Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Role of PYK2 in the development of obesity and insulin resistance

Non-receptor proline-rich tyrosine kinase-2 (PYK2), which is activated by phosphorylation of one or more of its tyrosine residues, has been implicated in the regulation of GLUT4 glucose transporter translocation and glucose transport. Some data favor a positive role of PYK2 in stimulating glucose transport, whereas other studies suggest that PYK2 may participate in the induction of insulin resistance. To ascertain the importance of PYK2 in the setting of obesity and insulin resistance, we (1) evaluated the regulation of PYK2 in mice fed a high-fat diet and (2) characterized body and glucose homeostasis in wild type (WT) and PYK2(-/-) mice on different diets. We found that both PYK2 expression and phosphorylation were significantly increased in liver and adipose tissues harvested from high-fat diet fed mice. Wild type and PYK2(-/-) mice were fed a high-fat diet for 8 weeks to induce insulin resistance/obesity. Surprisingly, in response to this diet PYK2(-/-) mice gained significantly more weight than WT mice (18.7+/-1.2g vs. 9.5+/-0.6g). Fasting serum leptin and insulin and blood glucose levels were significantly increased in high-fat diet fed mice irrespective of the presence of PYK2 protein. There was a close correlation between serum leptin and body weight. Intraperitoneal glucose tolerance tests revealed that as expected, the high-fat diet resulted in increased blood glucose levels following glucose administration in wild type mice compared to those fed normal chow. An even greater increase in blood glucose levels was observed in PYK2(-/-) mice compared to wild type mice. These results demonstrate that a lack of PYK2 exacerbates weight gain and development of glucose intolerance/insulin resistance induced by a high-fat diet, suggesting that PYK2 may play a role in slowing the development of obesity, insulin resistance, and/or frank diabetes.     

Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding

Human pulmonary alveolar macrophages synthesized and secreted several characteristic high molecular weight proteins for at least 7 d in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronectin IgG contained one major labeled polypeptide of molecular weight 440,000 (unreduced) or 220,000 (reduced). An identical polypeptide in conditioned medium from radiolabeled macrophages bound specifically to gelatin-Sepharose, demonstrating that alveolar macrophages synthesized and secreted a molecule immunologically and functionally similar to fibronectin. Fibronectin was the major newly synthesized and secreted polypeptide of freshly harvested alveolar macrophages. Pulse-chase experiments revealed that newly synthesized fibronectin was rapidly secreted into medium, approximately 50 percent appearing by 1 h and 80 percent by 8 h. Immunoperoxidase staining using antifibronectin F(ab’)(2)-peroxidase conjugates revealed the majority of immunoreactive fibronectin to be intracellular, localized to endoplasmic reticulum and Golgi apparatus. No extracellular matrix fibronectin was visualized, and cell surface staining was rarely seen, usually appearing only at sites where cells were closely apposed and not at sites of macrophage-substrate attachment. Similar immunostaining of fibroblast cultures revealed cell surface-associated fibrillar fibronectin. Ultrastructural localization of fibronectin during binding and phagocytosis of gelatin-coated and plain latex particles revealed fibronectin only on gelatin-latex beads and at their cell binding sites. Neigher plain latex beads nor their cell membrane binding sites stained for fibronectin. These results demonstrate that fibronectin is a major product of human alveolar macrophages, is rapidly secreted, and is localized at cell membrane binding sites for gelatin-coated particles. In view of the known binding properties of fibronectin, it may serve as an endogenous opsonic factor promoting the binding of staphylococcus, denatured collagen, fibrin, or other macromolecules to macrophages in the lower respiratory tract.     

Phylogenetic screening of the human genome: identification of differentially hybridizing repetitive sequence families

The phi-screen, a method of phylogenetic screening, can be employed to detect repetitive sequence families that differentially hybridize between closely related species. Such differences may involve sequence divergence or variations in copy number, including total presence versus absence of a family of repeated DNA. We present the results of a phi-screen comparing the human genome to that of the prosimian, Galago crassicaudatus. Three human repetitive families that are divergent or not present in galago have been detected. One of these families is described in detail; it is similar among the anthropoids but is present in a lower copy number and/or divergent form in prosimians. The family is clearly related to the transposon-like human element (THE) described by Paulson et al. (1985). THEs have long terminal repeats reminiscent of retroviruses but are unique in that they have no sequence similarity to known mammalian retroviruses. The sequence of a solo long terminal repeat, found unassociated with THE internal sequence, is presented. This family member, THE p2, is bordered by a 5-bp target-site repeat and is interrupted by the insertion of an Alu element. A solo THE element sequenced by Wiginton et al. (1986) contains an insertion of Alu at precisely the same position as does THE p2.