Toward fully automated genotyping: allele assignment, pedigree construction, phase determination, and recombination detection in Duchenne muscular dystrophy.

Human genetic maps have made quantum leaps in the past few years, because of the characterization of > 2,000 CA dinucleotide repeat loci: these PCR-based markers offer extraordinarily high PIC, and within the next year their density is expected to reach intervals of a few centimorgans per marker. These new genetic maps open new avenues for disease gene research, including large-scale genotyping for both simple and complex disease loci. However, the allele patterns of many dinucleotide repeat loci can be complex and difficult to interpret, with genotyping errors a recognized problem. Furthermore, the possibility of genotyping individuals at hundreds or thousands of polymorphic loci requires improvements in data handling and analysis. The automation of genotyping and analysis of computer-derived haplotypes would remove many of the barriers preventing optimal use of dense and informative dinucleotide genetic maps. Toward this end, we have automated the allele identification, genotyping, phase determinations, and inheritance consistency checks generated by four CA repeats within the 2.5-Mbp, 10-cM X-linked dystrophin gene, using fluorescein-labeled multiplexed PCR products analyzed on automated sequencers. The described algorithms can deconvolute and resolve closely spaced alleles, despite interfering stutter noise; set phase in females; propagate the phase through the family; and identify recombination events. We show the implementation of these algorithms for the completely automated interpretation of allele data and risk assessment for five Duchenne/Becker muscular dystrophy families. The described approach can be scaled up to perform genome-based analyses with hundreds or thousands of CA-repeat loci, using multiple fluorophors on automated sequencers.     

The involvement of human RECQL4 in DNA double‐strand break repair

Rothmund–Thomson syndrome (RTS) is an autosomal recessive hereditary disorder associated with mutation in RECQL4 gene, a member of the human RecQ helicases. The disease is characterized by genomic instability, skeletal abnormalities and predisposition to malignant tumors, especially osteosarcomas. The precise role of RECQL4 in cellular pathways is largely unknown; however, recent evidence suggests its involvement in multiple DNA metabolic pathways. This study investigates the roles of RECQL4 in DNA double‐strand break (DSB) repair. The results show that RECQL4‐deficient fibroblasts are moderately sensitive to γ‐irradiation and accumulate more γH2AX and 53BP1 foci than control fibroblasts. This is suggestive of defects in efficient repair of DSB’s in the RECQL4‐deficient fibroblasts. Real time imaging of live cells using laser confocal microscopy shows that RECQL4 is recruited early to laser‐induced DSBs and remains for a shorter duration than WRN and BLM, indicating its distinct role in repair of DSBs. Endogenous RECQL4 also colocalizes with γH2AX at the site of DSBs. The RECQL4 domain responsible for its DNA damage localization has been mapped to the unique N‐terminus domain between amino acids 363–492, which shares no homology to recruitment domains of WRN and BLM to the DSBs. Further, the recruitment of RECQL4 to laser‐induced DNA damage is independent of functional WRN, BLM or ATM proteins. These results suggest distinct cellular dynamics for RECQL4 protein at the site of laser‐induced DSB and that it might play important roles in efficient repair of DSB’s.     

Systematics of the parasitic wasp genus Oxyscelio Kieffer (Hymenoptera,Platygastridae s.l.), part II: the?Australian and southwest Pacific fauna

The Australasian and southwest Pacific species of Oxyscelio (Hymenoptera: Platygastridae s.l.) are revised. A total of 80 species are recognized as valid, 13 of which are redescribed: O. atricoxa (Dodd), O. concoloripes (Dodd), O. flavipes (Kieffer), O. grandis (Dodd), O. hyalinipennis (Dodd), O. magniclava (Dodd), O. mirellus (Dodd), O. montanus (Dodd), O. nigriclava (Dodd), O. nigricoxa (Dodd), O. rugulosus (Dodd), O. shakespearei (Girault), and O. solitarius (Dodd). Oxyscelio glabriscutellum (Dodd) syn. n. is placed as a subjective junior synonym of O. rugulosus. Sixty-seven new species are described, many representing new distributional records for the genus - O. aciculae Burks, sp. n., O. anfractus Burks, sp. n., O. bellariorum Burks, sp. n., O. bicoloripedis Burks, sp. n., O. brevitas Burks, sp. n., O. catenae Burks, sp. n., O. caudarum Burks, sp. n., O. circulorum Burks, sp. n., O. clivi Burks, sp. n., O. clupei Burks, sp. n., O. conjuncti Burks, sp. n., O. contusionis Burks, sp. n., O. corrugationis Burks, sp. n., O. croci Burks, sp. n., O. cuspidis Burks, sp. n., O. densitatis Burks, sp. n., O. dissimulationis Burks, sp. n., O. divisionis Burks, sp. n., O. exiguitatis Burks, sp. n., O. fluctuum Burks, sp. n., O. foliorum Burks, sp. n., O. funis Burks, sp. n., O. gressus Burks, sp. n., O. hamorum Burks, sp. n., O. incisurae Burks, sp. n., O. lenitatis Burks, sp. n., O. leviventris Burks, sp. n., O. limbi Burks, sp. n., O. liminis Burks, sp. n., O. linguae Burks, sp. n., O. lintris Burks, sp. n., O. livens Burks, sp. n., O. mystacis Burks, sp. n., O. nasi Burks, sp. n., O. nitoris Burks, sp. n., O. obliquiatis Burks, sp. n., O. oblongiclypei Burks, sp. n., O. obturationis Burks, sp. n., O. oculi Burks, sp. n., O. palati Burks, sp. n., O. pectinis Burks, sp. n., O. pollicis Burks, sp. n., O. proceritatis Burks, sp. n., O. productionis Burks, sp. n., O. radii Burks, sp. n., O. rami Burks, sp. n., O. rupturae Burks, sp. n., O. sarcinae Burks, sp. n., O. scismatis Burks, sp. n., O. sciuri Burks, sp. n., O. scutorum Burks, sp. n., O. sepisessor Burks, sp. n., O. sinuationis Burks, sp. n., O. sordes Burks, sp. n., O. spatula Burks, sp. n., O. stipulae Burks, sp. n., O. stringerae Burks, sp. n., O. tenuitatis Burks, sp. n., O. truncationis Burks, sp. n., O. tubi Burks, sp. n., O. umbonis Burks, sp. n., O. uncinorum Burks, sp. n., O. valdecatenae Burks, sp. n., O. velamenti Burks, sp. n., O. verrucae Burks, sp. n., O. viator Burks, sp. n., and O. wa Burks, sp. n. The fauna is divided into nine diagnostic species groups, with five species unplaced to group.     

Delta opioid receptor-selective ligands: [D-Pen2,D-Pen5]enkephalin-dermenkephalin chimeric peptides.

A number of DPDPE-dermenkephalin chimeric peptides have been synthesized in which the putative C-terminal delta-address of dermenkephalin has been linked to the highly delta opioid selective cyclic peptide [D-Pen2,D-Pen5]enkephalin (DPDPE). Asp, Met-Asp and Leu-Met-Asp have been added to the C-terminus of DPDPE and both the carboxyl terminal and the carboxamide terminal series have been prepared. The bioassays using the mouse vas deferens and guinea pig ileum preparations have revealed a steady decrease in potency (compared to DPDPE) at delta and mu receptors as the dermenkephalin sequences were added. Some of the analogues, however, retained high delta selectivity. Similar results were obtained using radioligand binding assays. These findings suggest that the C-terminal amino acid sequence of dermenkephalin plays a role of delta-address which is specific to dermenkephalin itself, and is not additive with another delta selective ligand such as DPDPE.     

Keratinocyte cadherin desmoglein 1 controls melanocyte behavior through paracrine signaling

The epidermis is the first line of defense against ultraviolet (UV) light from the sun. Keratinocytes and melanocytes respond to UV exposure by eliciting a tanning response dependent in part on paracrine signaling, but how keratinocyte:melanocyte communication is regulated during this response remains understudied. Here, we uncover a surprising new function for the keratinocyte‐specific cell–cell adhesion molecule desmoglein 1 (Dsg1) in regulating keratinocyte:melanocyte paracrine signaling to promote the tanning response in the absence of UV exposure. Melanocytes within Dsg1‐silenced human skin equivalents exhibited increased pigmentation and altered dendrite morphology, phenotypes which were confirmed in 2D culture using conditioned media from Dsg1‐silenced keratinocytes. Dsg1‐silenced keratinocytes increased melanocyte‐stimulating hormone precursor (Pomc) and cytokine mRNA. Melanocytes cultured in media conditioned by Dsg1‐silenced keratinocytes increased Mitf and Tyrp1 mRNA, TYRP1 protein, and melanin production and secretion. Melanocytes in Dsg1‐silenced skin equivalents mislocalized suprabasally, reminiscent of early melanoma pagetoid behavior. Together with our previous report that UV reduces Dsg1 expression, these data support a role for Dsg1 in controlling keratinocyte:melanocyte paracrine communication and raise the possibility that a Dsg1‐deficient niche contributes to pagetoid behavior, such as occurs in early melanoma development.     

Regulation of gastric emptying

Studies carried out in the years since William Beaumont's direct observations of gastric motility have provided increased understanding of the physiological roles of the stomach and of the mechanisms for the regulation of gastric motility. Tonic contractions of the proximal stomach are of primary importance for transfer of liquids from the stomach to the duodenum. Peristaltic contractions of the distal stomach are of primary importance for reducing the size of solid food particles and for transfer of solids to the duodenum. Because gastric emptying requires a net antral-duodenal pressure gradient, contractions of the duodenum also influence the rate of gastric emptying. Gastrointestinal hormones, including gastrin, cholecystokinin, secretin, somatostatin, and others, are released by contact of chyme with the intestinal mucosa, and affect contractions of the proximal stomach, distal stomach, and duodenum. Neural reflexes that arise from the stomach act through autonomic motor nerves to allow regulation by the central nervous system of gastric motility. gamma-Aminobutyric acid, opioids, and bombesin may serve as central neurochemical regulators of gastric motility.     

Expression profiling of herpesvirus and vaccinia virus proteins using a high-throughput baculovirus screening system

We have developed a high-throughput system for generating baculoviruses and testing the expression, solubility, and affinity column purification of encoded proteins. We have used this system to generate baculoviruses for and analyze the expression of 337 proteins from three different herpesviruses (HSV-1, EBV, and CMV) and vaccinia virus. Subsets of these proteins were also tested for expression and solubility in E. coli. Comparisons of the results in the two systems are presented for each virus.     

Apoptosis repressor with caspase recruitment domain is regulated by MAPK/PI3K and confers drug resistance and survival advantage to AML

The apoptosis repressor with caspase recruitment domain (ARC) protein is known to suppress both intrinsic and extrinsic apoptosis. We previously reported that ARC expression is a strong, independent adverse prognostic factor in acute myeloid leukemia (AML). Here, we investigated the regulation and role of ARC in AML. ARC expression is upregulated in AML cells co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and suppressed by inhibition of MAPK and PI3K signaling. AML patient samples with RAS mutations (N = 64) expressed significantly higher levels of ARC than samples without RAS mutations (N = 371) (P = 0.016). ARC overexpression protected and ARC knockdown sensitized AML cells to cytarabine and to agents that selectively induce intrinsic (ABT-737) or extrinsic (TNF-related apoptosis inducing ligand) apoptosis. NOD–SCID mice harboring ARC-overexpressing KG-1 cells had significantly shorter survival than mice injected with control cells (median 84 vs 111 days) and significantly fewer leukemia cells were present when NOD/SCID IL2Rγ null mice were injected with ARC knockdown as compared to control Molm13 cells (P = 0.005 and 0.03 at 2 and 3 weeks, respectively). Together, these findings demonstrate that MSCs regulate ARC in AML through activation of MAPK and PI3K signaling pathways. ARC confers drug resistance and survival advantage to AML in vitro and in vivo, suggesting ARC as a novel target in AML therapy.     

Importin-alpha-16 is a translocon-associated protein involved in sorting membrane proteins to the nuclear envelope

A viral inner nuclear membrane-sorting motif sequence (INM-SM) was used to identify proteins that recognize integral membrane proteins destined for the INM. Herein we describe importin-alpha-16, a membrane-associated isoform of Spodoptera frugiperda importin-alpha that contains the C-terminal amino acid residues comprising armadillo helical-repeat domains 7-10. In the endoplasmic reticulum (ER) membrane, importin-alpha-16 is adjacent to the translocon protein Sec61alpha. Importin-alpha-16 cross-links to the INM-SM sequence as it emerges from the ribosomal tunnel and remains adjacent to the INM-SM after INM-SM integration into the ER membrane and release from the translocon. Cross-linking results suggest that importin-alpha-16 discriminates between INM- and non-INM-directed proteins. Thus, it seems that during and after cotranslational membrane integration, importin-alpha-16 is involved in the trafficking of integral membrane proteins to the INM.