Autoradiographic studies of efferent nerves from the celiac ganglion in the cat

Summary Postganglionic sympathetic fibers originating in the celiac ganglion can be traced by autoradiography to the stomach, spleen, liver and pancreas.     

Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein

Fraser syndrome (OMIM 219000) is a multisystem malformation usually comprising cryptophthalmos, syndactyly and renal defects. Here we report autozygosity mapping and show that the locus FS1 at chromosome 4q21 is associated with Fraser syndrome, although the condition is genetically heterogeneous. Mutation analysis identified five frameshift mutations in FRAS1, which encodes one member of a family of novel proteins related to an extracellular matrix (ECM) blastocoelar protein found in sea urchin. The FRAS1 protein contains a series of N-terminal cysteine-rich repeat motifs previously implicated in BMP metabolism, suggesting that it has a role in both structure and signal propagation in the ECM. It has been speculated that Fraser syndrome is a human equivalent of the blebbed phenotype in the mouse, which has been associated with mutations in at least five loci including bl. As mapping data were consistent with homology of FRAS1 and bl, we screened DNA from bl/bl mice and identified a premature termination of mouse Fras1. Thus, the bl mouse is a model for Fraser syndrome in humans, a disorder caused by disrupted epithelial integrity in utero.     

Evidence for kinks in DNA folding in the nucleosome

The nucleosome subunit of chromatin consists of DNA folded around a histone core as a 1.8-turn left-handed solenoid. The crystal structure of the nucleosome core particle revealed that it has a dyad symmetry axis and that the minor helix groove faces outwards from the protein core. Richmond et al. noticed that the path traversed by the helix has severe bends at sites approximately one and four helix turns from the dyad axis. We have developed two photochemical methods to study the structure of DNA, and in particular that wrapped around the nucleosome core. One method depends on the sensitization of singlet oxygen production by an eosin analogue. We have monitored the rate at which excited state oxygen diffuses into contact with DNA base planes, and find that it attacks the nucleosome with high specificity. We have also mapped the DNA binding of the intercalating dye methylene blue, and conclude that it binds to the same sites accessible to oxygen by diffusion. On the basis of these results we suggest that the DNA in the nucleosome is bent or kinked at two sites, 1.5 helix turns from the dyad axis.     

Ammoniacal silver staining of normal and inflamed rat synovial membrane

Summary An ammoniacal silver technique was used to detect changes in histone profiles in normal rats and rats with adjuvant-induced arthritis. Under these conditions histone staining reactions change at times when synovial cells are becoming more metabolically active.Acknowledgments: Supported by Biomedical Research Development Grant No. ISO08RR09016-01 from the National Institutes of Health, by grants to both authors from the Marquette University Committee on Research and by Marquette University School of Dentistry.     

CD1d-lipid-antigen recognition by the semi-invariant NKT T-cell receptor

The CD1 family is a large cluster of non-polymorphic, major histocompatibility complex (MHC) class-I-like molecules that bind distinct lipid-based antigens that are recognized by T cells. The most studied group of T cells that interact with lipid antigens are natural killer T (NKT) cells, which characteristically express a semi-invariant T-cell receptor (NKT TCR) that specifically recognizes the CD1 family member, CD1d. NKT-cell-mediated recognition of the CD1d-antigen complex has been implicated in microbial immunity, tumour immunity, autoimmunity and allergy. Here we describe the structure of a human NKT TCR in complex with CD1d bound to the potent NKT-cell agonist alpha-galactosylceramide, the archetypal CD1d-restricted glycolipid. In contrast to T-cell receptor-peptide-antigen-MHC complexes, the NKT TCR docked parallel to, and at the extreme end of the CD1d-binding cleft, which enables a lock-and-key type interaction with the lipid antigen. The structure provides a basis for the interaction between the highly conserved NKT TCR alpha-chain and the CD1d-antigen complex that is typified in innate immunity, and also indicates how variability of the NKT TCR beta-chain can impact on recognition of other CD1d-antigen complexes. These findings provide direct insight into how a T-cell receptor recognizes a lipid-antigen-presenting molecule of the immune system.     

Nesting ecology of waterbirds at Grays Lake, Idaho

Montane wetlands provide valuable habitat for nesting waterfowl and other waterbirds in the western United States, but relatively little information is available about the nesting ecology of their waterbird communities. We describe the general nesting ecology of breeding waterbirds at a large, shallow, montane wetland in southeastern Idaho during 1997-2000. Habitats include upland grasslands and intermittently to semipermanently flooded wetland habitats. We located a total of 1207 nests of 23 bird species: Eared Grebe ( Podiceps nigricollis ), Canada Goose ( Branta canadensis ), Mallard ( Anas platyrhynchos ), Gadwall ( A. strepera ), American Wigeon ( A. americana ), Green-winged Teal ( A. crecca ), Blue-winged Teal ( A. discors ), Cinnamon Teal ( A. cyanoptera ), Northern Shoveler ( A. clypeata ), Northern Pintail ( A. acuta ), Redhead ( Aythya americana ), Canvasback ( A. valisineria ), Lesser Scaup ( A. affinis ), Ruddy Duck ( Oxyura jamaicensis ), Northern Harrier ( Circus cyaneus ), American Coot ( Fulica americana ), Virginia Rail ( Rallus limicola ), Greater Sandhill Crane ( Grus canadensis tabida ), American Avocet ( Recurvirostra americana ), Long-billed Curlew ( Numenius americanus ), Wilson's Snipe ( Gallinago delicata ), Wilson's Phalarope ( Phalaropus tricolor ), and Short-eared Owl ( Asio flammeus ). Most nests were initiated in May-early June and were terminated (hatched or destroyed) by the 3rd week of June. Mean daily survival rate (DSR) for Canada Goose nests was 0.954 ± 0.005 ( s ￣x ; n = 127 nests), equivalent to Mayfield nest success of 21%. Mean DSR for dabbling duck nests over all 4 years was 0.938 ± 0.006 ( n = 41), equivalent to Mayfield nest success of 11%. For all other species where we found > 10 nests each year (Eared Grebe, Redhead, Canvasback, Coot, Sandhill Crane, American Avocet, and Wilson's Snipe), > 50% of nests found hatched at least 1 young. Success rates for geese, cranes, and ducks were lower than reported for Grays Lake during 1949-1951 and lower than most other wetlands in the region.     

Loss of huntingtin-associated protein 1 impairs insulin secretion from pancreatic β-cells

Hap1 was originally identified as a neuronal protein that interacts with huntingtin, the Huntington’s disease (HD) protein. Later studies revealed that Hap1 participates in intracellular trafficking in neuronal cells and that this trafficking function can be adversely affected by mutant huntingtin. Hap1 is also present in pancreatic β-cells and other endocrine cells; however, the role of Hap1 in these endocrine cells remains unknown. Using the Cre-loxP system, we generated conditional Hap1 knockout mice to selectively deplete the expression of Hap1 in mouse pancreatic β-cells. Mutant mice with Hap1 deficiency in pancreatic β-cells had impaired glucose tolerance and decreased insulin release in response to intraperitoneally injected glucose. Using cultured pancreatic β-cell lines and isolated mouse pancreatic islets, we confirmed that decreasing Hap1 could reduce glucose-mediated insulin release. Electron microscopy suggested that there was a reduced number of insulin-containing vesicles docked at the plasma membrane of pancreatic islets in Hap1 mutant mice following intraperitoneal glucose injection. Glucose treatment decreased the phosphorylation of Hap1A in cultured β-cells and in mouse pancreatic tissues. Moreover, this glucose treatment increased Hap1’s association with kinesin light chain and dynactin p150, both of which are involved in microtubule-dependent trafficking. These studies suggest that Hap1 is important for insulin release from β-cells via dephosphorylation that can regulate its intracellular trafficking function.     

Frequent somatic mutations in MAP3K5 and MAP3K9 in metastatic melanoma identified by exome sequencing

We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.