Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling

Protein kinase C (PKC) family members transduce an abundance of diverse intracellular signals. Here we address the role of spatial and temporal segregation in signal specificity by measuring the activity of endogenous PKC at defined intracellular locations in real time in live cells. We targeted a genetically encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter (CKAR) (Violin, J. D., Zhang, J., Tsien, R. Y., and Newton, A. C. (2003) J. Cell Biol. 161, 899-909), to the plasma membrane, Golgi, cytosol, mitochondria, or nucleus by fusing appropriate targeting sequences to the NH2 or COOH terminus of CKAR. Measuring the phosphorylation of the reporter in the presence of PKC inhibitors, activators, and/or phosphatase inhibitors shows that activity at each region is under differential control by phosphatase activity; nuclear activity is completely suppressed by phosphatases, whereas membrane-associated activity is the least suppressed by phosphatases. UTP stimulation of endogenous P2Y receptors in COS 7 cells reveals spatiotemporally divergent PKC responses. Imaging the second messengers Ca2+ and diacylglycerol (DAG) reveal that PKC activity at each location is driven by an initial spike in Ca2+, followed by location-specific diacylglycerol generation. In response to UTP, phosphorylation of GolgiCKAR was sustained the longest, driven by the persistence of DAG, whereas phosphorylation of CytoCKAR was of the shortest duration, driven by high phosphatase activity. Our data reveal that the magnitude and duration of PKC signaling is location-specific and controlled by the level of phosphatase activity and persistence of DAG at each location.     

In vitro expressed dystrophin fragments do not associate with each other

Dystrophin, a component of the muscle membrane cytoskeleton, is the protein altered in Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD). Dystrophin shares significant homology with other cytoskeletal proteins, such as α-actinin and spectrin. On the basis of its sequence similarity with α-actinin and spectrin, dystrophin has been proposed to function as dimer. However, the existence of both dimers and monomers have been observed by electron microscopy. To address this apparent discrepancy, we expressed dystrophin fragments composed of different domains in an in vitro translation system. The expressed fragments were tested for their ability to interact with each other and full-length dystrophin by both immunoprecipitation and blot overlay assays. These assays were successfully used to demonstrate the dimerization of α-actinin and spectrin, yet failed to detect any interaction between dystrophin fragments. Although these in vitro results do not prove that dystrophin is not a dimer in vivo, they do indicate that this interaction is not like that of the α-actinin and spectrin.     

The Bloom's syndrome protein (BLM) interacts with MLH1 but is not required for DNA mismatch repair

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre- and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair.     

Thylakoid centers: Structures associated with the cyanobacterial photosynthetic membrane system

An ultrastructural study of four cyanobacteria (Anabaena cylindrica, Dermocarpa violaceae, Gleocapsa alpicola, Pleurocapsa minor) indicates the presence of previously undescribed thylakoid centers from which photosynthetic membranes (thylakoids) radiate. These peripherally located thylakoid centers are cylinders 30 nm wide by 320 nm long, consisting of globular subunits oriented in nonparallel stacked arrays. Thylakoids are attached to the outer surface of the cylinder along its longitudinal axis. Thylakoid centers appear to be functionally significant due to their structure, location and thylakoid association.     

Skewed X inactivation in a female MZ twin results in Duchenne muscular dystrophy.

One of female MZ twins presented with muscular dystrophy. Physical examination, creatine phosphokinase levels, and muscle biopsy were consistent with Duchenne muscular dystrophy (DMD). However, because of her sex she was diagnosed as having limb-girdle muscular dystrophy. With cDNA probes to the DMD gene, a gene deletion was detected in the twins and their mother. The de novo mutation which arose in the mother was shown by novel junction fragments generated by HindIII, PstI, or TaqI when probed with cDNA8. Additional evidence of a large gene deletion was given by novel SfiI junction fragments detected by probes p20, J-Bir, and J-66 on pulsed-field gel electrophoresis (PFGE). Immunoblot analysis of muscle from the affected twin showed dystrophin of normal size but of reduced amount. Immunofluorescent visualization of dystrophin revealed foci of dystrophin-positive fibers adjacent to foci of dystrophin-negative fibers. These data indicate that the affected twin is a manifesting carrier of an abnormal DMD gene, her myopathy being a direct result of underexpression of dystrophin. Cytogenetic analysis revealed normal karyotypes, eliminating the possibility of a translocation affecting DMD gene function. Both linkage analysis and DNA fingerprint analysis revealed that each twin has two different X chromosomes, eliminating the possibility of uniparental disomy as a mechanism for DMD expression. On the basis of methylation differences of the paternal and maternal X chromosomes in these MZ twins, we propose uneven lyonization (X chromosome inactivation) as the underlying mechanism for disease expression in the affected female.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Expression and contribution of endogenous IL-13 in an experimental model of sepsis

IL-13 has been shown to exert potent anti-inflammatory properties. In this study, we elucidated the functional role of endogenous IL-13 in a murine model of septic peritonitis induced by cecal ligation and puncture (CLP). Initial studies demonstrated that the level of IL-13 increased in tissues including liver, lung, and kidney, whereas no considerable increase was found in either peritoneal fluid or serum after CLP. Immunohistochemically, IL-13-positive cells were Kupffer cells in liver, alveolar macrophages in lung, and epithelial cells of urinary tubules in kidney. IL-13 blockade with anti-IL-13 Abs significantly decreased the survival rate of mice after CLP from 53% to 14% on day 7 compared with control. To determine the potential mechanisms whereby IL-13 exerted a protective role in this model, the effects of anti-IL-13 Abs on both local and systemic inflammation were investigated. Administration of anti-IL-13 Abs did not alter the leukocyte infiltration and bacterial load in the peritoneum after CLP but dramatically increased the neutrophil influx in tissues after CLP, an effect that was accompanied by significant increases in the serum levels of aspartate transaminase, alanine transaminase, blood urea nitrogen, and creatinine. Tissue injury caused by IL-13 blockade was associated with increases in mRNA and the protein levels of CXC chemokines macrophage inflammatory protein-2 and KC as well as the CC chemokine macrophage inflammatory protein-1alpha and the proinflammatory cytokine TNF-alpha. Collectively, these results suggest that endogenous IL-13 protected mice from CLP-induced lethality by modulating inflammatory responses via suppression of overzealous production of inflammatory cytokines/chemokines in tissues.