Effects of exposure to 17alpha-ethinylestradiol during early development on sexual differentiation and induction of vitellogenin in zebrafish (Danio rerio)

The effect of acute stress on plasma beta-corticosterone (B), testosterone (T) and estradiol-17beta (E2) concentrations in juvenile alligators collected from sites with varying sediment contaminants was examined in this study. Dramatic increases in plasma B concentrations were observed in alligators from all of the sites after 2 h of capture although females from the intermediate contaminant site exhibited a significantly lower percentage increase in B than females from the other two sites. Males from the site with the highest contaminant levels exhibited elevated initial B concentrations relative to the other sites. This pattern was not observed after 2 h of restraint. Females from the highest contaminant site exhibited depressed initial T when compared to the other sites although this pattern was not observed after 2 h of restraint. Neither E2 nor T decreased after 2 h in females, whereas T concentrations decreased in all males over the same time period. The variance associated with these endpoints was also examined to determine whether it could serve as a more sensitive marker for perturbations of the endocrine system and stress response. Females from the higher and intermediate contaminant sites exhibited the lowest and highest standard errors (respectively) associated with 2 h plasma B concentrations with no differences among mean concentrations suggesting a perturbation of the stress response in these animals that was not detected by examining the means. We concluded that the environmental contaminants could be acting as stressors, leading to the observed differences.     

The ability to form full-length intron RNA circles is a general property of nuclear group I introns

In addition to splicing, group I intron RNA is capable of an alternative two-step processing pathway that results in the formation of full-length intron circular RNA. The circularization pathway is initiated by hydrolytic cleavage at the 3′ splice site and followed by a transesterification reaction in which the intron terminal guanosine attacks the 5′ splice site presented in a structure analogous to that of the first step of splicing. The products of the reactions are full-length circular intron and unligated exons. For this reason, the circularization reaction is to the benefit of the intron at the expense of the host. The circularization pathway has distinct structural requirements that differ from those of splicing and appears to be specifically suppressed in vivo. The ability to form full-length circles is found in all types of nuclear group I introns, including those from the Tetrahymena ribosomal DNA. The biological function of the full-length circles is not known, but the fact that the circles contain the entire genetic information of the intron suggests a role in intron mobility.     

Conditional expression of Smad7 in pancreatic beta cells disrupts TGF-beta signaling and induces reversible diabetes mellitus

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-beta (TGF-beta) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-beta signaling, to identify distinct roles for this pathway in adult and embryonic beta cells. Smad7 expression in Pdx1+ embryonic pancreas cells resulted in striking embryonic beta cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1+ cells reduced detectable beta cell expression of MafA, menin, and other factors that regulate beta cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-beta signaling. Thus, our studies reveal that TGF-beta signaling is crucial for establishing and maintaining defining features of mature pancreatic beta cells.     

Transforming growth factor-alpha in the mammalian brain. Immunohistochemical detection in neurons and characterization of its mRNA

In this communication, we demonstrate that adult mammalian brain neurons express transforming growth factor-alpha (TGF-alpha). We used the anti-TGF-alpha monoclonal antibody, MF9, to immunohistochemically localize TGF-alpha in human and rat brain. We found specific immunoreactivity in neurons throughout the brain which was not a result of cross-reactivity of MF9 with the neuropeptide, synenkephalin. Northern blot analysis of bovine and rat brain RNA using human and rat TGF-alpha cDNA probes, respectively, revealed a single 4.8-kilobase pair mRNA with approximately equal abundance in the bovine brainstem, cerebellum, hypothalamus, and cerebral cortex. Fetal rat brain had about 2-fold more TGF-alpha mRNA than did adult rat. The brain TGF-alpha cDNA was cloned from a human neonatal brainstem library. Four identical clones were isolated after screening 10(6) recombinant lambda gt11 phage. The sequence of the 894-base pair cDNA was virtually identical with the cDNA isolated from a human renal cell carcinoma. A single alanine codon was deleted in the brain cDNA at an exon-exon junction. The alanine deletion is within the amino-terminal region of the TGF-alpha precursor that is thought to be removed by proteolytic processing of the precursor to the mature growth factor. These studies indicate that the normal mammalian brain neurons express TGF-alpha.     

Identification of local selective sweeps in human populations since the exodus from Africa

Selection on the human genome has been studied using comparative genomics and SNP architecture in the lineage leading to modern humans. In connection with the African exodus and colonization of other continents, human populations have adapted to a range of different environmental conditions. Using a new method that jointly analyses haplotype block length and allele frequency variation (F(ST)) within and between populations, we have identified chromosomal regions that are candidates for having been affected by local selection. Based on 1.6 million SNPs typed in 71 individuals of African American, European American and Han Chinese descent, we have identified a number of genes and non-coding regions that are candidates for having been subjected to local positive selection during the last 100 000 years. Among these genes are those involved in skin pigmentation (SLC24A5) and diet adaptation (LCT). The list of genes implicated in these local selective sweeps overlap partly with those implicated in other studies of human populations using other methods, but show little overlap with those postulated to have been under selection in the 5-7 myr since the divergence of the ancestors of human and chimpanzee. Our analysis provides focal points in the genome for detailed studies of evolutionary events that have shaped human populations as they explored different regions of the world.     

Parallel post-source decay for increasing protein identification confidence levels from 2-D gels

Peptide mass fingerprinting (PMF) has over the years become one of the most commonly used tools for high-throughput analysis and identification of proteins. This method is applicable when relatively simple samples have to be analysed and it is commonly used for analysing proteins previously separated by 2-DE. The most common type of instrument used for this approach is the MALDI-TOF that has proved to be particularly suitable for the PMF analysis because of its characteristics of speed, robustness, sensitivity and automation. We have used a MALDI-TOF equipped with a novel parallel PSD capability (MALDI micro MX), to perform the analysis of two sets of different biological samples isolated by 2-DE. By using a method that integrates the data obtained by PMF analysis with the PSD data obtained in the same experiment, we show that the new multiplexed PSD solution increases the protein identification rate compared to the normal PMF approach. We also investigated the use of a charge-directed fragmentation modification reagent to improve the identification rate and confidence levels.     

Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis

2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) prior to separation with 2-DE. Due to the high extinction coefficient of the Alexa-fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2-DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II-tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten-fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1-4 ng) and dynamic ranges (10(3)). ALIS is a powerful normalization method for quantitative 2-DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance-variance compared to normalization through division by the total spot volume.     

RNAi-mediated Hip1R silencing results in stable association between the endocytic machinery and the actin assembly machinery

Actin filaments transiently associate with the endocytic machinery during clathrin-coated vesicle formation. Although several proteins that might mediate or regulate this association have been identified, in vivo demonstration of such an activity has not been achieved. Huntingtin interacting protein 1R (Hip1R) is a candidate cytoskeletal-endocytic linker or regulator because it binds to clathrin and actin. Here, Hip1R levels were lowered by RNA interference (RNAi). Surprisingly, rather than disrupting the transient association between endocytic and cytoskeletal proteins, clathrin-coated structures (CCSs) and their endocytic cargo became stably associated with dynamin, actin, the Arp2/3 complex, and its activator, cortactin. RNAi double-depletion experiments demonstrated that accumulation of the cortical actin-endocytic complexes depended on cortactin. Fluorescence recovery after photobleaching showed that dynamic actin filament assembly can occur at CCSs. Our results provide evidence that Hip1R helps to make the interaction between actin and the endocytic machinery functional and transient.     

Cutting Edge: TLR13 Is a Receptor for Bacterial RNA

Bacterial RNA (bRNA) can induce cytokine production in macrophages and dendritic cells (DCs) through a previously unidentified receptor. Gene expression analysis of murine DCs showed that bRNA induced gene regulation similar to that induced by stimulation of TLR7 with R848. Although TLR7 was dispensable for cytokine induction by bRNA, TLR-associated proteins MyD88 and UNC93B were required. TLR13 is an endosomal murine TLR that has been described to interact with UNC93B with, so far, no characterized ligand. Small interfering RNA against TLR13 reduced cytokine induction by bRNA in DCs. Moreover, Chinese hamster ovary cells transfected with TLR13, but not with TLR7 or 8, could activate NF-κB in response to bRNA or Streptococcus pyogenes in an RNA-specific manner. TLR7 antagonist IRS661 could, in addition, inhibit TLR13 signaling and reduced recognition of whole Gram-positive bacteria by DCs, also in the absence of TLR7. The results identify TLR13 as a receptor for bRNA.