Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP.

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

The influence of surface charges on quaternary ammonium block of shaker K+ channels.

Block of K+ channels can be influenced by the ability of charged residues on the protein surface to accumulate cationic blocking ions to concentrations greater than those in bulk solution. We examined the ionic strength dependence of extracellular block of Shaker K+ channels by tetraethylammonium ions (TEA+) and by a trivalent quaternary ammonium ion, gallamine3+. Wild-type and mutant channels were expressed in Xenopus oocytes and currents recorded with the cut-open oocyte technique. Channel block by both compounds was substantially increased when the bathing electrolyte ionic strength was lowered, but with a much larger effect for trivalent gallamine. These data were quantitatively well described by a simple electrostatic model, accounting for accumulation of blocking ions near the pore of the channel by surface charges. The surface charge density of the wild-type channel consistent with the results was -0.1 e nm-2. Shaker channels with T449Y mutations have an increased affinity for both TEA and gallamine but the ionic strength dependence of block was described with the same surface charge density as wild-type channels. Much of the increased sensitivity of Shaker K+ channels to gallamine may be due to a larger local accumulation of the trivalent ion. The negative charge at position 431 contributes to the sensitivity of channels to TEA (MacKinnon & Yellen, 1990). A charge reversal mutation at this location had little effect on the ionic strength dependence of quaternary ammonium ion block, suggesting that the charge on this amino acid may directly affect binding affinity but not local ion accumulation.

The Ras/Rac guanine nucleotide exchange factor mammalian Son-of-sevenless interacts with PACSIN 1/syndapin I, a regulator of endocytosis and the actin cytoskeleton.

Mammalian Son-of-sevenless (mSos) functions as a guanine nucleotide exchange factor for Ras and Rac, thus regulating signaling to mitogen-activated protein kinases and actin dynamics. In the current study, we have identified a new mSos-binding protein of 50 kDa (p50) that interacts with the mSos1 proline-rich domain. Mass spectrometry analysis and immunodepletion studies reveal p50 as PACSIN 1/syndapin I, a Src homology 3 domain-containing protein functioning in endocytosis and regulation of actin dynamics. In addition to PACSIN 1, which is neuron-specific, mSos also interacts with PACSIN 2, which is expressed in neuronal and nonneuronal tissues. PACSIN 2 shows enhanced binding to the mSos proline-rich domain in pull-down assays from brain extracts as compared with lung extracts, suggesting a tissue-specific regulation of the interaction. Proline to leucine mutations within the Src homology 3 domains of PACSIN 1 and 2 abolish their binding to mSos, demonstrating the specificity of the interactions. In situ, PACSIN 1 and mSos1 are co-expressed in growth cones and actin-rich filopodia in hippocampal and dorsal root ganglion neurons, and the two proteins co-immunoprecipitate from brain extracts. Moreover, epidermal growth factor treatment of COS-7 cells causes co-localization of PACSIN 1 and mSos1 in actin-rich membrane ruffles, and their interaction is regulated through epidermal growth factor-stimulated mSos1 phosphorylation. These data suggest that PACSINs may function with mSos1 in regulation of actin dynamics.

Residues and mechanisms for slow activation and Ba2+ block of the cardiac muscarinic K+ channel, Kir3.1/Kir3.4.

Mechanisms and residues responsible for slow activation and Ba(2+) block of the cardiac muscarinic K(+) channel, Kir3.1/Kir3.4, were investigated using site-directed mutagenesis. Mutagenesis of negatively charged residues located throughout the pore of the channel (in H5, M2, and proximal C terminus) reduced or abolished slow activation. The strongest effects resulted from mutagenesis of residues in H5 close to the selectivity filter; mutagenesis of residues in M2 and proximal C terminus equivalent to those identified as important determinants of the activation kinetics of Kir2.1 was less effective. In giant patches, slow activation was present in cell-attached patches, lost on excision of the patch, and restored on perfusion with polyamine. Mutagenesis of residues in H5 and M2 close to the selectivity filter also decreased Ba(2+) block of the channel. A critical residue for Ba(2+) block was identified in Kir3.4. Mutagenesis of the equivalent residue in Kir3.1 failed to have as pronounced an effect on Ba(2+) block, suggesting an asymmetry of the channel pore. It is concluded that slow activation is principally the result of unbinding of polyamines from negatively charged residues close to the selectivity filter of the channel and not an intrinsic gating mechanism. Ba(2+) block involves an interaction with the same residues.

Matrix glycosaminoglycans in the growth phase of fibroblasts: more of the story in wound healing.

BACKGROUND: Understanding wound healing and ways to accelerate the healing process includes understanding the factors that influence the synthesis of granulation tissue, which fills the wound before epithelialization. An important phase of early wound healing involves secretion of glycosaminoglycans (GAGs) by fibroblasts which form a hydrophilic matrix suitable for remodeling during healing. The complexity of GAG structure and function in the extracellular matrix (ECM) remains poorly studied in wound healing. There is no established model for cutaneous wound healing due to variations in donor age, anatomic site, or stage of organ development. Rat embryo fibroblasts (REF) developed as a model to study malignant changes in fibroblasts were used as a model for fibroblasts in early wound healing because they lack the confounding variations based on age, site, and stage present in other fibroblasts used to study early wound healing. The purpose of this study was to identify and characterize the sulfated GAGs synthesized by REF-D. MATERIALS AND METHODS: Rat embryo fibroblasts (REF-D) were cultured in serum-based medium and radiolabeled during their growth phase with (35)S to identify the GAG chains usually associated with proteoglycans (PGs). The sites of attachment (ECM-rich) were collected with detergent in sodium acetate buffer, pH 5.8, in the presence of protease inhibitors. Sulfated molecules were collected by ion-exchange chromatography and then assayed for GAGs. Nitrous acid deamination was used to determine heparan sulfate GAGs, and chondroitinase was used for chondroitin/dermatan sulfate GAGs. The proportion of individual GAGs was expressed with respect to sulfated molecules isolated. In addition, RNA was isolated from subconfluent REF-D, and core proteins for proteoglycans (decorin, biglycan, syndecan-2, and perlecan) were assayed by reverse transcription polymerase chain reaction. RESULTS: There were two major configurations of GAGs: free GAG chains (79.7% of sulfated molecules) and GAGs attached to the core protein of a proteoglycan (15.6%). The free GAG chains were composed of chondroitin sulfate (79.1% +/- 3.5) and heparan sulfate (28.7% +/- 2.1). In the smaller group of PGs, both heparan sulfate (94.8% +/- 7.3) and chondroitin sulfate (88.9% +/- 3.2) chains were attached to a core protein. REF-D expressed mRNA for biglycan and decorin, which are chondroitin sulfate-containing PGs. In addition, REF-D expressed mRNA for syndecan-2 and perlecan, which are PGs that contain primarily heparan sulfate chains. CONCLUSIONS: A majority of GAG chains synthesized by subconfluent REF-D are chondroitin sulfate. A smaller proportion of chondroitin sulfate chains associate with a core protein as part of a PG (e.g., biglycan, decorin, syndecan-2). Heparan sulfate chains are also present, with a small proportion associated with a core protein (e.g., the PGs syndecan-2, perlecan). The greater presence of free GAG chains forming weak interactions with surrounding molecules may assist fibroblasts that are moving and replicating during this phase. Therefore, REF-D are particularly well suited to study early wound healing by their expression of chondroitin sulfate chains and associated PGs without the influence of donor age, stage, or anatomic site.

The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain.

We recently identified intersectin, a protein containing two EH and five SH3 domains, as a component of the endocytic machinery. The N-terminal SH3 domain (SH3A), unlike other SH3 domains from intersectin or various endocytic proteins, specifically inhibits intermediate events leading to the formation of clathrin-coated pits. We have now identified a brain-enriched, 170 kDa protein (p170) that interacts specifically with SH3A. Screening of combinatorial peptides reveals the optimal ligand for SH3A as Pp(V/I)PPR, and the 170 kDa mammalian son-of-sevenless (mSos1) protein, a guanine-nucleotide exchange factor for Ras, con- tains two copies of the matching sequence, PPVPPR. Immunodepletion studies confirm that p170 is mSos1. Intersectin and mSos1 are co-enriched in nerve terminals and are co-immunoprecipitated from brain extracts. SH3A competes with the SH3 domains of Grb2 in binding to mSos1, and the intersectin-mSos1 complex can be separated from Grb2 by sucrose gradient centrifugation. Overexpression of the SH3 domains of intersectin blocks epidermal growth factor-mediated Ras activation. These results suggest that intersectin functions in cell signaling in addition to its role in endocytosis and may link these cellular processes.

A family of proteins implicated in axon guidance and outgrowth.

Rapid progress in the identification and characterization of axon guidance molecules and their receptors has left the field poised to explore the intracellular mechanisms by which signals are transduced into growth cone responses. The TUC (TOAD/Ulip/CRMP) family of proteins has emerged as a strong candidate for a role in growth cone signaling. The TUC family members reach their highest expression levels in all neurons during their peak periods of axonal growth and are strongly down-regulated afterward. When axonal regrowth in the adult is triggered by axotomy, TUC-4 is reexpressed during the period of regrowth. Mutations in unc-33, a homologous nematode gene, lead to severe axon guidance errors in all neurons. Furthermore, the TUC family is required for the growth cone-collapsing activity of collapsin-1. An important role for the TUC family is also suggested by its high degree of interspecies amino acid sequence identity, with the rat TUC-2 protein showing 98% identity with its chick ortholog and 89% identity with its Xenopus ortholog. Information gained from the study of the TUC family will be of key importance in understanding how growth cones find their targets.

Effect of extracellular cations on the inward rectifying K+ channels Kir2.1 and Kir3.1/Kir3.4.

The effects of Ba2+, Mg2+, Ca2+ and Na+ as blocking ions were investigated in 90 and 10 mM extracellular K+ solutions on the cloned inward rectifying K+ channel Kir2.1 expressed in Xenopus oocytes. Some data were also obtained using another inward rectifying K+ channel Kir3.1/Kir3.4. The addition of Ba2+ caused a concentration-, voltage- and time-dependent block of both channels. Decreasing the extracellular K+ concentration augmented the block. The data suggest that Ba2+ blocks the channels by binding to a site within the channel pore and that the electrical binding distance, delta, of the site is significantly different for Kir2.1 and Kir3. 1/Kir3.4 (0.38 and 0.22, respectively). Mg2+ and Ca2+ caused an instantaneous concentration- and voltage-dependent block of both channels. With Kir2.1, decreasing the K+ concentration augmented the block. The voltage dependence of the block was less than that of Ba2+ ([delta], 0.1), indicating a more superficial binding site for these ions within the channel pore. The affinity of the channels for Mg2+ and Ca2+ was 1000-fold lower than that for Ba2+. Addition of Na+ resulted in a concentration-, voltage- and time-dependent block of Kir2.1, similar to that observed with Ba2+. The competition between the blocking cations (for Kir2.1: Ba2+, Mg2+, Ca2+; for Kir3. 1/Kir3.4: Ba2+) and extracellular K+ suggests that the binding sites for the blocking cations may be sites to which K+ binds as part of the normal passage of K+ through the channels. It is possible that under normal physiological conditions naturally occurring extracellular cations may partly block the two inward rectifying K+ channels.

Cartridge discharge residue contamination--the search for the source.

The detection of cartridge discharge residue (CDR) particles on some control samples in a firearms case started an extensive search to discover their origin. Investigation of potential sources of the contamination showed that it was airborne but random in nature, and it is recommended that frequent examination of air control samples should be made in contamination-sensitive areas.

The role of the forensic scientist at pseudo-military incidents.

Most forensic scientists called to a scene are briefed by a police officer and requested to carry out a specific function. The investigation of terrorist incidents in Northern Ireland provided a major challenge for scientists, involving large numbers of participants and several separate scenes. In two such cases, the areas initially sealed off were confined to the obvious focus of interest, the bodies and the damaged building, but the scenes expanded to other areas, including firing points and escape routes, house take-overs, hostage situations, and abandoned vehicles thought to have been used in the incidents. Because of security factors, all these scenes had to be examined quickly to preserve any evidence yet carefully to avoid contamination and to ensure the safety of all the personnel involved.

Use of an adjustable tourniquet to reverse cyanosis in the newborn pig.

BACKGROUND: Previous surgical models of cyanosis have been permanent. Because normal oxygenation was not restored in these models, it is unclear whether the metabolic changes produced by prolonged exposure to hypoxemia are irreversible. We therefore designed an experimental model of cyanosis that is reversible. METHODS: The left atrial appendage was anastomosed directly to the main pulmonary artery in 8 piglets, aged 2 to 4 weeks. RESULTS: The oxygen saturation fell from 95.3% +/- 0.8% to 72.4% +/- 3.9% (p < 0.001). A tourniquet was placed around the anastomosis to produce incremental changes in the level of cyanosis. Complete tourniquet occlusion resulted in obliteration of the right to left shunt, with return of systemic oxygen saturation to baseline levels. Systemic, left atrial, and pulmonary pressures did not change during the study. CONCLUSIONS: In this acute preparation, stable hemodynamic conditions were maintained despite substantial variations in systemic levels of oxygenation. Most important, this model allows reversal of cyanosis with the return of normal oxygenation. Application of this experimental design in a chronic model may help to determine whether the metabolic effects of prolonged hypoxemia are potentially reversible.

Human prostate carcinoma cells produce extracellular heparanase.

The degradation of heparan sulfate proteoglycan (HSPG) in basement membranes (BM) has been previously suggested to be accomplished by an endoglycosidase activity called heparanase which has not been isolated outside of platelets. HSPG degradation by heparanase has been associated with tumor cell invasion, angiogenesis, and growth factor function. In this study, we identify heparanase activity biochemically and immunologically in malignant human prostate carcinoma cells (PC-3M), linking platelet heparanase probes with the tumor heparanase activity observed. Concentrated conditioned medium from PC-3M cells was analyzed by a heparin-Sepharose affinity column. Three peaks eluted with 0.15, 0.35, and 0.5 M NaCl. Each peak was analyzed by incubation with 3H-labeled heparin as well as [3H]HSPG from EHS tumor BM. The 0.5 M peak material degraded [3H]-heparin by 17.2%, with little additional degradation by the other peaks in comparison to the conditioned medium from which they were obtained. Likewise, the same amount of the 0.5 M peak accounted for the majority of degradation (30.8%) of 3H-labeled HSPG. Interestingly, for the same amount of 0.5 M peak material, significantly more HSPG was degraded than heparin under the same conditions. In addition, carrageenan-lambda, an inhibitor of glycanase, completely inhibited the degradation of heparin and heparan sulfate proteoglycan by the 0.5 M peak. Using antibody to the N-terminus domain of platelet heparanase, a 60-kDa protein was identified by immunoblot in 0.5 M peak material. Additionally, immunohistochemical staining of human prostate carcinoma specimens showed granular staining at or near the cell membrane and near the luminal surface using antibody to the N-terminus and C-terminus domains of platelet heparanase. In summary, human prostate carcinoma cells show heparanase activity in conditioned medium that degrades heparin and BM HSPG and is detected by antibody to platelet heparanase. In addition, the membrane-associated staining in tissue sections of prostate cancer strongly correlates with the biochemical and immunological detection in conditioned medium of human PC-3M cells.

Sorting of heparan sulfate proteoglycan into matrix compartments of prostate adenocarcinoma cells.

There is increasing evidence that the association of prostate carcinoma cells with the surrounding extracellular matrix is important for their growth. Proteoglycans (PGs) are components of matrix that form important associations with other molecules. Heparan sulfate proteoglycan (HSPG), for example, associates with several matrix components such as fibronectin, laminin, and basic fibroblast growth factor. The purpose of this study was to determine if human prostate carcinoma cells (PC-3) synthesized HSPG in tissue culture. Three areas of sulfate-labeled matrix were collected from PC-3 cells and analyzed: attachment sites, nonattachment cell surfaces, and media. Charged groups were separated by ion-exchange columns, and PGs identified by gel filtration chromatography after chemical and enzymatic treatment. The media fraction contained the greatest proportion of sulfated PGs (79.3%), of which 45.5% were HSPG. The greatest concentration of HSPG was in the attachment site fraction where 88.6% of PGs were HSPG, although only 11.7% of sulfated molecules were PGs. In nonattachment surfaces, only 19.6% were PGs, of which 19.4% were HSPG. When PGs were assessed for hydrophobic binding to octyl-Sepharose beads, only a proportion of HSPG in the media fraction contained hydrophobic domains (18.8%). In summary, PC-3 cells synthesize at least two types of HSPG, sorting them into different matrix compartments. The major PG in attachment sites is HSPG without a hydrophobic domain, while an HSPG in the media fraction has a hydrophobic domain. The localization of different types of HSPG may be functionally important and may be altered during the metastatic process for prostate carcinoma when in association with specific stroma.

Metastatic renal cell carcinoma to the right cardiac ventricle without contiguous vena caval involvement.

We report an unusual metastatic renal cell carcinoma in the right cardiac ventricle in a 53-year-old man who presented with increasing dyspnea. Diagnostic evaluation revealed a left renal tumor without contiguous extension into the inferior vena cava and a right ventricular mass. To our knowledge this is only the second reported case of metastatic renal cell carcinoma to the right cardiac ventricle diagnosed ante mortem. We discuss the case and review the literature.