Denigrins H-L: Sulfated Derivatives of Denigrins D and E from a New Zealand Dictyodendrilla c.f. dendyi Marine Sponge.

Five new sulfated arylpyrrole and arylpyrrolone alkaloids, denigrins H-L (1-5), along with two known compounds, dictyodendrin B and denigrin G, were isolated from an extract of a New Zealand Dictyodendrilla c.f. dendyi marine sponge. Denigrins H-L represent the first examples of sulfated denigrins, with denigrins H and I (1-2), as derivatives of denigrin D, containing a pyrrolone core, and denigrins J-L (3-5), as derivatives of denigrin E (6), containing a pyrrole core. Their structures were elucidated by interpretation of 1D and 2D NMR spectroscopic data, ESI, and HR-ESI-MS spectrometric data, as well as comparison with literature data. Compounds 1-5, along with six known compounds previously isolated from the same extract, showed minimal cytotoxicity against the HeLa cervical cancer cell line.

Mutations in Grxcr1 are the basis for inner ear dysfunction in the pirouette mouse.

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.

Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function.

Positional cloning of hereditary deafness genes is a direct approach to identify molecules and mechanisms underlying auditory function. Here we report a locus for dominant deafness, DFNA36, which maps to human chromosome 9q13-21 in a region overlapping the DFNB7/B11 locus for recessive deafness. We identified eight mutations in a new gene, transmembrane cochlear-expressed gene 1 (TMC1), in a DFNA36 family and eleven DFNB7/B11 families. We detected a 1.6-kb genomic deletion encompassing exon 14 of Tmc1 in the recessive deafness (dn) mouse mutant, which lacks auditory responses and has hair-cell degeneration. TMC1 and TMC2 on chromosome 20p13 are members of a gene family predicted to encode transmembrane proteins. Tmc1 mRNA is expressed in hair cells of the postnatal mouse cochlea and vestibular end organs and is required for normal function of cochlear hair cells.

Chitosan: A review of sources and preparation methods.

Chitosan, derived from chitin, has many desirable biomedical attributes. This review aims to explore different sources of chitin and methods of chitosan production with industrial consideration. This article first discussed different sources of chitin for industrial scale production, with considerations given to both their environmental impacts and commercialization potential. Secondly, this article reviews the two categories of chitosan preparation - chemical methods and biological methods - based on existing publications which used lobster by-products as a feedstock source. The mechanisms of the chemical methods are firstly summarized, and then the different chemical agents and reaction parameters used are discussed. Next, both enzymatic and fermentation-based approaches are reviewed under biological methods and compared with chemical methodologies, with lactic fermentation methods as the major focus. This article concludes that lobster cephalothorax could be an ideal source for chitosan preparation on an industrial scale; and chemical methods involve simpler processing overall, while producing chitosan with stronger bioactivities because of the lower molecular weight (MW) and higher degree of deacetylation (DD) achieved by the products. Moreover, due to biological methods inevitably necessitating further chemical processing, an approach involving some unconventional chemical methods has been regarded as a more suitable strategy for industrial scale chitosan production.

Development of a qPCR Method to Measure Mitochondrial and Genomic DNA Damage with Application to Chemotherapy-Induced DNA Damage and Cryopreserved Cells.

DNA damage quantitation assays such as the comet assay have focused on the measurement of total nuclear damage per cell. The adoption of PCR-based techniques to quantify DNA damage has enabled sequence- and organelle-specific assessment of DNA lesions. Here we report on an adaptation of a qPCR technique to assess DNA damage in nuclear and mitochondrial targets relative to control. Novel aspects of this assay include application of the assay to the Rotor-Gene platform with optimized DNA polymerase/fluorophore/primer set combination in a touchdown PCR protocol. Assay validation was performed using ultraviolet C radiation in A549 and THP1 cancer cell lines. A comparison was made to the comet assay applied to peripheral blood mononuclear cells, and an estimation of the effects of cryopreservation on ultraviolet C-induced DNA damage was carried out. Finally, dose responses for DNA damage were measured in peripheral blood mononuclear cells following exposure to the cytotoxic agents bleomycin and cisplatin. We show reproducible experimental outputs across the tested conditions and concordance with published findings with respect to mitochondrial and nuclear genotoxic susceptibilities. The application of this DNA damage assay to a wide range of clinical and laboratory-derived samples is both feasible and resource-efficient.

Signatures from tissue-specific MPSS libraries identify transcripts preferentially expressed in the mouse inner ear.

Specialization in cell function and morphology is influenced by the differential expression of mRNAs, many of which are expressed at low abundance and restricted to certain cell types. Detecting such transcripts in cDNA libraries may require sequencing millions of clones. Massively parallel signature sequencing (MPSS) is well suited to identifying transcripts that are expressed in discrete cell types and in low abundance. We have made MPSS libraries from microdissections of three inner ear tissues. By comparing these MPSS libraries to those of 87 other tissues included in the Mouse Reference Transcriptome online resource, we have identified genes that are highly enriched in, or specific to, the inner ear. We show by RT-PCR and in situ hybridization that signatures unique to the inner ear libraries identify transcripts with highly specific cell-type localizations. These transcripts serve to illustrate the utility of a resource that is available to the research community. Utilization of these resources will increase the number of known transcription units and expand our knowledge of the tissue-specific regulation of the transcriptome.

Mutation of a transcription factor, TFCP2L3, causes progressive autosomal dominant hearing loss, DFNA28.

We ascertained a large American family with an autosomal dominant form of progressive non-syndromic sensorineural hearing loss. After excluding linkage to known deafness loci, we performed a genome-wide scan and found linkage to marker GAAT1A4 on chromosome 8q22 (LOD=5.12 at theta=0), and this locus was designated DFNA28. Sequencing of six candidate genes in the 1.4 cM linked region identified a frameshift mutation (1609-1610insC) resulting in a premature translation stop codon in exon 14 of the gene TFCP2L3 (transcription factor cellular promoter 2-like 3). TFCP2L3 is a member of a family of transcription factor genes whose archetype is TFCP2, a mammalian homolog of the Drosophila gene grainyhead. Northern blot analyses and in situ hybridization studies show that mouse Tfcp2l3 is expressed in many epithelial tissues, including cells lining the cochlear duct, at embryonic day 18.5 and postnatal day 5.