Frameshift deletions of exons 3-7 and revertant fibers in Duchenne muscular dystrophy: mechanisms of dystrophin production.

Duchenne muscular dystrophy (DMD) patients with mutations that disrupt the translational reading frame produce little or no dystrophin. Two exceptions are the deletion of exons 3-7 and the occurrence of rare dystrophin-positive fibers (revertant fibers) in muscle of DMD patients. Antibodies directed against the amino-terminus and the 5' end of exon 8 did not detect dystrophin in muscle from patients who have a deletion of exons 3-7. However, in all cases, dystrophin was detected with an antibody directed against the 3' end of exon 8. The most likely method of dystrophin production in these cases is initiation at a new start codon in exon 8. We also studied two patients who have revertant fibers: one had an inherited duplication of exons 5-7, which, on immunostaining, showed two types of revertant fibers; and the second patient had a 2-bp nonsense mutation in exon 51, which creates a cryptic splice site. An in-frame mRNA that uses this splice site in exon 51 was detected. Immunostaining demonstrated the presence of the 3' end of exon 51, which is in agreement with the use of this mRNA in revertant fibers. The most likely method of dystrophin production in these fibers is a second mutation that restores the reading frame.     

A species in decline: genetic diversity and conservation of the Victorian eastern barred bandicoot, Perameles gunnii

The eastern barred bandicoot, Perameles gunnii, has undergone a dramatic decline in distribution and abundance on the mainland of Australia during the twentieth century. In 1988 a captive breeding program was initiated to reduce the chance of extinction. With the extinction of the last wild mainland population in the early 1990s, reintroductions from captive-bred P. gunnii have met limited success, and currently only two extant populations persist in predator proof enclosures in the State of Victoria. With ~20 years of breeding, there are concerns that the genetic diversity within the breeding program has declined and may inhibit current and future success of the program. We have used ten nuclear microsatellite loci and sequencing of two partial mitochondrial genes (cytochrome oxidase I and ATPase 6) to determine genetic diversity within current Victorian P. gunnii. These diversity estimates are compared with historic samples from the captive breeding program dating back to 1995, historic samples from the last wild mainland population found at Hamilton in 1992 and contemporary Tasmanian wild populations. Results indicate that the captive P. gunnii population in the State of Victoria has lost significant genetic diversity through time. Genetic diversity is also reduced in populations at Hamilton Community Parklands and Mount Rothwell. Samples from the last wild population at Hamilton collected in 1992, along with samples from Tasmanian P. gunnii, had significantly greater genetic diversity than contemporary mainland populations. The results are discussed with reference to management options for maintaining genetic diversity within Victorian P. gunnii, including crossing Victorian and Tasmanian P. gunnii to increase genetic diversity, adaptability and evolutionary potential.     

Regulation of monocyte survival in vitro by deposited IgG: role of macrophage colony-stimulating factor

IgG deposition at tissue sites characteristically leads to macrophage accumulation and organ injury. Although the mechanism by which deposited IgG induces tissue injury is not known, we have recently demonstrated that deposited IgG stimulates the release of IL-8 and monocyte chemoattractant protein-1 from normal human monocytes, which may drive inflammation. Since IgG also induces macrophage accumulation in these diseases, we hypothesized that deposited IgG protects monocytes from apoptosis. As an in vitro model of the effect of deposited IgG on monocyte survival, monocyte apoptosis was studied after FcgammaR cross-linking. Monocytes cultured on immobilized IgG, which induces FcgammaR cross-linking, were protected from apoptosis, whereas monocytes cultured with equivalent concentrations of F(ab')2 IgG or 50 times higher concentrations of soluble IgG, neither of which induces FcgammaR cross-linking, were not protected. Moreover, this protection was transferable, as supernatants from immobilized IgG-stimulated monocytes protected freshly isolated monocytes from apoptosis and contained functional M-CSF, a known monocyte survival factor. M-CSF mediated the monocyte survival induced by FcgammaR cross-linking, as neutralizing anti-human M-CSF Abs blocked the monocyte protection provided by either immobilized IgG or IgG-stimulated monocyte supernatants. These findings demonstrate a novel mechanism by which deposited IgG targets tissue macrophage accumulation through FcgammaR-mediated M-CSF release. This pathway may play an important role in promoting and potentiating IgG-mediated tissue injury.     

Teleost introns are characterized by a high A+T content

We previously observed that Antarctic fish genes contain intron sequences of high A+T content (60-70% average A+T) which are in stark contrast with adjacent protein coding-sequences. Here, we report that this disparity in intron/exon base composition is a common feature among teleosts. We analyzed 483 teleost genomic DNA sequences, containing 2583 introns, from 80 teleost genera that populate polar, temperate, or tropical habitats. Eighty-nine percent of teleost introns display an A+T content between 50-84% A+T with a mean of 60% A+T. In contrast, only 37% of teleost exons have an A+T content greater-than 50% with a mean of 48% A+T. A comparison to homologous mammalian genes showed a striking difference; in this case, introns and exons have similar base compositions, averaging 45-47% A+T. This indicates that most teleost genes exhibit a large difference in base composition between their introns and exons. There was no correlation of teleost intron A+T content to intron length or habitat temperature range. Thus, teleost intron sequences tend to show the common feature of being much higher in A+T content then neighboring exons.     

Real time non-invasive imaging of receptor-ligand interactions in vivo

Non-invasive longitudinal detection and evaluation of gene expression in living animals can provide investigators with an understanding of the ontogeny of a gene's biological function(s). Currently, mouse model systems are used to optimize magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and optical imaging modalities to detect gene expression and protein function. These molecular imaging strategies are being developed to assess tumor growth and the tumor microenvironment. In addition, pre-labeling of progenitor cells can provide invaluable information about the developmental lineage of stem cells both in organogenesis and tumorigenesis. The feasibility of this approach has been extensively tested by targeting of endogenous tumor cell receptors with labeled ligand (or ligand analog) reporters and targeting enzymes with labeled substrate (or substrate analog). We will primarily discuss MRI, PET, and SPECT imaging of cell surface receptors and the feasibility of non-invasive imaging of gene expression using the tumor microenvironment (e.g., hypoxia) as a conditional regulator of gene expression.     

Artificial reporter gene providing MRI contrast based on proton exchange

Existing magnetic resonance reporter genes all rely on the presence of (super)paramagnetic substances and employ water relaxation to gain contrast. We designed a nonmetallic, biodegradable, lysine rich-protein (LRP) reporter, the prototype of a potential family of genetically engineered reporters expressing artificial proteins with frequency-selective contrast. This endogenous contrast, based on transfer of radiofrequency labeling from the reporter's amide protons to water protons, can be switched on and off.     

Cross-organ interactions between reproductive, gastrointestinal, and urinary tracts: modulation by estrous stage and involvement of the hypogastric nerve

Central nervous system neurons process information converging from the uterus, colon, and bladder, partly via the hypogastric nerve. This processing is influenced by the estrous cycle, suggesting the existence of an estrous-modifiable central nervous system substrate by which input from one pelvic organ can influence functioning of other pelvic organs. Here, we tested predictions from this hypothesis that acute inflammation of colon, uterine horn, or bladder would produce signs of inflammation in the other uninflamed organs (increase vascular permeability) and that cross-organ effects would vary with estrous and be eliminated by hypogastric neurectomy (HYPX). Under urethane anesthesia, the colon, uterine horn, or bladder of rats in proestrus or metestrus, with or without prior HYPX, was treated with mustard oil or saline. Two hours later, Evans Blue dye extravasation was measured to assess vascular permeability. Extravasation was increased in all inflamed organs, regardless of estrous stage. For rats in proestrus, but not metestrus, either colon or uterine horn inflammation significantly increased extravasation in the uninflamed bladder. Much smaller cross-organ effects were seen in colon and uterine horn. HYPX reduced extravasation in the inflamed colon and inflamed uterine horn, but not the inflamed bladder. HYPX eliminated the colon-to-bladder and uterine horn-to-bladder effects. These results demonstrate that inflaming one pelvic organ can produce estrous-modifiable signs of inflammation in other pelvic organs, particularly bladder, and suggest that the cross-organ effects involve the hypogastric nerve and are at least partly centrally mediated. Such effects could contribute to co-occurrence and cyclicity of distressing pelvic disorders in women.     

The effect of heavy metal speciation in sediment on bioavailability to tubificid worms

The bioavailability of heavy metals in sediment to freshwater tubificid worms was compared with measures of chemical extractability using a sequential extraction procedure. In order to provide a range of test sediments of different quality, various mineral phases were prepared, in which the metals were spiked by adsorption or coprecipitation and these were then mixed with a bulk base sediment in known proportions. Results indicated good correlation between worm metal burden and metal mobilised from the sediments in the first (exchangeable) sequential extraction step for Cd, Cu and Pb. Of the other metals tested, Zn levels in the worms were found to be constant, suggesting regulation, and Ni uptake was too small for accurate measurement. In general, metals spiked to the sediment directly, or adsorbed on the clay mineral phase were found to be much more available than those bound to sewage sludge, carbonate or hydrous ferric oxide phases.