Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection

The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex-pression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.     

PERMANENT GENETIC RESOURCES: Twelve new microsatellite markers for the Chinese shrimp Fenneropenaeus chinensis

Twelve new microsatellite markers were isolated by sequencing random clones from a genomic library of Fenneropenaeus chinensis. The number of alleles per locus ranged from five to 15. The polymorphism information content ranged from 0.568 to 0.898, and observed and expected heterozygosities from 0.344 to 0.882 and from 0.691 to 0.915, respectively. All loci except one conformed to Hardy-Weinberg equilibrium. These markers, described here for Chinese shrimp, will be further used to analyse the species' population genetics.     

A-type GABA receptor as a central target of TRPM8 agonist menthol

Menthol is a widely-used cooling and flavoring agent derived from mint leaves. In the peripheral nervous system, menthol regulates sensory transduction by activating TRPM8 channels residing specifically in primary sensory neurons. Although behavioral studies have implicated menthol actions in the brain, no direct central target of menthol has been identified. Here we show that menthol reduces the excitation of rat hippocampal neurons in culture and suppresses the epileptic activity induced by pentylenetetrazole injection and electrical kindling in vivo. We found menthol not only enhanced the currents induced by low concentrations of GABA but also directly activated GABA(A) receptor (GABA(A)R) in hippocampal neurons in culture. Furthermore, in the CA1 region of rat hippocampal slices, menthol enhanced tonic GABAergic inhibition although phasic GABAergic inhibition was unaffected. Finally, the structure-effect relationship of menthol indicated that hydroxyl plays a critical role in menthol enhancement of tonic GABA(A)R. Our results thus reveal a novel cellular mechanism that may underlie the ambivalent perception and psychophysical effects of menthol and underscore the importance of tonic inhibition by GABA(A)Rs in regulating neuronal activity.     

Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes

The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this study is to identify candidate virulence genes by systematic comparative analysis of the attenuated strain with the published genome sequences of two virulent and closely related strains of B. abortus, 9-941 and 2308. The two S19 chromosomes are 2,122,487 and 1,161,449 bp in length. A total of 3062 genes were identified and annotated. Pairwise and reciprocal genome comparisons resulted in a total of 263 genes that were non-identical between the S19 genome and any of the two virulent strains. Amongst these, 45 genes were consistently different between the attenuated strain and the two virulent strains but were identical amongst the virulent strains, which included only two of the 236 genes that have been implicated as virulence factors in literature. The functional analyses of the differences have revealed a total of 24 genes that may be associated with the loss of virulence in S19. Of particular relevance are four genes with more than 60 bp consistent difference in S19 compared to both the virulent strains, which, in the virulent strains, encode an outer membrane protein and three proteins involved in erythritol uptake or metabolism.     

45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp

Background

In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported.

Methods and Results

During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions) at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region.

Conclusions

The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.     

In vivo patellofemoral forces in high flexion total knee arthroplasty

This study compares the in vivo patellofemoral contact forces generated in high flexion fixed bearing posterior cruciate retaining Nexgen CR-Flex (PCR) and high flexion posterior stabilized Nexgen LPS-Flex (LPS) TKAs with that of normal knees from full knee extension to maximum weight bearing flexion. Ten patients with the PCR total knee arthroplasty (TKA), ten with the LPS TKA and seven patients having normal knees were fluoroscoped while performing a deep knee bend activity. In vivo femorotibial kinematics, obtained from 3D-to-2D registration technique, and patellar kinematics obtained by direct measurements from the fluoroscopic images were entered into a 3D inverse dynamics mathematical model to determine the in vivo contact forces at the knee. The variation in the patellofemoral and quadriceps forces with flexion were found to be similar across the three groups-increasing from full extension to 90 degrees of flexion, reaching a maximum between 90 degrees and 120 degrees of flexion and then decreasing until maximum flexion. At maximum knee flexion, these forces were found to be significantly lower in the normal knees than in the TKAs. The patellar ligament to quadriceps force ratio decreased with the increase in knee flexion while the patellofemoral to quadriceps force ratio increased. A strong correlation was found to exist between the patellofemoral forces, the femorotibial contact forces and the forces in the extensor mechanism. The PCR TKA in this study exhibited greater resemblance to the normal patients with respect to the patellofemoral forces than the LPS TKA though significant differences in the two implant types were not observed.     

Extracellular superoxide dismutase inhibits inflammation by preventing oxidative fragmentation of hyaluronan

Extracellular superoxide dismutase (EC-SOD) is expressed at high levels in lungs. EC-SOD has a polycationic matrix-binding domain that binds to polyanionic constituents in the matrix. Previous studies indicate that EC-SOD protects the lung in both bleomycin- and asbestos-induced models of pulmonary fibrosis. Although the mechanism of EC-SOD protection is not fully understood, these studies indicate that EC-SOD plays an important role in regulating inflammatory responses to pulmonary injury. Hyaluronan is a polyanionic high molecular mass polysaccharide found in the extracellular matrix that is sensitive to oxidant-mediated fragmentation. Recent studies found that elevated levels of low molecular mass hyaluronan are associated with inflammatory conditions. We hypothesize that EC-SOD may inhibit pulmonary inflammation in part by preventing superoxide-mediated fragmentation of hyaluronan to low molecular mass fragments. We found that EC-SOD directly binds to hyaluronan and significantly inhibits oxidant-induced degradation of this glycosaminoglycan. In vitro human polymorphic neutrophil chemotaxis studies indicate that oxidative fragmentation of hyaluronan results in polymorphic neutrophil chemotaxis and that EC-SOD can completely prevent this response. Intratracheal injection of crocidolite asbestos in mice leads to pulmonary inflammation and injury that is enhanced in EC-SOD knock-out mice. Notably, hyaluronan levels are increased in the bronchoalveolar lavage fluid after asbestos-induced pulmonary injury, and this response is markedly enhanced in EC-SOD knock-out mice. These data indicate that inhibition of oxidative hyaluronan fragmentation probably represents one mechanism by which EC-SOD inhibits inflammation in response to lung injury.     

The characterization of color mutations in Bangiaceae (Bangiales, Rhodophyta)

The color mutations in Bangiaceae were investigated by treating the blades, conchocelis and conchospores phase of Bangia sp., Porphyra yezoensis, and P. haitanensis sampled in China with mutagen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). A high percentage of mutation in different expression characteristics in all three phases were shown within optimum mutagen concentrations. Among mutagenized blades, mutations occurred on single cells, which is a direct outcome of mutation of haploid cells. The mutation of mutagenized conchocelis resulted in a two-step process: low-level expression in conchocelis phase, and high-level expression in progeny, explaining that mutation took place in diploid cells. The mutations of conchospores were expressed immediately at germination of spores, indicating a change in ploidy. This paper reports the process of meiosis and its effect on frond development, and the relation between color mutations and morphological characteristics expressed by mutations in Bangiaceae.     

Shox2-deficiency leads to dysplasia and ankylosis of the temporomandibular joint in mice

The temporomandibular joint (TMJ) is a unique synovial joint whose development differs from the formation of other synovial joints. Mutations have been associated with the developmental defects of the TMJ only in a few genes. In this study, we report the expression of the homeobox gene Shox2 in the cranial neural crest derived mesenchymal cells of the maxilla-mandibular junction and later in the progenitor cells and undifferentiated chondrocytes of the condyle as well as the glenoid fossa of the developing TMJ. A conditional inactivation of Shox2 in the cranial neural crest-derived cells causes developmental abnormalities in the TMJ, including dysplasia of the condyle and glenoid fossa. The articulating disc forms but fuses with the fibrous layers of the condyle and glenoid fossa, clinically known as TMJ ankylosis. Histological examination indicates a delay in development in the mutant TMJ, accompanied by a significantly reduced rate of cell proliferation. In situ hybridization further demonstrates an altered expression of several key osteogenic genes and a delayed expression of the osteogenic differentiation markers. Shox2 appears to regulate the expression of osteogenic genes and is essential for the development and function of the TMJ. The Shox2 conditional mutant thus provides a unique animal model of TMJ ankylosis.     

Mechanisms involved in the regulation of histone lysine demethylases

Since the first histone lysine demethylase KDM1 (LSD1) was discovered in 2004, a great number of histone demethylases have been recognized and shown to play important roles in gene expression, as well as cellular differentiation and animal development. The chemical mechanisms and substrate specificities have already been extensively discussed elsewhere. This review focuses primarily on regulatory mechanisms that modulate demethylase recruitment and activity.