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Biomedical subjects

S L Campbell

Publications and source records attributed to S L Campbell.

At least 19 recordsLinked to original sources
PubMed2001

The insert region of Rac1 is essential for membrane ruffling but not cellular transformation.

The Rho family of Ras-related proteins, which includes Rac1, RhoA, and Cdc42, is distinguished from other members of the Ras superfamily of small GTPases in that its members possess additional sequences positioned between beta-strand 5 and alpha-helix 4, designated the insert region. Previous studies have established the importance of an intact insert region for the transforming, but not actin cytoskeletal reorganization, activities of Cdc42 and RhoA. Similarly, the insert region was determined to be essential for Rac1-mediated mitogenesis. Additionally, an intact insert region was also determined to be required for the antiapoptotic activity of Rac1 as well as for Rac1 activation of reactive oxygen species and the NF-kappaB transcription factor. However, it has not been determined whether the insert region is important for Rac1-mediated growth transformation. In this study, we assessed the requirement for the insert region in Rac1 transformation and signaling in NIH 3T3 cells. Unexpectedly, we found that a mutant of constitutively activated Rac1 that lacked the insert region retained potent transforming activity. The insert region of Rac1 was dispensable for Rac1 stimulation of transcription from the cyclin D1 promoter and for activation of the c-Jun, NF-kappaB, and E2F-1 transcription factors but was essential for Rac1 induction of serum response factor activity. While an intact insert region was dispensable for inducing reactive oxygen species production in vivo, it was required for Rac1 induction of lamellipodia. When taken together, these results show that the insert region of Rac1 serves roles in regulating actin organization and cell growth that are distinct from those of the analogous regions of Cdc42 and RhoA and support its involvement in regulating specific downstream effector interactions.

3T3 Cells↗
PubMed2000

Elucidation of binding determinants and functional consequences of Ras/Raf-cysteine-rich domain interactions.

Raf-1 is a critical downstream target of Ras and contains two distinct domains that bind Ras. The first Ras-binding site (RBS1) in Raf-1 has been shown to be essential for Ras-mediated translocation of Raf-1 to the plasma membrane, whereas the second site, in the Raf-1 cysteine-rich domain (Raf-CRD), has been implicated in regulating Raf kinase activity. While recognition elements that promote Ras.RBS1 complex formation have been characterized, relatively little is known about Ras/Raf-CRD interactions. In this study, we have characterized interactions important for Ras binding to the Raf-CRD. Reconciling conflicting reports, we found that these interactions are essentially independent of the guanine nucleotide bound state, but instead, are enhanced by post-translational modification of Ras. Specifically, our findings indicate that Ras farnesylation is sufficient for stable association of Ras with the Raf-CRD. Furthermore, we have also identified a Raf-CRD variant that is impaired specifically in its interactions with Ras. NMR data also suggests that residues proximal to this mutation site on the Raf-CRD form contacts with Ras. This Raf-CRD mutant impairs the ability of Ras to activate Raf kinase, thereby providing additional support that Ras interactions with the Raf-CRD are important for Ras-mediated activation of Raf-1.

Animals↗
PubMed2000

Vav2 is an activator of Cdc42, Rac1, and RhoA.

Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity in their Dbl homology domains, it has been reported that they are active GEFs with distinct substrate specificities toward Rho family members. Whereas Vav displayed GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was reported to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42. Consistent with their distinct substrate targets, it was found that constitutively activated versions of Vav and Vav2 caused distinct transformed phenotypes when expressed in NIH 3T3 cells. In contrast to the previous findings, we found that Vav2 can act as a potent GEF for Cdc42, Rac1, and RhoA in vitro. Furthermore, we found that NH(2)-terminally truncated and activated Vav and Vav2 caused indistinguishable transforming actions in NIH 3T3 cells that required Cdc42, Rac1, and RhoA function. In addition, like Vav and Rac1, we found that Vav2 activated the Jun NH(2)-terminal kinase cascade and also caused the formation of lamellipodia and membrane ruffles in NIH 3T3 cells. Finally, Vav2-transformed NIH 3T3 cells showed up-regulated levels of Rac-GTP. We conclude that Vav2 and Vav share overlapping downstream targets and are activators of multiple Rho family proteins. Therefore, Vav2 may mediate the same cellular consequences in nonhematopoietic cells as Vav does in hematopoietic cells.

3T3 Cells↗
PubMed1999

Dependence of Dbl and Dbs transformation on MEK and NF-kappaB activation.

Dbs was identified initially as a transforming protein and is a member of the Dbl family of proteins (>20 mammalian members). Here we show that Dbs, like its rat homolog Ost and the closely related Dbl, exhibited guanine nucleotide exchange activity for the Rho family members RhoA and Cdc42, but not Rac1, in vitro. Dbs transforming activity was blocked by specific inhibitors of RhoA and Cdc42 function, demonstrating the importance of these small GTPases in Dbs-mediated growth deregulation. Although Dbs transformation was dependent upon the structural integrity of its pleckstrin homology (PH) domain, replacement of the PH domain with a membrane localization signal restored transforming activity. Thus, the PH domain of Dbs (but not Dbl) may be important in modulating association with the plasma membrane, where its GTPase substrates reside. Both Dbs and Dbl activate multiple signaling pathways that include activation of the Elk-1, Jun, and NF-kappaB transcription factors and stimulation of transcription from the cyclin D1 promoter. We found that Elk-1 and NF-kappaB, but not Jun, activation was necessary for Dbl and Dbs transformation. Finally, we have observed that Dbl and Dbs regulated transcription from the cyclin D1 promoter in a NF-kappaB-dependent manner. Previous studies have dissociated actin cytoskeletal activity from the transforming potential of RhoA and Cdc42. These observations, when taken together with those of the present study, suggest that altered gene expression, and not actin reorganization, is the critical mediator of Dbl and Rho family protein transformation.

3T3 Cells↗
PubMed1998

Increasing complexity of Ras signaling.

The initial discovery that ras genes endowed retroviruses with potent carcinogenic properties and the subsequent determination that mutated ras genes were present in a wide variety of human cancers, prompted a strong suspicion that the growth-promoting actions of mutated Ras proteins contribute to their aberrant regulation of growth stimulatory signaling pathways. In 1993, a remarkable convergence of experimental observations from genetic analyses of Drosophila, S. cerevisiae and C. elegans as well as biochemical and biological studies in mammalian cells came together to define a clear role for Ras in signal transduction. What emerged was an elegant linear signaling pathway where Ras functions as a relay switch that is positioned downstream of cell surface receptor tyrosine kinases and upstream of a cytoplasmic cascade of kinases that included the mitogen-activated protein kinases (MAPKs). Activated MAPKs in turn regulated the activities of nuclear transcription factors. Thus, a signaling cascade where every component between the cell surface and the nucleus was defined and conserved in worms, flies and man. This was a remarkable achievement in our efforts to appreciate how the aberrant function of Ras proteins may contribute to the malignant growth properties of the cancer cell. However, the identification of this pathway has proven to be just the beginning, rather than the culmination, of our understanding of Ras in signal transduction. Instead, we now appreciate that this simple linear pathway represents but a minor component of a very complex signaling circuitry. Ras signaling has emerged to involve a complex array of signaling pathways, where cross-talk, feedback loops, branch points and multi-component signaling complexes are recurring themes. The simplest concept of a signaling cascade, where each component simply relays the same message to the next, is clearly not the case. In this review, we summarize our current understanding of Ras signal transduction with an emphasis on new complexities associated with the recognition and/or activation of cellular effectors, and the diverse array of signaling pathways mediated by interaction between Ras and Ras-subfamily proteins with multiple effectors.

Amino Acid Sequence↗
PubMed1998

Rho family proteins and Ras transformation: the RHOad less traveled gets congested.

The Rho family of small GTPases has attracted considerable research interest over the past 5 years. During this time, we have witnessed a remarkable increase in our knowledge of the biochemistry and biology of these Ras-related proteins. Thus, Rho family proteins have begun to rival, if not overshadow, interest in their more celebrated cousins, the Ras oncogene proteins. The fascination in Rho family proteins is fueled primarily by two major observations. First, like Ras, Rho family proteins serve as guanine nucleotide-regulated binary switches that control signaling pathways that in turn regulate diverse cellular processes. Rho family proteins are key components in cellular processes that control the organization of the actin cytoskeleton, activate kinase cascades, regulate gene expression, regulate membrane trafficking, promote growth transformation and induce apoptosis. Second, at least five Rho family proteins have been implicated as critical regulators of oncogenic Ras transformation. Thus, it is suspected that Rho family proteins contribute significantly to the aberrant growth properties of Ras-transformed cells. Rho family proteins are also critical mediators of the transforming actions of other transforming proteins and include Dbl family oncogene proteins, G protein-coupled receptors and G protein alpha subunits. Thus, Rho family proteins may be key components for the transforming actions of diverse oncogene proteins. Major aims of Rho family protein studies are to define the molecular mechanism by which Rho family proteins regulate such a diverse spectrum of cellular behavior. These efforts may reveal novel targets for the development of anti-Ras and anti-cancer drugs.

Actins↗
PubMed1997

14-3-3 zeta negatively regulates raf-1 activity by interactions with the Raf-1 cysteine-rich domain.

Although Raf-1 is a critical effector of Ras signaling and transformation, the mechanism by which Ras promotes Raf-1 activation is complex and remains poorly understood. We recently reported that Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139-184) may be required for Raf-1 activation. The Raf-CRD is located in the NH2-terminal negative regulatory domain of Raf-1 and is highly homologous to cysteine-rich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. In the present study, we demonstrate that 14-3-3 zeta binds directly to the isolated Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1.

14-3-3 Proteins↗
PubMed1997

Structural and functional analysis of a mutant Ras protein that is insensitive to nitric oxide activation.

Ras proteins cycle between active, guanosine triphosphate (GTP)-bound and inactive, guanosine diphospate (GDP)-bound states to mediate signal transduction pathways that promote cell growth and differentiation. It is believed that the major physiological mechanism for Ras activation is via interaction with guanine-nucleotide exchange factors (GEFs). This interaction is highly regulated and results in elevated levels of Ras-GTP by facilitating GDP dissociation. Recently, a novel mechanism of Ras activation has been proposed, whereby nitric oxide (NO) modification of Cys-118, like GEF interaction, populates Ras in its biologically active form by stimulating GDP release. Here, we describe characterization of a variant of Ras, C118S, that is insensitive to NO modification. We have measured the GTPase activity and the GDP dissociation rate of the C118S mutant and found them to be similar to wild-type Ras. We have also analyzed the structure of this mutant using multidimensional heteronuclear NMR methods. Analysis of chemical shifts and distance restraints demonstrates that this mutation has not disrupted the structure of the protein. These results suggest that NO modification of Cys-118 may not alter Ras structure and that the basis of Ras activation by NO is destabilization of a crucial interaction between residues in the GDP-binding pocket and the nucleotide. We have also found that this mutant is a more stable form of Ras at concentrations required for NMR studies, probably due to the removal of a surface-accessible cysteine residue. This stable variant may facilitate structural and biochemical investigations of Ras and other guanine-nucleotide-binding proteins containing a cysteine at this position.

GTP Phosphohydrolases↗
PubMed1996

The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phospholipid binding site.

The Raf-1 protein kinase is the best-characterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals. The details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the involvement of both Ras-binding sites in effective Raf-1-mediated transformation provides insight into the molecular aspects and consequences of Ras-Raf interactions. The Raf-1 CRD is a member of an emerging family of domains, many of which are found within signal transducing proteins. Several contain binding sites for diacylglycerol (or phorbol esters) and phosphatidylserine and are believed to play a role in membrane translocation and enzyme activation. The CRD from Raf-1 does not bind diacylglycerol but interacts with Ras and phosphatidylserine. To investigate the ligand-binding specificities associated with CRDs, we have determined the solution structure of the Raf-1 CRD using heteronuclear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PKC). The differences are confined to regions of the CRDs involved in binding phorbol ester in the PKC domains. Since phosphatidylserine is a common ligand, we expect its binding site to be located in regions where the structures of the Raf-1 and PKC domains are similar. The structure of the Raf-1 CRD represents an example of this family of domains that does not bind diacylglycerol and provides a framework for investigating its interactions with other molecules.

Amino Acid Sequence↗
PubMed1996

Involvement of the switch 2 domain of Ras in its interaction with guanine nucleotide exchange factors.

While Ras proteins are activated by stimulated GDP release, which enables acquisition of the active GTP-bound state, little is known about how guanine nucleotide exchange factors (GEFs) interact with Ras to promote this exchange reaction. Here we report that mutations within the switch 2 domain of Ras (residues 62-69) inhibit activation of Ras by the mammalian GEFs, Sos1, and GRF/CDC25Mm. While mutations in the 62-69 region blocked upstream activation of Ras, they did not disrupt Ras effector functions, including transcriptional activation and transformation of NIH 3T3 cells. Biochemical analysis indicated that the loss of GEF responsiveness of a Ras(69N) mutant was due to a loss of GEF binding, with no change in intrinsic nucleotide exchange activity. Furthermore, structural analysis of Ras(69N) using NMR spectroscopy indicated that mutation of residue 69 had a very localized effect on Ras structure that was limited to alpha-helix 2 of the switch 2 domain. Together, these results suggest that the switch 2 domain of Ras forms a direct interaction with GEFs.

3T3 Cells↗
PubMed1996

Ras interaction with two distinct binding domains in Raf-1 may be required for Ras transformation.

Although Raf-1 is a critical Ras effector target, how Ras mediates Raf-1 activation remains unresolved. Raf-1 residues 55-131 define a Ras-binding domain essential for Raf-1 activation. Therefore, our identification of a second Ras-binding site in the Raf-1 cysteine-rich domain (residues 139-184) was unexpected and suggested a more complex role for Ras in Raf-1 activation. Both Ras recognition domains preferentially associate with Ras-GTP. Therefore, mutations that impair Ras activity by perturbing regions that distinguish Ras-GDP from Ras-GTP (switch I and II) may disrupt interactions with either Raf-1-binding domain. We observed that mutations of Ras that impaired Ras transformation by perturbing its switch I (T35A and E37G) or switch II (G60A and Y64W) domain preferentially diminished binding to Raf-1-(55-131) or the Raf-1 cysteine-rich domain, respectively. Thus, these Ras-binding domains recognize distinct Ras-GTP determinants, and both may be essential for Ras transforming activity. Finally, since Ha-Ras T35A and E37G mutations prevent Ras interaction with full-length Raf-1, we suggest that Raf-Cys is a cryptic binding site that is unmasked upon Ras interaction with Raf-1-(55-131).

3T3 Cells↗
PubMed1996

Assessing the efficacy of three dentifrices in the treatment of dentinal hypersensitivity.

A multicenter clinical trial conducted by the authors compared the desensitizing efficacy of a new 5 percent potassium nitrate: 0.243 percent sodium fluoride dentifrice along with two clinically proven, commercially available desensitizing dentifrices to a placebo dentifrice. Sensitivity to cold air and tactile stimulation, along with patients' subjective assessments, were evaluated to assess the dentinal desensitizing efficacy of the test dentifrices. Results demonstrated that after four weeks, participants who used the new dentifrice formulation experienced significant decreases in dentinal sensitivity compared to the placebo group for all measured indexes.

Adult↗
PubMed1996

Triple compliance: FMLA, ADA, and workers' comp.

The major nightmare many employers have encountered is that they fail to notify an employee out on workers' comp leave in writing that the FMLA leave period began when they stopped working.

Disability Evaluation↗
PubMed1996

The comparative efficacy of two commercial tartar control dentifrices in preventing calculus development and facilitating easier dental cleanings.

Tartar control dentifrices have been proven effective in reducing the build-up of supragingival calculus deposits between professional tooth cleanings. In addition to providing consumers with a cleaner dentition, these formulations can, in principle, contribute to easier professional tooth cleaning. Until recently, clinical methods for evaluating the effects of tartar control dentifrices were limited to the assessment of area coverage of calculus on the tooth surface. The development of the Quanticalc dental scaler permits the quantitative clinical measurement of force and stroke number associated with supragingival calculus debridement. In this clinical study, two commercial tartar control dentifrices, one containing 5.0% pyrophosphate as the tartar control agent and the other containing a combination of 1.3% pyrophosphate and 1.5% Gantrez copolymer, were compared for efficacy in reducing the development of supragingival calculus between prophylaxes, and in facilitating easier calculus removal in subsequent scaling. Results showed that the two commercial dentifrices were equally effective in reducing calculus extent between prophylaxes. In contrast, the 5.0% pyrophosphate dentifrice was observed to be almost twice as effective on a percentage basis as the lower dosage pyrophosphate dentifrice in facilitating easier calculus removal. The enhanced activity of the 5.0% pyrophosphate dentifrice may be postulated to be due to elevated dosage of tartar control crystallization inhibitor or to potential side effects of copolymer in the other commercial dentifrice. Importantly, these results substantiate that the clinical benefits of tartar control dentifrices are not completely described by actions in reducing calculus build-up as assessed by VMI, and that important clinical benefits and differences in efficacy may be provided by these formulations in facilitating easier dental cleaning of supragingival calculus.

Adult↗
PubMed1995

Chylothorax associated with constrictive pericarditis in a dog.

Chylothorax was associated with constrictive pericarditis in a 6-year-old mixed-breed dog. Clinical signs included hepatomegaly, bilateral jugular pulses, muffled heart sounds, and dyspnea. Pleural effusion was identified on thoracic radiographs. Thoracentesis yielded 3 L of chylous effusion, confirmed by the cholesterol/triglyceride ratio (0.14). Echocardiography revealed a thickened pericardium, with numerous fibrin tags, which was suggestive of constrictive pericarditis. Central venous, right atrial, and right ventricular pressures were high and on right ventricular diastolic pressure tracings, the square-root sign, which is indicative of constrictive pericarditis, was evident. Exploratory thoracotomy and pericardiectomy were performed. Histopathologic findings were consistent with chronic non-suppurative pericarditis. The dog's condition improved after surgery. On reevaluation 11 months later, central venous pressure and results of physical examination and thoracic radiography were normal. On the basis of these findings, we concluded that chylothorax was caused by constrictive pericarditis in this dog. Chylothorax is often associated with disorders that have a poor prognosis, but if constrictive pericarditis can be identified as the cause of the chylothorax, exploratory thoracotomy and pericardiectomy can provide a cure.

Animals↗
PubMed1994

Longitudinal measurements of spontaneous otoacoustic emissions in infants.

It has previously been shown [E. M. Burns, K. H. Arehart, and S. L. Campbell, J. Acoust. Soc. Am. 91, 1575-1581 (1992)] that both the overall prevalence of spontaneous otoacoustic emissions (SOAEs) and most of the various gender- and ear-related prevalence tendencies are not significantly different in 1-month-olds and adults. However, large differences were found between the neonates and adults in the distributions of the frequencies and levels of SOAEs. Both the average level and the median frequency were significantly higher in infants. To obtain longitudinal SOAE data, infants from this original group of 1-month-olds were tested at ages 3, 6, 12, and 24 months. In general, individual SOAEs decrease in level with age, and high-frequency SOAEs tend to show the largest decreases. No substantial shifts occur in the frequencies of individual SOAEs. The frequency and level distributions at 24 months of age are still not adult-like. SOAEs which show short-term instabilities in frequency and/or amplitude at 1 month of age typically continue to evidence such instabilities at later ages. These results suggest the cochlea is adult-like at birth, and imply that the observed SOAE changes reflect developmental changes in the external and middle ear.

Age Factors↗