Census of orthologous genes and self-organizing maps of biologically relevant transcriptional patterns in chickens (Gallus gallus)

The launch of large-scale chicken expressed sequence tags (EST) projects has placed the chicken in the lead for the number of EST sequences in agriculturally important animals. More than 451,000 chicken ESTs derived from over 158 libraries have been deposited in the NCBI dbEST database as of December 2003. But how many genes these ESTs represent and how they are expressed in different chicken tissues/organs remain undetermined. In the present research, we developed a human gene-based strategy for census of chicken orthologous genes and identification of their expression patterns. Among 34,157 human coding genes used in the study, BLAST analysis revealed that 11,066 genes provisionally matched 248,628 chicken ESTs. Based on the average EST abundance of the orthologous genes, the current public repository of chicken ESTs could represent 20,000 provisional genes. Analysis of gene expression in 14 single tissues/organs showed that approximately 15% of genes were expressed exclusively in single tissue/organ whereas the remaining 85% of genes were co-expressed in two or more tissues/organs. A majority (91.15%) of genes expressed in chicken embryos were also expressed at post-hatch stages, indicating that most genes activated in chicken embryos could serve housekeeping functions. Self-organizing maps (SOM) analysis organized 8807 provisional genes in selected chicken tissues into 98 clusters with each cluster being indicative of common regulatory factors and pathways. A total of 969 provisional orthologous genes were identified as preferentially expressed genes (PEGs) in various chicken tissues/organs (LOD>3.0). No doubt, the present study on gene expression patterns will provide insight into dynamics of metabolic pathways and tissue/organ programming and reprogramming in chickens.     

Hydrophobic sequences target and anchor perilipin A to lipid droplets

Perilipins regulate triacylglycerol storage and hydrolysis in adipocytes. The central 25% of the perilipin A sequence, including three hydrophobic sequences (H1, H2, and H3) and an acidic region, targets and anchors perilipins to lipid droplets. Thus, we hypothesized that H1, H2, and H3 are targeting and anchoring motifs. We now show that deletion of any single hydrophobic sequence or combinations of H1 and H3 or H2 and H3 does not prevent targeting of the mutated perilipin to lipid droplets. In contrast, mutated perilipin lacking H1 and H2 showed reduced targeting, whereas perilipin lacking H1, H2, and H3 targeted poorly to lipid droplets; thus, H3 is a weak targeting signal and either H1 or H2 is required for optimal targeting. Complete elimination of perilipin targeting was observed only when all three hydrophobic sequences were deleted in combination with either the acidic region or N-terminal sequences predicted to form amphipathic beta-strands. Unlike intact perilipin A, mutated perilipin lacking either H1 and H2 or H1, H2, and H3 was released from lipid droplets after alkaline carbonate treatment, suggesting that these forms are loosely associated with lipid droplets. The three hydrophobic sequences play a major role in targeting and anchoring perilipins to lipid droplets.     

Efficacy of linear and convex transducers for ultrasound-guided transvaginal follicle aspiration

A new device was developed to hold linear transducers for transvaginal follicle aspiration. Efficacy of follicle aspiration was compared using a linear 6 MHz and a convex 5 MHz transducer. Fifty-five cows were submitted to follicle aspiration at random days of the estrous cycle. Aspirations were conducted with linear (n=28) and convex (n=38) transducers with 18 G needles at a negative pressure corresponding to 13 ml H(2)O/min. A greater number of follicles were aspirated using convex than to linear probe (12.4 versus 7.8, respectively, P<0.05). Mean number of oocytes and recovery rates were similar for convex (5.4 and 48.6%) and linear (4.6 and 59.3%) transducers. Limited space between the linear transducer and needle guide restricted access to some portions of the ovary, reducing the number of follicles aspirated using a linear transducer. The newly developed adaptor allowed greater stability, holding the ovaries firmly against the linear transducer. This diminished mobility permitted a similar number of oocytes to be recovered with both transducers. In conclusion, this new adaptor provided a low cost alternative for routine follicle aspiration and oocyte recovery in cattle.     

Pertussis toxin directly activates endothelial cell p42/p44 MAP kinases via a novel signaling pathway

Bordetella pertussisgenerates a bacterial toxin utilized in signal transductioninvestigation because of its ability to ADP ribosylate specific Gproteins. We previously noted that pertussis toxin (PTX) directlyactivates endothelial cells, resulting in disruption of monolayerintegrity and intercellular gap formation via a signaling pathway thatinvolves protein kinase C (PKC). We studied the effect of PTX on theactivity of the 42- and 44-kDa extracellular signal-regulated kinases(ERK), members of a kinase family known to be activated by PKC. PTXcaused a rapid time-dependent increase in bovine pulmonary arteryendothelial cell ERK activity that was significantly attenuated by1) pharmacological inhibition of MEK, the upstream ERKactivating kinase, 2) an MEK dominant-negative construct,and 3) PKC inhibition with bisindolylmaleimide. There waslittle evidence for the involvement of either G-subunits, RasGTPases, Raf-1, p60^src, or phosphatidylinositol 3'-kinasesin PTX-mediated ERK activation. Both the purified -oligomer bindingsubunit of the PTX holotoxin and a PTX holotoxin mutant geneticallyengineered to eliminate intrinsic ADP ribosyltransferase activitycompletely reproduced PTX effects on ERK activation, suggesting thatPTX-induced ERK activation involves a novel PKC-dependent signalingmechanism that is independent of either Ras or Raf-1 activities anddoes not require G protein ADP ribosylation.

     

Next‐generation sequencing,FISH mapping and synteny‐based modeling reveal mechanisms of decreasing dysploidy in Cucumis

In the large Cucurbitaceae genus Cucumis, cucumber (C. sativus) is the only species with 2n = 2x = 14 chromosomes. The majority of the remaining species, including melon (C. melo) and the sister species of cucumber, C. hystrix, have 2n = 2x = 24 chromosomes, implying a reduction from n = 12 to n = 7. To understand the underlying mechanisms, we investigated chromosome synteny among cucumber, C. hystrix and melon using integrated and complementary approaches. We identified 14 inversions and a C. hystrix lineage‐specific reciprocal inversion between C. hystrix and melon. The results reveal the location and orientation of 53 C. hystrix syntenic blocks on the seven cucumber chromosomes, and allow us to infer at least 59 chromosome rearrangement events that led to the seven cucumber chromosomes, including five fusions, four translocations, and 50 inversions. The 12 inferred chromosomes (AK1–AK12) of an ancestor similar to melon and C. hystrix had strikingly different evolutionary fates, with cucumber chromosome C1 apparently resulting from insertion of chromosome AK12 into the centromeric region of translocated AK2/AK8, cucumber chromosome C3 originating from a Robertsonian‐like translocation between AK4 and AK6, and cucumber chromosome C5 originating from fusion of AK9 and AK10. Chromosomes C2, C4 and C6 were the result of complex reshuffling of syntenic blocks from three (AK3, AK5 and AK11), three (AK5, AK7 and AK8) and five (AK2, AK3, AK5, AK8 and AK11) ancestral chromosomes, respectively, through 33 fusion, translocation and inversion events. Previous results (Huang, S., Li, R., Zhang, Z. et al., 2009 , Nat. Genet. 41, 1275–1281; Li, D., Cuevas, H.E., Yang, L., Li, Y., Garcia‐Mas, J., Zalapa, J., Staub, J.E., Luan, F., Reddy, U., He, X., Gong, Z., Weng, Y. 2011a, BMC Genomics, 12, 396) showing that cucumber C7 stayed largely intact during the entire evolution of Cucumis are supported. Results from this study allow a fine‐scale understanding of the mechanisms of dysploid chromosome reduction that has not been achieved previously.     

Determination of the lysosomal hydrolase activity in blood collected on filter paper,an alternative to screen high risk populations

This study aimed to determine the enzymatic activity in dried blood samples collected on filter paper (DBS) for the diagnosis of the following diseases: Fabry, Pompe, Mucopolysaccharidosis type I (MPS I) and Mucopolysaccharosis type VI (MPS VI). DBS was used for high risk patientscreening, according to clinical suspicion. Plasma, leukocytes and cultured fibroblasts were used to confirm the diagnosis when necessary. Among the 529 DBS samples sent to the laboratory, 164 had abnormal results. Confirmatory materials of 73 individuals were rerouted. The frequency of diagnosis for lysosomal storage disorders was 5.9%. DBS is an alternative screening technique used in high risk populations, which should lead to earlier diagnosis for lysosomal storage disorders (LSDs), help patients get treatment sooner and improve the outcome of the disease.     

Understanding the function of bacterial and eukaryotic thiolases II by integrating evolutionary and functional approaches

Acetoacetyl-CoA thiolase (EC 2.3.1.9), commonly named thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA and CoA. This enzyme acts in anabolic processes as the first step in the biosynthesis of isoprenoids and polyhydroxybutyrate in eukaryotes and bacteria, respectively. We have recently reported the evolutionary and functional equivalence of these enzymes, suggesting that thiolase II could be the rate limiting enzyme in these pathways and presented evidence indicating that this enzyme modulates the availability of reducing equivalents during abiotic stress adaptation in bacteria and plants. However, these results are not sufficient to clarify why thiolase II was evolutionary selected as a critical enzyme in the production of antioxidant compounds. Regarding this intriguing topic, we propose that thiolase II could sense changes in the acetyl-CoA/CoA ratio induced by the inhibition of the tricarboxylic acid cycle under abiotic stress. Thus, the high level of evolutionary and functional constraint of thiolase II may be due to the connection of this enzyme with an ancient and conserved metabolic route.     

Off-Target Effects of the Septin Drug Forchlorfenuron on Nonplant Eukaryotes

The septins are a family of GTP-binding proteins that form cytoskeletal filaments. Septins are highly conserved and evolutionarily ancient but are absent from land plants. The synthetic plant cytokinin forchlorfenuron (FCF) was shown previously to inhibit budding yeast cell division and induce ectopic septin structures (M. Iwase, S. Okada, T. Oguchi, and A. Toh-e, Genes Genet. Syst. 79:199–206, 2004, http://dx.doi.org/10.1266/ggs.79.199). Subsequent studies in a wide range of eukaryotes have concluded that FCF exclusively inhibits septin function, yet the mechanism of FCF action in nonplant cells remains poorly understood. Here, we report that the cellular effects of FCF are far more complex than previously described. The reported growth arrest of budding yeast cells treated with 1 mM FCF partly reflects sensitization caused by a bud4 mutation present in the W303 strain background. In wild-type (BUD4⁺) budding yeast, growth was inhibited at FCF concentrations that had no detectable effect on septin structure or function. Moreover, FCF severely inhibited the proliferation of fission yeast cells, in which septin function is nonessential. FCF induced fragmentation of budding yeast mitochondrial reticula and the loss of mitochondrial membrane potential. Mitochondria also fragmented in cultured mammalian cells treated with concentrations of FCF that previously were assumed to target septins only. Finally, FCF potently inhibited ciliation and motility and induced mitochondrial disorganization in Tetrahymena thermophila without apparent alterations in septin structure. None of these effects was consistent with the inhibition of septin function. Our findings point to nonseptin targets as major concerns when using FCF.