protein and red fluorescence protein, R-COS7 cells that stably express red fluorescence protein, and AG-COS7 cells that stably express both ACE2 and green fluorescence protein, respectively. When SR-COS7 cells or R-COS7 cells were cocultured with AG-COS7 cells, syncytia with yellow fluorescence were conveniently observed after 12 h in SR-COS7 cells plus AG-COS7 cells, but not in R-COS7 cells plus AG-COS7 cells. The cell-to-cell fusion efficiency was simply determined for quantitative analysis based on the number of syncytium detected by flow cytometry. Such new cell-to-cell fusion model was further assessed by the potent HR2 peptide inhibitor, which led to the obvious decrease of the cell-to-cell fusion efficiency. The successful fusion and inhibition of cell-based binding assay shows that it can be well used for the study of SARS-CoV entry and inhibition.     

The crystal structure of the peptide-binding fragment from the yeast Hsp40 protein Sis1

BACKGROUND: Molecular chaperone Hsp40 can bind non-native polypeptide and facilitate Hsp70 in protein refolding. How Hsp40 and other chaperones distinguish between the folded and unfolded states of proteins to bind nonnative polypeptides is a fundamental issue. RESULTS: To investigate this mechanism, we determined the crystal structure of the peptide-binding fragment of Sis1, an essential member of the Hsp40 family from Saccharomyces cerevisiae. The 2.7 A structure reveals that Sis1 forms a homodimer in the crystal by a crystallographic twofold axis. Sis1 monomers are elongated and consist of two domains with similar folds. Sis1 dimerizes through a short C-terminal stretch. The Sis1 dimer has a U-shaped architecture and a large cleft is formed between the two elongated monomers. Domain I in each monomer contains a hydrophobic depression that might be involved in binding the sidechains of hydrophobic amino acids. CONCLUSIONS: Sis1 (1-337), which lacks the dimerization motif, exhibited severe defects in chaperone activity, but could regulate Hsp70 ATPase activity. Thus, dimer formation is critical for Sis1 chaperone function. We propose that the Sis1 cleft functions as a docking site for the Hsp70 peptide-binding domain and that Sis1-Hsp70 interaction serves to facilitate the efficient transfer of peptides from Sis1 to Hsp70.     

Molecular Cloning and Characterization of Two 9-Lipoxygenase Genes from Taxus chinensis

Plant lipoxygenases (LOXs) are functionally diverse class of dioxygenases involved in multiple physiological processes such as plant growth, biotic and abiotic stress responses, and secondary metabolite accumulation. In this paper, two LOX genes, TcLOX1 and TcLOX2, were cloned from Taxus chinensis cells. Multiple alignment of the deduced amino acid sequences with those of other plants demonstrated the putative LH2/PLAT domain, lipoxygenase iron-binding catalytic domain, lipoxygenase_1 and lipoxygenase_2 signature sequences. Phylogenetic analysis suggested that TcLOX1 and TcLOX2 putative proteins are most probably 9-LOXs, and shared the highest identity with the tea plant CsLOX1 and Picea sitchensis LOX genes, respectively. Semiquantitative RT-PCR analysis showed that TcLOX1 was preferentially expressed in stem and root, while TcLOX2 was preferentially expressed in root. Real-time quantitative PCR analysis showed that a strong upregulation of TcLOX1 was observed in response to methyl jasmonate and abscisic acid (ABA), while TcLOX2 was strongly upregulated by ABA. However, TcLOX1 and TcLOX2 were nearly not responding to salicylic acid. These data suggest both TcLOX1 and TcLOX2 play an important role in T. chinensis, and they are required in different physiological processes involved in different plant signals in vivo.     

The mechanism involved in the loss of PTEN expression in NSCLC tumor cells

Loss of PTEN expression is observed in most non-small cell lung cancers (NSCLC). However, the mechanism by which PTEN expression is regulated in NSCLC has not been fully elucidated. In this study, we investigated the role of DNA methyltransferases (Dnmts), microRNA-29b (miR-29b), and anti-miR-29b inhibitor in PTEN promoter methylation and PTEN gene expression in H358 NSCLC cells in vitro and in vivo. PTEN mRNA was measured by RT-PCR. PTEN and Dnmts protein levels were measured by Western blot. miR-29b expression was detected by Northern blot. A xenograft H358 tumor mouse model was established by subcutaneously inoculating H358 cells into the right hind limbs of nude mice. We found that radiation induced cell apoptosis and hypomethylation in PTEN promoter, PTEN and miR-29b expression, and downregulation of Dnmt1, 3a and 3b expression in H358 tumor cells. The effect of radiation on gene expression and apoptosis was blocked by anti-miR-29b inhibitor. In the xenograft H358 tumor model, anti-miR-29b inhibitor reversed radiation-induced tumor growth delay, PTEN reexpression and downregulation of Dnmts expression. Our study suggested that miR-29b is an upstream molecule of PTEN. miR-29b regulates PTEN gene expression through downregulating Dnmts expression and subsequently induces hypomethylation in PTEN promoter. Targeting therapy could be established in NSCLC by upregulating miR-29b expression.