Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.

Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.

Effects of chemotherapy on osseointegration of implants: a case report.

A patient underwent mandibular resection for high-grade osteosarcoma with immediate reconstruction with a microvascular fibula free bone graft and simultaneous placement of osseointegrated implants. Following initial healing, she underwent six cycles of chemotherapy and had further revision surgery prior to implant exposure and construction of a prosthesis. The chemotherapy appears to have had no deleterious effects on implant osseointegration or survival.

Gore-Tex tubing as a conduit for repair of lingual and inferior alveolar nerve continuity defects: a preliminary report.

PURPOSE: This report describes the results of using a Gore-Tex (Gore Company, Flagstaff, AZ) tube as a conduit for repair of continuity defects in the inferior alveolar or lingual nerves. PATIENTS AND METHODS: Seven nerve repairs were performed in five patients (M:F = 1:4) with an age range of 16 to 56 years. The duration from injury to repair ranged from 4 to 30 months. Two inferior alveolar and five lingual nerves were repaired. RESULTS: All seven patients had anesthesia by objective testing preoperatively and had a continuity defect at the time of operation. The size of the defects ranged from 2 to 15 mm. Two of the seven patients had some return of sensation, occurring in defects of 3 mm or smaller. CONCLUSION: The results of this pilot study indicate that Gore-Tex tubing may not be effective in the repair of continuity defects except in those defects 3 mm or smaller, in which it may act as a protective barrier membrane rather than as a conduit.

Noninvasive somatosensory monitoring of the injured inferior alveolar nerve using magnetic source imaging.

PURPOSE: This study evaluates magnetoencephalography (MEG) as an objective monitor for the evaluation of post-traumatic inferior alveolar nerve injuries. MATERIALS AND METHODS: Six patients with unilateral inferior alveolar nerve injuries were assessed using conventional sensory examination techniques. All damaged nerves, and their contralateral controls, were then reexamined using MEG technology. Regions of somatosensory-induced magnetic activity were superimposed on three-dimensional magnetic resonance imaging (MRI) scans to give cortical magnetic source images (MSI). All patients subsequently underwent surgical exploration of the damaged nerves. RESULTS: All six patients had no sensitivity to conventional testing on the damaged side. On evaluation with MEG, all six control nerves had an appropriate cortical signal in response to repetitive lip stimulation. Somatosensory stimulation of two of the six damaged nerves resulted in cortical magnetic field changes. Surgical exploration showed that the four nerves with negative MEG tests had discontinuity defects. In contrast, the two patients with positive MEG signals had intact nerves. CONCLUSION: This technology may differentiate between intact but damaged nerves and transected nerves.

Rhodamine-phalloidin staining of F-actin in rhodophyta.

Rhodamine-phalloidin was used to label F-actin in unfixed cells of 13 species of filamentous and blade-forming red algae from the three families Ceramiaceae, Acrochaetiaceae and Bangiaceae. Labelling was achieved only after treatment with either beta-glucuronidase or a combination of cellulase and an extract of snail gut enzyme. Different species required different enzyme treatments and incubation times for successful labelling. All species examined showed extensive arrays of F-actin which generally are confined to the peripheral cytoplasm and are oriented longitudinally. Transverse arrays are present beside the crosswalls of Griffithsia pacifica, and Audouinella species show actin concentrations at the tips of apical cells and in developing branch initials.

Gamma-tubulin is associated with a cortical-microtubule-organizing zone in the developing guard cells of Allium cepa L.

A key event in the differentiation of elliptically shaped guard cells such as those in Allium is the formation of a radial array of cortical microtubules (Mts) which, by controlling the orientation of wall microfibrils, plays an important role in cell shaping. Previous experiments strongly indicated that the array is nucleated in a zone adjacent to the new ventral wall soon after cytokinesis. In order to further clarify the function of this zone, we performed dual immunolocalizations on Allium guard cells with anti-beta-tubulin, to detect Mts, and an antibody to gamma-tubulin, a protein known to be present at Mt-organizing centers in other species and recently identified in plants as well. gamma-Tubulin antibody stained the cortical zone adjacent to the ventral wall, while little or no fluorescence was present elsewhere along the radial Mt array or at other sites in the cell. The antibody also stained the mitotic poles and phragmoplast in guard mother cells, as it does in other material. No staining was seen when the primary antibody was omitted. The results are consistent with nucleation of the radial array at a cortical-Mt-organizing zone next to the ventral wall, and set the stage for more in-depth studies on the spatial and temporal control of Mt formation in differentiating cells.

Regulating insulin-receptor-gene expression by differentiation and hormones.

Insulin regulates cell function by first binding to the insulin receptor (IR) localized on the cell surface. With the cloning of IR cDNA and the IR-gene promoter, the regulation of the IR gene during differentiation and by various hormones can be studied. Muscle is a major target tissue for insulin action. BC3H1 cells, a mouse muscle cell line in culture, are a model cell type for studying insulin action. Differentiation in these cells results in a 5- to 10-fold increase in IR binding and a 5- to 10-fold increase in IR content. Studies of IR mRNA by Northern and slot-blot analyses reveal a 10-fold increase in IR mRNA after differentiation. These studies indicate that there is a selective increase in IR-gene expression during muscle differentiation. A similar increase in IR-gene expression is observed for the IR during pancreatic acinar cell differentiation. Glucocorticoids increase IR content in several target tissues. Studies in cultured IM-9 lymphocytes indicate that glucocorticoids induce a 5-fold increase in IR mRNA levels. Studies of IR mRNA half-life indicate that glucocorticoids do not alter IR mRNA stability. When the transcription of the IR is measured by elongation assays, glucocorticoids directly stimulate IR transcription 5- to 10-fold. The effect is detectable within 30 min of glucocorticoid treatment and is maximal within 2 h. Therefore, these studies demonstrate that the IR gene is under the direct regulation of glucocorticoids. Insulin downregulates the IR in various target tissues. Prior studies indicate that this downregulation was partly because of accelerated IR degradation. Studying AR42J pancreatic acinar cells, we also found that insulin accelerates IR degradation. Moreover, in these cells, insulin decreases IR biosynthesis by approximately 50%. Studies of IR mRNA indicate there is a concomitant decrease in IR mRNA levels after insulin treatment. Thus, insulin decreases IR-gene expression. The genomic structure of the IR promoter has been elucidated. Primer extension and nuclease S1 analysis indicate that IR mRNA has multiple start sites. The promoter fragment was ligated to a promoterless "reporter" plasmid containing the bacterial gene chloramphenicol acetyltransferase (CAT). When this plasmid is transfected into cultured cells, CAT activity is detected, indicating promoter activity. Various portions of a genomic fragment were ligated to a promoter to study glucocorticoid regulation of the IR promoter. These studies indicate that IR-gene expression is regulated by differentiation and hormonal agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms of insulin-induced insulin-receptor downregulation. Decrease of receptor biosynthesis and mRNA levels.

The influence of insulin on the downregulation of its receptor was studied in AR42J cultured pancreatic acinar cells, a cell line that has been demonstrated to be metabolically responsive to insulin. Downregulation induced by insulin was time and dose dependent. After a 20-h incubation with 1 microM insulin, Scatchard analysis revealed approximately 80% loss of insulin receptors. Studies of receptor half-life indicated that treatment with insulin accelerated the degradation of both the alpha- and beta-subunits of the insulin receptor by 30-60%. In addition, biosynthetic-labeling studies indicated that insulin inhibited the biosynthesis of the insulin-receptor precursor by greater than 30%. This decreased biosynthesis of the precursor was associated with decreased production of mature receptor subunits. Poly(A)+ RNA was extracted from control cells and cells treated for 24 h with 100 nM insulin. Slot blots and Northern transfers revealed that insulin induced an approximately 50% decrease in insulin-receptor mRNA levels. Therefore, these studies indicate that insulin may diminish the concentration of its receptors in target cells by at least two mechanisms: acceleration of receptor degradation and inhibition of receptor biosynthesis at the level of mRNA.

Sequence and analysis of promoter region of human insulin-receptor gene.

The promoter region of the human insulin-receptor (HINSR) gene was isolated from a human chromosome 19 bacteriophage library. With S1 nuclease mapping and primer-extension analysis, we identified multiple transcription-initiation sites. Dexamethasone, a known inducer of HINSR transcription, enhanced transcription of all major transcription-initiation sites. DNA sequence analysis indicated that the HINSR promoter has neither a TATA box nor a CAAT box. The HINSR promoter region contains six GGGCGG sequences that may be binding sites for the transcription factor Sp1. In addition, there were three TCCC sequences that were putative promoter regulatory regions. The HINSR gene promoter has structural similarity to the epidermal growth factor receptor gene promoter and has some features of the promoter of the meglutol (hydroxymethylglutaryl, HMG) CoA reductase gene and the early promoter of simian virus 40.

Dietary regulation of rat intestinal cholecystokinin gene expression.

Cholecystokinin (CCK) is a gastrointestinal hormone produced by discrete endocrine cells in the upper small intestine and released after ingestion of a meal. The present study was designed to determine if enhanced CCK secretion is associated with increases in intestinal CCK mRNA levels. Rats, prepared with indwelling intraduodenal cannulae, were first fed an elemental diet that did not stimulate CCK release. Next, as a means of stimulating CCK secretion, soybean trypsin inhibitor was perfused for up to 24 h. Trypsin inhibitor administration increased plasma CCK levels from 0.9 +/- 0.1 to approximately 5 pmol/liter. RNA was prepared from the proximal small intestine at various times after trypsin inhibitor perfusion and mRNA levels analyzed by hybridization with a CCK cDNA probe. After 12 and 24 h of trypsin inhibitor treatment there were three- and fourfold increases, respectively, in CCK mRNA levels. In comparison, there was no change in beta-actin mRNA levels. To determine if regulation of CCK mRNA was at the level of CCK gene transcription, labeled transcripts from nuclear run-on incubations were hybridized to immobilized CCK cDNA. In trypsin inhibitor-treated rats, a two- to threefold increase in transcriptional activity was observed, whereas beta-actin gene transcription levels were unaltered. These studies indicate that stimulation of CCK secretion is associated with an increase in intestinal CCK mRNA content resulting from an increase in CCK gene transcription.

Mechanism of glucocorticoid receptor down-regulation by glucocorticoids.

The effect of glucocorticoids on the regulation of glucocorticoid receptor mRNA was studied in two different cell lines, human IM-9 lymphocytes and rat pancreatic acinar AR42J cells. Using a glucocorticoid receptor cDNA probe, glucocorticoid receptor mRNA was examined by Northern blot hybridization and quantitated by slot-blot hybridization. In IM-9 and AR42J cells, dexamethasone decreased steady-state glucocorticoid receptor mRNA levels to approximately 50% of control. This decrease occurred with a one-half time of 3 h for IM-9 cells and 6 h for AR42J cells. Dexamethasone was the most potent steroid tested with a one-half maximal effect occurring at 10 nM and a maximal effect occurring at 100 nM. Glucocorticoid receptor mRNA half-life and gene transcription were then studied to determine the mechanism of decreased mRNA levels. The glucocorticoid mRNA half-life was approximately 120 min in IM-9 cells and 240 min in AR42J cells; these rates were not affected by dexamethasone treatment. In contrast, the rate of glucocorticoid gene transcription as measured by run-on assays in IM-9 cells was decreased to 50 +/- 6% of control by dexamethasone. These results indicate therefore that glucocorticoids regulate glucocorticoid receptor mRNA levels by influencing gene transcription.

Glucocorticoid regulation of insulin receptor gene transcription in IM-9 cultured lymphocytes.

We have reported that glucocorticoids increase steady state insulin receptor mRNA levels in target cells. In the present study using IM-9 cultured human lymphocytes, we investigated the mechanism responsible for this glucocorticoid mediated increase in insulin receptor mRNA levels. Incubation of IM-9 cells with 100 nM dexamethasone for 4 h stimulated a parallel increase in both polysomal and nuclear insulin receptor RNAs indicating that glucocorticoids did not alter the nuclear transport of insulin receptor RNA. Dexamethasone did not alter insulin receptor mRNA half life (t 1/2 = 140 +/- 20 min), indicating that glucocorticoids did not influence mRNA stability. Furthermore, the dexamethasone-induced increase in insulin receptor mRNA levels was not blocked by pretreatment of cells with cycloheximide indicating that the glucocorticoid effect was independent of new protein synthesis. When the labeled transcripts from nuclear run-off incubations were then hybridized to immobilize human insulin receptor cDNA, a three- to fourfold increase in transcriptional activity was observed. This transcriptional effect occurred before the increase in steady state insulin receptor mRNA levels and over the same range of dexamethasone concentrations. These studies indicate therefore a direct effect of glucocorticoids on insulin receptor gene transcription, and demonstrate that the insulin receptor gene is under hormonal control.

Mechanism of glucocorticoid-induced increase in pancreatic amylase gene transcription.

To determine the mechanism(s) responsible for glucocorticoid-induced increases in amylase content in pancreatic acinar AR42J cells, we examined the effects of dexamethasone on amylase protein biosynthesis, steady-state mRNA levels, and gene transcription. Dexamethasone treatment led to a dose-dependent increase in amylase synthesis which was one-half maximal at 2 nM and maximal at 100 nM where a 6-fold increase was achieved. This dexamethasone-induced increase in amylase synthesis was detectable after 12 h, one-half maximal after 19 h, and approached maximal after 72 h. Dexamethasone treatment also increased amylase mRNA levels in a time- and dose-dependent manner in parallel with the changes in amylase synthesis. Nuclear RNA transcript elongation (run-on) assays indicated that amylase gene transcription was also increased in a time- and dose-dependent manner. Glucocorticoid enhancement of amylase gene transcription occurred relatively slowly, with a 6-fold increase occurring after 48 h of treatment with 100 nM dexamethasone. Thus, the effects of glucocorticoids on pancreatic amylase gene transcription fully accounted for the increased levels of amylase mRNA, synthesis, and content. However, due to the slow time course of dexamethasone induction of amylase gene expression we evaluated the possibility of glucocorticoid induction of a regulatory protein. We found that inclusion of cycloheximide or puromycin during dexamethasone treatment blocked the induction of amylase mRNA. These data suggest that the glucocorticoid-induced increase in amylase gene transcription requires induction of an unidentified regulatory protein(s).

Regulation of insulin-receptor mRNA levels by glucocorticoids.

We found with IM-9 human cultured lymphocytes, that the glucocorticoid dexamethasone increased insulin-receptor mRNA levels. This increase correlated in a time- and dose-dependent manner with the increase in the biosynthesis of the insulin-receptor precursor. In addition, in AR42J cultured rat pancreatic acinar cells, dexamethasone increased insulin-receptor mRNA levels. These studies suggest, therefore, that an increase in mRNA levels is an early step in the regulation of the insulin receptor by glucocorticoids in several cell types.

Proteins from rat liver cytosol which stimulate mRNA transport. Purification and interactions with the nuclear envelope mRNA translocation system.

Two polysome-associated proteins with particular affinities for poly(A) have been purified from rat liver. These proteins stimulate the efflux of mRNA from isolated nuclei in conditions under which such efflux closely stimulates mRNA transport in vivo, and they are therefore considered as mRNA-transport-stimulatory proteins. Their interaction with the mRNA-translocation system in isolated nuclear envelopes has been studied. The results are generally consistent with the most recently proposed kinetic model of mRNA translocation. One protein, P58, has not been described previously. It inhibits the protein kinase that down-regulates the NTPase, it enhances the NTPase activity in both the presence and the absence of poly(A) and it seems to increase poly(A) binding in unphosphorylated, but not in phosphorylated, envelopes. The other protein, P31, which probably corresponds to the 35,000-Mr factor described by Webb and his colleagues, enhances the binding of poly(A) to the mRNA-binding site in the envelope, thus stimulating the phosphoprotein phosphatase and, in consequence, the NTPase. The possible physiological significance of these two proteins is discussed.