Analysis of UvrABC endonuclease reaction intermediates on cisplatin-damaged DNA using mobility shift gel electrophoresis.

One of the least understood steps in the UvrABC mediated excision repair process is the recognition of lesions in the DNA. The isolation of different reaction intermediates is of vital importance for the unraveling of the mechanism. A mobility shift gel electrophoresis assay is described which visualizes such intermediates. After incubation of a DNA substrate containing a specific cisplatin adduct with UvrA alone or with UvrA and UvrB, UvrA.DNA, UvrAB.DNA and UvrB.DNA complexes were observed which could be identified using specific antibodies. At low UvrA concentrations in the presence of UvrB only the UvrB.DNA complex is observed. Bands corresponding to the UvrAB.DNA complex and also other nonspecific bands are found at relatively high UvrA concentrations. The DNase-I footprint for the UvrAB.- and UvrB.DNA complex are very similar and protect about 20 bases. Both complexes are incised in the presence of UvrC with comparable efficiency. The UvrAB.- and the UvrB.DNA complex were both incised at the 8th phosphodiester bond 5' to a specific cisplatin adduct. In addition the UvrAB.DNA complex could also be incised at the 15th phosphodiesterbond 5' to the damaged site. The results suggest that the UvrB.DNA complex is the natural substrate for UvrC-induced incision.     

Rapid effects of nerve growth factor on the Na,K-pump in rat pheochromocytoma cells

Nerve growth factor (NGF) induces neuronal differentiation of rat pheochromocytoma cells (PC12). Here we show that NGF causes a stimulation of Na⁺,K⁺-pump mediated K⁺ influx, with a maximum at 30 min after addition of NGF. The stimulation of the Na⁺,K⁺-pump is completely blocked by the Na⁺-flux inhibitor amiloride (0.2 mM) and can be mimicked by the Na⁺ ionophore monensin. These results suggest that NGF causes a rapid enhancement of Na⁺ influx leading to an activation of the Na⁺,K⁺-pump, a mechanism similar to the action of other growth factors.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

Steroid radiommuoassay: contribution of standards to blank values

The sensitivity of the radioimmunoassay of steroids is considerably reduced by high blank values which may be derived in part from co-chromatographed standards. Blank levels approach the detection limit of the radioimmunoassay of aldosterone, testosterone and androstenedione when 10,000 dpm (30–35 pg) labeled steroids are used as reference standard. When 20 μg aldosterone, testosterone, or androstenedione is used as standard, blank levels of up to 12,800 pg were measured in the radioimmunoassay. Application of the standards on a separate strip does not improve the results. From our experiments it seems clear that contamination is transported by the solvent.     

Metabolic conversion of the biologically active phospholipid, lysophosphatidic acid, in fibroblasts.

Lysophosphatidic acid (3-sn-lysophosphatidic acid; LPA) can activate cells similar to hormones and growth factors. We have considered the question whether metabolic conversion of LPA taken up by the cell could be of any importance in this activation. Addition of [14C-glycerol]LPA to quiescent Rat-1 fibroblasts resulted in rapid formation of [14C]monoacylglycerol (MG), closely followed by accumulation of [14C]triacylglycerol. Only very little [14C]diacylglycerol and [14C]phosphatidic acid was formed (approx. 100-fold less than MG). MG, when added exogenously to cells, lacks detectable biological activity. The results suggest that LPA itself, rather than one of its metabolites is the biologically active principle.     

Classification and nomenclature of all human homeobox genes

Background

The homeobox genes are a large and diverse group of genes, many of which play important roles in the embryonic development of animals. Increasingly, homeobox genes are being compared between genomes in an attempt to understand the evolution of animal development. Despite their importance, the full diversity of human homeobox genes has not previously been described.

Results

We have identified all homeobox genes and pseudogenes in the euchromatic regions of the human genome, finding many unannotated, incorrectly annotated, unnamed, misnamed or misclassified genes and pseudogenes. We describe 300 human homeobox loci, which we divide into 235 probable functional genes and 65 probable pseudogenes. These totals include 3 genes with partial homeoboxes and 13 pseudogenes that lack homeoboxes but are clearly derived from homeobox genes. These figures exclude the repetitive DUX1 to DUX5 homeobox sequences of which we identified 35 probable pseudogenes, with many more expected in heterochromatic regions. Nomenclature is established for approximately 40 formerly unnamed loci, reflecting their evolutionary relationships to other loci in human and other species, and nomenclature revisions are proposed for around 30 other loci. We use a classification that recognizes 11 homeobox gene 'classes' subdivided into 102 homeobox gene 'families'.

Conclusion

We have conducted a comprehensive survey of homeobox genes and pseudogenes in the human genome, described many new loci, and revised the classification and nomenclature of homeobox genes. The classification scheme may be widely applicable to homeobox genes in other animal genomes and will facilitate comparative genomics of this important gene superclass.     

Interaction of c-Src with gap junction protein connexin-43. Role in the regulation of cell-cell communication

Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by certain mitogens, but the underlying regulatory mechanisms are incompletely understood. Our previous studies have implicated the c-Src tyrosine kinase in mediating transient closure of Cx43-based gap junctions in normal fibroblasts. Here we show that activated c-Src (c-SrcK(+)) phosphorylates the COOH-terminal tail of Cx43, both in vitro and in intact cells. Coimmunoprecipitation experiments reveal that Cx43 associates with c-SrcK(+) and, to a lesser extent, with wild-type c-Src, but not with kinase-dead c-Src. Mutation of residue Cx43 Tyr(265) (Cx43-Y265F mutant) abolishes both tyrosine phosphorylation of Cx43 and its coprecipitation with c-Src. Expression of c-SrcK(+) in Rat-1 cells disrupts gap junctional communication. Strikingly, the communication-defective phenotype is bypassed after coexpression of the Cx43-Y265F mutant or a COOH-terminally truncated version of Cx43 (Cx43Delta263) that lacks residue Tyr(265). Our results support a model in which activated c-Src phosphorylates the COOH-terminal tail of Cx43 on residue Tyr(265), resulting in a stable interaction between both proteins leading to inhibition of gap junctional communication.     

Dynamin is required for the activation of mitogen-activated protein (MAP) kinase by MAP kinase kinase

Internalization of activated receptors from the plasma membrane has been implicated in the activation of mitogen-activated protein (MAP) kinase. However, the mechanism whereby membrane trafficking may regulate mitogenic signaling remains unclear. Here we report that dominant-negative dynamin (K44A), an inhibitor of endocytic vesicle formation, abrogates MAP kinase activation in response to epidermal growth factor, lysophosphatidic acid, and protein kinase C-activating phorbol ester. In contrast, dynamin-K44A does not affect the activation of Ras, Raf, and MAP kinase kinase (MEK) by either agonist. Through immunofluorescence and subcellular fractionation studies, we find that activated MEK is present both at the plasma membrane and in intracellular vesicles but not in the cytosol. Our findings suggest that dynamin-regulated endocytosis of activated MEK, rather than activated receptors, is a critical event in the MAP kinase activation cascade.     

p116Rip is a novel filamentous actin-binding protein

p116Rip is a ubiquitously expressed protein that was originally identified as a putative binding partner of RhoA in a yeast two-hybrid screen. Overexpression of p116Rip in neuroblastoma cells inhibits RhoA-mediated cell contraction induced by lysophosphatidic acid (LPA); so far, however, the function of p116Rip is unknown. Here we report that p116Rip localizes to filamentous actin (F-actin)-rich structures, including stress fibers and cortical microfilaments, in both serum-deprived and LPA-stimulated cells, with the N terminus (residues 1-382) dictating cytoskeletal localization. In addition, p116Rip is found in the nucleus. Direct interaction or colocalization with RhoA was not detected. We find that p116Rip binds tightly to F-actin (Kd approximately 0.5 microm) via its N-terminal region, while immunoprecipitation assays show that p116Rip is complexed to both F-actin and myosin-II. Purified p116Rip and the F-actin-binding region can bundle F-actin in vitro, as shown by electron microscopy. When overexpressed in NIH3T3 cells, p116Rip disrupts stress fibers and promotes formation of dendrite-like extensions through its N-terminal actin-binding domain; furthermore, overexpressed p116Rip inhibits growth factor-induced lamellipodia formation. Our results indicate that p116Rip is an F-actin-binding protein with in vitro bundling activity and in vivo capability of disassembling the actomyosin-based cytoskeleton.