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By the mid 1980's, it was clear that the transforming activity of oncogenic Src was linked to the activity of its tyrosine kinase domain and attention turned to identifying substrates, the putative next level of control in the pathway to transformation. Among the first to recognize the potential of phosphotyrosine-specific antibodies, Parsons and colleagues launched a risky shotgun-based approach that led ultimately to the cDNA cloning and functional characterization of many of today's best-known Src substrates (for example, p85-Cortactin, p110-AFAP1, p130Cas, p125FAK and p120-catenin). Two decades and over 6000 citations later, the original goals of the project may be seen as secondary to the enormous impact of these protein substrates in many areas of biology. At the request of the editors, this review is not restricted to the current status of the substrates, but reflects also on the anatomy of the project itself and some of the challenges and decisions encountered along the way.

Objectives: Recreational erectile enhancing medication (EEM) use has been associated with a number of health risk behaviours among gay and bisexual men. This study aims to extend previous findings about the associations between recent EEM use and illegal drug use, incident sexually transmitted infections (STIs) and unprotected sex, as well as to report on motivations for EEM use., Methods: A cross-sectional, street-intercept survey method was used to collect data from 912 gay/bisexual men at two large lesbian, gay and bisexual community events in New York City in 2006., Results: Lifetime EEM use was reported by 28.0% of the men; 17.4% used EEM in the past 3 months. EEM users were more likely to be white and HIV positive. EEM users were more likely to engage in unprotected anal insertive sex with seroconcordant and serodiscordant partners. EEM users who were HIV negative were more likely to report using alcohol and other drugs before and during sex, especially crystal methamphetamine (AOR 18.66; 95% CI 6.82 to 51.02) as well as to endorse incident STIs. The most frequent responses for EEM use were to "add to the fun", "maintain an erection while using a condom" and "to have sex for hours". Men with HIV were 2.93 times (95% CI 1.24 to 6.88) more likely to endorse using EEMs to bareback., Conclusions: Gay and bisexual men use EEMs to enhance their sexual experiences among other motives. Different motives and correlates emerged by HIV status. Overall, EEM use was correlated with multiple health risk behaviours. EEM users who were HIV negative appear to be at particularly high risk of acquiring HIV.

The purpose of the study was to test constructs of the Transtheoretical Model (TTM) for predicting alcohol and other drug use in HIV-positive youth (ages 16-25). Questionnaires and interviews about alcohol and other drug use, stage of change, self-efficacy, emotional distress and social support were obtained from 64 HIV-positive youth. Structural equation modeling with standard errors determined by methods appropriate to small samples, demonstrated that self-efficacy mediated the relationship between stage of change and alcohol use and between social support and alcohol use. The same pattern of results emerged for marijuana use. The models predicted 47% of the variance in alcohol use and 69% of the variance in marijuana use. Results supported the TTM and highlight the potential of interventions that seek to boost self-efficacy and social support specific to reducing substance use.

The objective of this study was to examine the knowledge and misinformation about human papillomavirus (HPV) and differences in sexual risk practices. Self-report surveys assessing the history of HPV/genital warts and sexual practices were completed by 1065 gay/bisexual men in New York City. Of the men reporting a history of HPV, genital warts, or both, the majority reported having warts but not HPV, demonstrating inaccurate knowledge. A significantly greater percentage of men who reported both HPV and warts (HPV+) reported having unsafe sex compared with men reporting neither (HPV-) and men reporting warts but not HPV (HPV+ uninformed). HPV+ and HPV+ uninformed men reported significantly more men non-primary sex partners in the past three months than HPV- men. Findings indicate that many gay/bisexual men, a population at risk for HPV, are misinformed about its various permutations. Men who are HPV+ report increased sexual risk practices and more sexual partners. Comprehensive gay men's health programmes must include HPV education.

While prior studies have targeted street-based male sex workers as potential vectors of disease transmission, the number of men who work independently through Internet chat-rooms and other online endeavors has steadily increased. It is likely that these men differ substantially from their street-based counterparts in terms of sexual risk behaviors with their clients. The purpose of this study was to explore the ways in which the Internet has impacted the work of male escorts and their sexual practices with clients. Semi-structured qualitative interviews and quantitative surveys were administered to 46 such men. Less than half the men reported unprotected anal sex with clients. The qualitative data lend support to this finding, in that the majority talked about refusing any unsafe sex with clients, and many reported taking the extra step of educating their clients about the dangers of risky sex. Some of the escorts described the methods used to incorporate safer sex practices into sessions with their clients. Internet-based male escorts can play an important role as potential sex educators on the front lines of the fight against HIV and other sexually transmitted infections.

Using cross-sectional data from an ethnically diverse sample of HIV-positive injection drug users (IDUs), we sought to identify correlates of depressive symptoms. We were particularly interested in whether perceived social support was associated with depression among HIV-positive IDUs and whether social support buffered adverse effects of other correlates. Data were collected from a sample of HIV-positive IDUs recruited from a variety of venues in the New York City and San Francisco metropolitan areas in the USA. Multiple regression analysis identified four significant correlates of depressive symptoms. Perceived social support and having a regular place for HIV medical care were significantly associated with lower levels of depressive symptoms, while history of mental health problems and non-injection polydrug use were significantly associated with higher levels of depressive symptoms. Moreover, a significant interaction effect was found between social support and non-injection polydrug use, indicating that social support buffers the association between non-injection polydrug use and depression. These results suggest that increasing social support might be a useful tool for HIV-positive IDUs in reducing depression and the adverse effect of non-injection polydrug use.

While unsafe sex has been reported throughout the HIV epidemic, the underlying assumption has been that most persons do not seek to purposely ham unprotected sex. Within the gay community, the term 'barebacking' has emerged to refer to intentional unsafe anal sex. The prevalence of barebacking is evidenced among gay men, particularly those who are HIV-positive, by the number of internet sites devoted to barebacking and the number of men seeking sexual partners through the use of the internet. To gain insight into barebacking, a sample of 112 HIV-positive gay men were recruited from internet sites where men seek to meet each other for sex. The major it of participants (84%)reported engaging in barebacking in the past three months, and 43% of the men reported recent bareback sex with a partner of unknown serostatus. These results indicate the potential for widespread transmission of HIV to uninfected men by the partners they meet on the internet. Analyses revealed that men who reported bareback sex only with HIV-positive partners scored lower in sexual adventurism than those who had bareback sex regardless of partner serostatus. A significant correlation was observed between defining masculinity as sexual prowess and intentional unprotected anal sex. There are serious implications for HIV prevention efforts, in that internet-based education should be a priority in order to reach men who rely on this mechanism to find sexual partners.

The goal of the project was to develop and evaluate theory-based interventions designed to change sexual behaviour and promote safer sex practices of HIV seropositive young men and adolescents with haemophilia to prevent transmission to sexual partners and offspring. Safer sex was defined as abstinence, consistent condom use, or 'outercourse' (intimate, non-intercourse sexual behaviour). This project utilized the Transtheoretical Model developed by Prochaska and DiClemente, which describes behaviour change as an incremental, stage-based process. The 1-year intervention protocol consisted of two individual sessions and two peer-centred activities. One hundred and four adolescents, residing in 22 states, participated. Pre- and post-intervention evaluations were conducted to measure stage progression for participants. The number who were in the action or maintenance stage of change for safer sex was significantly greater at post-test than at pre-test (79 vs. 62%, P < 0.0001). Participants also reported an increased use of outercourse. In addition, significant increases in self-efficacy and knowledge regarding safer sex were demonstrated. Following these stage-based interventions, participants were significantly more likely to be engaging in safer sex behaviours than they were previously. These intervention activities can be adapted for use with other adolescent populations and for other behaviour change goals in adolescents with haemophilia.

Data regarding HIV antiretroviral treatment regimens, access to treatment and medical care, and adherence to medications were collected as part of the Seropositive Urban Men's Study, a formative study of HIV-positive men who have sex with men. Participants (N = 456) were recruited from AIDS service organizations, mainstream gay venues and public/commercial sex environments. The sample was 94% gay or bisexually-identified; 29% were African American, 24% Latino and 30% white. The majority (71%) indicated being on antiretroviral treatment, and most were taking a protease inhibitor/nucleoside reverse transcriptase inhibitor combination. African American men in New York City were less likely to be on treatment. Among those on treatment (n = 322), 51% reported at least one day in which they had missed a dose of their medication and the mean number of days in which a dose was missed (in the past 30 days) was 1.72. Multivariate analyses indicated that avoidant coping, frequency of drinking alcohol and difficulty in communicating with sex partners about HIV were related to days of missed doses, suggesting the need or desire to escape from the reality of life with HIV as a potential explanation for poor adherence.

An ethnically diverse sample (69.7% men of colour) of HIV-seropositive MSM from the New York City and San Francisco metropolitan areas were recruited from a variety of social settings. While only 27.0% of the participants were recruited from PSEs (public sex environments where men 'cruise' for potential sex partners, such as parks) and CSEs (commercial sex environments where an admission is paid for entrance, such as bathhouses and sex clubs), 49.6% reported attending PSEs and 40.7% reported attending CSEs. Only a minority of participants from the full sample reported sexual behaviours that would have placed a partner at highest risk for HIV seroconversion. However, differences between those who frequented CSEs and those who did not emerged on several psychosocial factors (sexual sensation seeking, depression, perceived responsibility towards protecting sexual partners from HIV infection), sexual risk behaviours (unprotected oral and anal sex) and types of recreational drugs used. Fewer differences were found between those who frequented PSEs and those who did not.

Exposure of cells to a variety of external signals causes rapid changes in plasma membrane morphology. Plasma membrane dynamics, including membrane ruffle and microspike formation, fusion or fission of intracellular vesicles, and the spatial organization of transmembrane proteins, is directly controlled by the dynamic reorganization of the underlying actin cytoskeleton. Two members of the Rho family of small GTPases, Cdc42 and Rac, have been well established as mediators of extracellular signaling events that impact cortical actin organization. Actin-based signaling through Cdc42 and Rac ultimately results in activation of the actin-related protein (Arp) 2/3 complex, which promotes the formation of branched actin networks. In addition, the activity of both receptor and non-receptor protein tyrosine kinases along with numerous actin binding proteins works in concert with Arp2/3-mediated actin polymerization in regulating the formation of dynamic cortical actin-associated structures. In this review we discuss the structure and role of the cortical actin binding protein cortactin in Rho GTPase and tyrosine kinase signaling events, with the emphasis on the roles cortactin plays in tyrosine phosphorylation-based signal transduction, regulating cortical actin assembly, transmembrane receptor organization and membrane dynamics. We also consider how aberrant regulation of cortactin levels contributes to tumor cell invasion and metastasis.

In the rat pilocarpine model, 1 h of status epilepticus caused significant inhibition of Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in cortex endoplasmic reticulum (microsomes) isolated immediately after the status episode. The rat pilocarpine model is also an established model of acquired epilepsy. Several weeks after the initial status epilepticus episode, the rats develop spontaneous recurrent seizures, or epilepsy. To determine whether inhibition of Ca(2+) uptake persists after the establishment of epilepsy, Ca(2+) uptake was studied in cortical microsomes isolated from rats displaying spontaneous recurrent seizures for 1 year. The initial rate and total Ca(2+) uptake in microsomes from epileptic animals remained significantly inhibited 1 year after the expression of epilepsy compared to age-matched controls. The inhibition of Ca(2+) uptake was not due to individual seizures nor an artifact of increased Ca(2+) release from epileptic microsomes. In addition, the decreased Ca(2+) uptake was not due to either selective isolation of damaged epileptic microsomes from the homogenate or decreased Mg(2+)/Ca(2+) ATPase protein in the epileptic microsomes. The data demonstrate that inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in the pilocarpine model may underlie some of the long-term plasticity changes associated with epileptogenesis.

Immunofluorescence studies with protein phosphatase-1 (PP1) isoforms-specific antibodies detected PP1delta, but not alpha or gamma1, at focal adhesions. PP1delta also co-immunoprecipitated with the focal adhesion kinase (FAK) and the alphav-integrin. In the present study glutathione S-transferase (GST)-PP1delta pulled-down FAK from fibroblasts extract and the interaction domain localized between residues 159 and 295 of delta. The association was confirmed by the ability to GST-FAK-related non-kinase (FRNK) to pull-down PP1delta from fibroblasts extract. GST-FRNK also pulled-down purified muscle PP1 catalytic subunit, thus indicating direct interaction between FAK and PP1. FAK displays consensus sequences for phosphorylation by cell division cycle kinase-2-cyclin B, and might be a PP1 substrate. In fact, FAK immunoprecipitated from metabolically-labelled mitotic HeLa cells without tyrosine phosphatase inhibitors was phosphorylated on Ser only and was dephosphorylated in vitro by purified muscle PP1, with loss of phospho-Ser. No PP1 was associated with FAK immunoprecipitated from mitotic HeLa cells. However, progressively more PP1 activity was assayed in FAK-immunoprecipitates obtained from cells released from mitosis. The associated activity was maximal at 2 h from the mitotic release (when 85-90% of the cells remained round) and decreased to basal level by 8 h (when cells were all polygonal). At the same time FAK underwent dephosphorylation, which was completed by 4 h. FAK obtained from cells at 1.5 h was Ser-phosphorylated, and underwent dephosphorylation during in vitro incubation, with loss of phospho-Ser, indicating the presence of active FAK-bound phosphatase. The only FAK-associated PP1 isoform between 1 and 8 h was PP1delta. The results suggest that FAK dephosphorylation by PP1delta occurs in cells released from mitosis, and confirmed the specific association of PP1delta, as detected previously in adherent cells.

Little is known about HIV-seropositive men's awareness and use of untested barrier methods during anal intercourse. A sample of 240 HIV-seropositive men (69.2% men of color) completed a self-administered survey that included items on nonoxynol-9 (N-9), female condoms, and the simultaneous use of two male condoms (double bagging). Most participants (79.6%) had heard of N-9 being used to prevent HIV transmission during anal intercourse. Of these, 20.0% rated N-9 as more effective than condoms, and 14.6% had used N-9 instead of condoms. Fewer men (35.4%) were aware of female condoms being used during anal intercourse. Overall, few respondents (5.4%) had used female condoms; 53.8% of whom rated the device as more pleasurable than male condoms. Most men (69.6%) had heard of double bagging, and 35.2% had engaged in this practice. Of these, 45.1% rated the practice as less pleasurable than using a single condom. Few associations were observed between participant characteristics and the awareness or use of these barrier methods. The widespread use of these untested methods emphasizes the urgent need to further educate HIV-seropositive men about the potential risks of N-9 use and to test the effectiveness of other strategies that may serve as alternatives to male condom use.

Purpose: Whereas there are many reports regarding treatment of early vocal cord cancer with cobalt 60 or 2-4-MV X-rays, there are still few reports on the results of treatment with 6-MV X-rays. Theoretically, 6-MV X-rays result in greater underdosage of tumor at the air-tissue interface and at the anterior commissure. This paper analyzes the results of irradiation of early and moderately advanced squamous cell carcinoma of the true vocal cord treated exclusively with 6-MV X-rays in a community hospital. The literature pertinent to the issue is reviewed., Methods and Materials: Eighty-three patients with Tis, T1, T2, or T3 squamous cell carcinoma of the true vocal cord were treated with curative intent at Bethesda Memorial Hospital in Boynton Beach, Florida between April 1986 and April 1998. The dose schedules most commonly used were 63 Gy in 28 fractions (2.25 Gy per fraction once a day) for T1 tumors or 74.40 Gy in 62 fractions (1.2 Gy per fraction twice a day) for T2 and T3 tumors. All patients have minimum 2-year follow-up; 63 (76%) have 5-year minimum follow-up., Results: Local control was achieved in 6 of 6 Tis, 53 of 54 (98%) T1, 8 of 8 T2, and 6 of 6 T3 lesions. No complications were encountered., Conclusions: A recent literature review indicates that the treatment of early vocal cord cancer with 6-MV X-rays remains controversial. The dose schedules used in the present paper produced a high rate of local control, a finding that is consistent with reports of other investigators who used dose schedules similar to those used in the present series. However, several other investigators have reported significantly lower rates of local control for T1 or T2 glottic cancer treated with 6-MV X-rays when compared to results obtained with cobalt 60 or 4 MV at their own institution. The latter institutions used lower total doses and/or lower dose per fraction than those institutions reporting high rates of local control with 6 MV. Data from the literature, as well as our own data, are consistent with the following hypotheses: (1) the lower rates of local control reported by several institutions when using 6 MV compared with cobalt or 2-4 MV, using the same radiation dose schedules for each beam energy, indicate that underdosage of mucosal surfaces in the laryngeal air cavity may be a clinically important phenomenon, and (2) time-dose factors, although certainly important for lower energy beams, may be even more important when using 6 MV.

This study focused on the effects of status epilepticus on the activity of calcineurin, a neuronally enriched, calcium-dependent phosphatase. Calcineurin is an important modulator of many neuronal processes, including learning and memory, induction of apoptosis, receptor function and neuronal excitability. Therefore, a status epilepticus-induced alteration of the activity of this important phosphatase would have significant physiological implications. Status epilepticus was induced by pilocarpine injection and allowed to continue for 60 min. Brain region homogenates were then assayed for calcineurin activity by dephosphorylation of p-nitrophenol phosphate. A significant status epilepticus-dependent increase in both basal and Mn(2+)-dependent calcineurin activity was observed in homogenates isolated from the cortex and hippocampus, but not the cerebellum. This increase was resistant to 150 nM okadaic acid, but sensitive to 50 microM okadaic acid. The increase in basal activity was also resistant to 100 microM sodium orthovanadate. Both maximal dephosphorylation rate and substrate affinity were increased following status epilepticus. However, the increase in calcineurin activity was not found to be due to an increase in calcineurin enzyme levels. Finally, increase in calcineurin activity was found to be NMDA-receptor activation dependent. The data demonstrate that status epilepticus resulted in a significant increase in both basal and maximal calcineurin activity.

The treatment of soft-tissue sarcomas has undergone significant changes over the past several decades. Previously, patients were often treated with surgery alone, which frequently necessitated amputation of the affected extremity. Less extensive, limb-sparing operations combined with adjuvant irradiation are now feasible for most patients without compromising the likelihood of cure.

Focal adhesion kinase (FAK) has been implicated in the regulation of cell migration. In addition, FAK expression is increased in a number of highly metastatic tumor cell lines. Therefore, we investigated the role of FAK in regulating migration of prostate carcinoma cell lines with increasing metastatic potential. We show that highly tumorigenic PC3 and DU145 cells exhibit intrinsic migratory capacity, while poorly tumorigenic LNCaP cells require a stimulus to migrate. Increased metastatic potential of PC3 and DU145 cells correlates with increased FAK expression, overall tyrosine phosphorylation and activity, as measured by autophosphorylation of tyrosine 397. However, in PC3 and DU145 cells, FAK autophosphorylation is adhesion dependent whereas a second site of tyrosine phosphorylation, tyrosine 861, a Src specific site, is uncoupled from adhesion-dependent signaling events. Finally, inhibiting the FAK/Src signal transduction pathway by over expressing FRNK (Focal adhesion kinase-Related Non-Kinase), an inhibitor of FAK activation, or treatment with PP2, a Src family kinase inhibitor, significantly inhibited migration of prostate carcinoma cell lines, demonstrating that tumor cell migration continues to be dependent on signals emanating from this pathway.

Cortactin is a c-src substrate associated with sites of dynamic actin assembly at the leading edge of migrating cells. We previously showed that cortactin binds to Arp2/3 complex, the essential molecular machine for nucleating actin filament assembly. In this study, we demonstrate that cortactin activates Arp2/3 complex based on direct visualization of filament networks and pyrene actin assays. Strikingly, cortactin potently inhibited the debranching of filament networks. When cortactin was added in combination with the active VCA fragment of N-WASp, they synergistically enhanced Arp2/3-induced actin filament branching. The N-terminal acidic and F-actin binding domains of cortactin were both necessary to activate Arp2/3 complex. These results support a model in which cortactin modulates actin filament dendritic nucleation by two mechanisms, (1) direct activation of Arp2/3 complex and (2) stabilization of newly generated filament branch points. By these mechanisms, cortactin may promote the formation and stabilization of the actin network that drives protrusion at the leading edge of migrating cells.

Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538-540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [(3)H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals.

We examined substance use in relationship to transmission risk behavior (unprotected insertive, UIAI, or receptive anal intercourse, URAI) between HIV-positive men who have sex with men (MSM) and their HIV-negative or unknown serostatus partners. Men who engaged in transmission risk behavior with casual partners were more likely than men who did not engage in such behavior to have used various substances. Users of certain drugs were specifically less likely to use condoms with HIV-negative or unknown status partners than users. Of men who drank alcohol, those who drank more frequently before or during sex engaged in significantly more UIAI with casual partners. Of men who used drugs, those who used more frequently before or during sex were more likely to engage in URAI with casual partners. In multivariate analyses, use of inhalants as well as drinking before or during sex predicted UIAI, while use of inhalants as well as noninjection drug use before or during sex predicted URAI. HIV prevention programs for HIV-positive MSM should focus on decreasing substance use and use specifically before or during sex. Developing prevention programs for substance-using MSM is critical to improve community health and decrease HIV transmissions.

Emerging research on methamphetamine use among gay men suggests that growth in the use of this drug could present serious problems for HIV/AIDS prevention within the gay community. This article summarizes current studies on the extent, role, and context of methamphetamine use among gay men and its relationship to high risk sexual behaviors related to HIV transmission. Methamphetamine is often used by gay men to initiate, enhance, and prolong sexual encounters. Use of the drug is, therefore, associated with particular environments where sexual contact among gay men is promoted, such as sex clubs and large "circuit" parties. Research with gay and bisexual men indicates that methamphetamine use is strongly associated with risky sexual behaviors that may transmit HIV. This relationship, coupled with emerging evidence that methamphetamine use is on the rise among gay men, suggests that the drug could exacerbate the HIV/AIDS epidemic among this community. The article offers recommendations for further research and suggestions for prevention programs regarding methamphetamine use by gay men.

An exploratory qualitative study of adult heterosexual men with haemophilia and HIV and women who were their sexual partners was conducted as formative research to better understand cognitive factors involved in behavioural intentions and practices which comprise HIV risk-reduction for sexual transmission. The study sought to generate hypotheses, uncover themes, and develop a broad perspective on possible determinants of behaviours related to HIV transmission risk reduction. Qualitative analysis of these data served as a basis for developing a subsequent quantitative, hypothesis-testing survey and an intervention. Face-to-face interviews were conducted with 23 single men and 28 married men with haemophilia and HIV infection, and 28 married women partners selected through stratified, purposeful sampling. The interviews identified beliefs, attitudes, and values underlying decisions regarding target behaviours related to preventing sexual transmission of HIV, including (1) using condoms consistently during vaginal intercourse and (2) talking to partners about risk reduction. The interviews elicited information about perceived advantages and disadvantages of performing each of the targeted behaviours, and factors that facilitate or prevent performing them. Qualitative analysis of coded responses yielded important themes regarding how choices are made about sexual activity and safer sex. Most notably, communication between partners (1) plays a direct, key role in facilitating condom use and (2) forms the basis for maintaining emotional intimacy in these relationships. The link between condom use and communicating about safer sex was viewed as pivotal in achieving HIV prevention for individuals in serodiscordant couples. Recommendations for risk reduction intervention development are discussed.

Focal adhesion kinase (FAK) is an important regulator of integrin signaling in adherent cells and accordingly its activity is significantly modulated during mitosis when cells detach from the extracellular matrix. During mitosis, FAK becomes heavily phosphorylated on serine residues concomitant with its inactivation and dephosphorylation on tyrosine. Little is known about the regulation of FAK activity by serine phosphorylation. In this report, we characterize two novel sites of serine phosphorylation within the C-terminal domain of FAK. Phosphorylation-specific antibodies directed to these sites and against two previously characterized sites of serine phosphorylation were used to study the regulated phosphorylation of FAK in unsynchronized and mitotic cells. Among the four major phosphorylation sites, designated pS1-pS4, phosphorylation of pS1 (Ser722) is unchanged in unsynchronized and mitotic cells. In contrast, pS3 and pS4 (Ser843 and Ser910) exhibit increased phosphorylation during mitosis. In vitro peptide binding experiments provide evidence that phosphorylation of pS1 (Ser722) may play a role in modulating FAK binding to the SH3 domain of the adapter protein p130(Cas).

The adapter molecule CAS is localized primarily within focal adhesions in fibroblasts. Because many of the cellular functions attributed to CAS are likely to be dependent on its presence in focal adhesions, this study was undertaken to identify regions of the protein that are involved in its localization. The SH3 domain of CAS, when expressed in isolation from the rest of the protein, was able to target to focal adhesions, whereas a variant containing a point mutation that rendered the SH3 domain unable to associate with FAK remained cytoplasmic. However, in the context of full-length CAS, this mutation did not prevent CAS localization to focal adhesions. Two other variants of CAS that contained deletions of either the SH3 domain alone, or the SH3 domain together with an adjoining proline-rich region, also retained the capacity to localize to focal adhesions. A second focal adhesion targeting region was mapped to the extreme carboxy terminus of CAS. The identification of this second focal adhesion targeting domain in CAS ascribes a previously unknown function to the highly conserved C terminus of CAS. The regulated targeting of CAS to focal adhesions by two independent domains may reflect the important role of CAS within this subcellular compartment.

Cellular signaling by small G-proteins is down-regulated by GTPase-activating proteins (GAPs), which increase the rate of GTP hydrolysis. The GTPase regulator associated with focal adhesion kinase (Graf) exhibits GAP activity toward the RhoA and Cdc42 GTPases, but is only weakly active toward the closely related Rac1. We determined the crystal structure of a 231-residue fragment of Graf (GrafGAP), a domain containing the GAP activity, at 2.4-A resolution. The structure clarifies the boundaries of the functional domain and yields insight to the mechanism of substrate recognition. Modeling its interaction with substrate suggested that a favorable interaction with Glu-95 of Cdc42 (Glu-97 of RhoA) would be absent with the corresponding Ala-95 of Rac1. Indeed, GrafGAP activity is diminished approximately 40-fold toward a Cdc42 E95A mutant, whereas a approximately 10-fold increase is observed for a Rac1 A95E mutant. The GrafGAP epitope that apparently interacts with Glu-95(Glu-97) contains Asn-225, which was recently found mutated in some myeloid leukemia patients. We conclude that position 95 of the GTPase is an important determinant for GrafGAP specificity in cellular function and tumor suppression.

Engagement of integrin receptors with extracellular ligands gives rise to the formation of complex multiprotein structures that link the ECM to the cytoplasmic actin cytoskeleton. These adhesive complexes are dynamic, often heterogeneous structures, varying in size and organization. In motile cells, sites of adhesion within filopodia and lamellipodia are relatively small and transient and are referred to as 'focal complexes,' whereas adhesions underlying the body of the cell and localized to the ends of actin stress fibers are referred to as 'focal adhesions'. Signal transduction through focal complexes and focal adhesions has been implicated in the regulation of a number of key cellular processes, including growth factor induced mitogenic signals, cell survival and cell locomotion. The formation and remodeling of focal contacts is a dynamic process under the regulation of protein tyrosine kinases and small GTPases of the Rho family. In this review, we consider the role of the focal complex associated protein tyrosine kinase, Focal Adhesion Kinase (FAK), in the regulation of cell movement with the emphasis on how FAK regulates the flow of signals from the ECM to the actin cytoskeleton.

Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its interaction with the cortical actin network: the fourth of six tandem repeats and the amino-terminal acidic region (NTA). Cortactin variants lacking either the fourth tandem repeat or the NTA failed to localize at the cell periphery. Tandem repeat four was necessary for cortactin to stably bind F-actin in vitro. The NTA region interacts directly with the Arp2/3 complex based on affinity chromatography, immunoprecipitation assays, and binding assays using purified components. Cortactin variants containing the NTA region were inefficient at promoting Arp2/3 actin nucleation activity. These data provide strong evidence that cortactin is specifically localized to sites of dynamic cortical actin assembly via simultaneous interaction with F-actin and the Arp2/3 complex. Cortactin interacts via its Src homology 3 (SH3) domain with ZO-1 and the SHANK family of postsynaptic density 95/dlg/ZO-1 homology (PDZ) domain-containing proteins, suggesting that cortactin contributes to the spatial organization of sites of actin polymerization coupled to selected cell surface transmembrane receptor complexes.

Status epilepticus is associated with sustained and elevated levels of cytosolic Ca(2+). To elucidate the mechanisms associated with changes of cytosolic Ca(2+) after status epilepticus, this study was initiated to evaluate the effect of pilocarpine-induced status epilepticus on Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in microsomes isolated from rat cortex, because the Ca(2+) uptake mechanism plays a major role in regulating intracellular Ca(2+) levels. The data demonstrated that the initial rate and overall Ca(2+) uptake in microsomes from pilocarpine treated animals were significantly inhibited compared with those in microsomes from saline-treated control animals. It was also shown that the inhibition of Ca(2+) uptake caused by status epilepticus was not an artifact of increased Ca(2+) release from microsomes, selective isolation of damaged microsomes from the homogenate, or decreased Mg(2+)/Ca(2+) ATPase protein in the microsomes. Pretreatment with the NMDA antagonist dizocilpine maleate blocked status epilepticus-induced inhibition of the initial rate and overall Ca(2+) uptake. The data suggest that inhibition of microsomal Mg(2+)/Ca(2+) ATPase Ca(2+) uptake is involved in NMDA-dependent deregulation of cytosolic Ca(2+) homeostasis associated with status epilepticus.

Purpose: To determine the factors associated with the adoption or maintenance of consistent safer sexual behaviors among human immunodeficiency virus-positive adolescents and young adults with hemophilia., Methods: One hundred eleven adolescents at 10 hemophilia care sites participated in an intervention program designed to increase safer sexual behaviors (abstinence, condom use, or nonpenetrative behavior). The theory-based intervention spanned 1 year. Adolescents attended individual sessions, small group activities, and an intensive group retreat., Results: Patients who maintained or improved safer sexual behaviors were compared with those who relapsed or did not improve. Logistic regression analyses found that improvement and maintenance of safer sexual behavior were significantly associated with perceived peer support for outercourse (odds ratio [OR]: 5.47; confidence interval [CI]: 1.4-20.8), perceived peer support for abstinence (OR: 5.08; CI: 1.2-20.1), and decreased general emotional distress (OR: 4.65; CI: 1.04-20.6). Perceived health status and previous sexual behavior were unrelated to change in safer sexual behavior., Conclusions: These longitudinal data indicate that improvement and maintenance of safer sexual behavior among adolescents during an intervention is strongly associated with perceptions of peer support for safer sex and lesser degrees of emotional distress. Programs for human immunodeficiency virus-infected adolescents may require developmentally appropriate social and psychological approaches to impact peer norms and emotional well-being.

To assess the differential effects of the perceived benefits and costs associated with both condom use and unprotected sex on sexual risk behaviors, data were collected from 704 ethnically diverse male and female sexually experienced late adolescent college students (aged 17-25). Perceived benefits and costs for condom use and perceived benefits and costs for unprotected sex were measured separately through an anonymous self-report survey. In addition, participants completed measures of self-efficacy for practicing safer sex and temptation for unsafe sex in various situations, and three measures of sexual risk-taking (stage of change for condom use, consistency of condom use during the past month, and whether or not a condom was used for the last act of intercourse). Univariate analyses indicated that benefits and costs of condom use, benefits of unprotected sex, self-efficacy and situational temptation were all related to sexual risk-taking. Gender differences were identified, with females reporting more benefits of condom use and costs of unprotected sex, fewer benefits of unprotected sex and costs of condom use, greater self-efficacy for practicing safer sex, and less situational temptation for unsafe sex. Multivariate analyses indicated that sexual risk behaviors were most related to situational temptation, self-efficacy for safer sex, and perceived benefits of unprotected sex. The results suggest that, among late adolescents, perceived benefits of the unhealthy behavior (unprotected sex) were better determinants of sexual risk-taking than were perceived benefits (or costs) associated with the healthy behavior (condom use). Perceived costs associated with unprotected sex were unrelated to sexual behaviors. These findings support previous work identifying adolescents as more driven by their perceptions of the positive benefits associated with risky behaviors, rather than knowledge of the costs or dangers involved in risk-taking., (Copyright 2000 The Association for Professionals in Services for Adolescents.)

A variety of agonists including phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Here we report that signals provided by extracellular matrix proteins (ECM) augment the PE-induced hypertrophic response of cardiomyocytes and provide evidence that ECM-dependent signaling is mediated in part by the protein tyrosine kinase, focal adhesion kinase (FAK). Addition of PE to cultured neonatal cardiomyocytes stimulated sarcomeric organization, increased cell size, and induced atrial natriuretic factor in cardiomyocytes plated on the ECM protein laminin or fibronectin. In contrast, cardiomyocytes plated on the non-adhesive substrate gelatin exhibited a reduced capacity to undergo these PE-stimulated hypertrophic changes. In cardiomyocytes cultured on ECM, PE stimulated a rapid increase in tyrosine phosphorylation of focal adhesion proteins including FAK, paxillin, and p130 Crk-associated substrate and subsequent formation of peripheral focal complexes. Inhibition of the PE-induced hypertrophic response by genistein and herbimycin-A indicated a requirement for protein tyrosine kinases in PE signaling. To determine whether activation of FAK is required for PE-induced hypertrophy, a dominant-interfering mutant form of FAK, termed FRNK (FAK-related non-kinase), was ectopically expressed in cardiomyocytes using a replication-defective adenovirus expression system. FRNK expression attenuated PE-stimulated hypertrophy as assessed by cell size, sarcomeric organization, and induction of atrial natriuretic factor. These data indicate that the signal transduction pathways leading to cardiomyocyte hypertrophy are strongly influenced by and/or dependent upon an integrin-mediated signaling process requiring FAK.

A theory-based HIV risk-reduction intervention was developed for HIV-positive men with haemophilia and their HIV-negative female romantic partners. The intervention was based on Prochaska and DiClemente's Transtheoretical Model which describes behaviour change as an incremental, stage-based process. The intervention targeted both communication about safer sex and safer sex behaviours (consistent condom use or abstinence from vaginal intercourse). A total of 255 males and 158 females from six funded haemophilia treatment centres or patient organizations (and 27 associated subsites) participated in the study. Baseline and follow-up (15 months after baseline) measures were administered to assess safer sexual behaviours, communication about safer sex and condom self-efficacy. A quasi-experimental, repeated measures design was utilized to compare two naturally occurring groups; those who received the full intervention package and those who received incomplete or no intervention components. Significant intervention effects for safer sex behaviours, communication about safer sex and condom self-efficacy were identified for the male participants, with those receiving the full intervention package demonstrating better outcomes at follow-up. Women who received the full intervention package were more likely to report the use of a condom by their male partner during the last act of vaginal intercourse.

The likelihood of local control after radiation therapy may be improved by increasing total dose or decreasing overall time. The probability of late complications increases with dose per fraction. Altered fractionation techniques usually employ two or more fractions per day using a dose per fraction that is similar or less than that employed in conventional fractionation. Altered fractionation may be broadly classified as hyperfractionation or accelerated fractionation. Data suggest that altered fractionation schedules may improve local control (and to a lesser extent, survival) compared with conventional irradiation.

Purpose: To review treatment and outcomes in 62 patients with clinical and/or gross evidence of perineural invasion from skin cancer of the head and neck., Methods and Materials: Sixty-two patients received radiotherapy at the University of Florida as part or all of their treatment between January 1965 and April 1995. All patients had clinical signs and symptoms of perineural involvement and/or documentation of tumor extending to grossly involve nerve(s). Twenty-one patients underwent therapy for previously untreated lesions, including 12 who received radiotherapy alone and nine who had surgery with postoperative radiotherapy. Forty-one patients underwent therapy for recurrent lesions, including 18 treated with radiotherapy alone and 23 who received preoperative or postoperative radiotherapy., Results: Factors on multivariate analysis that predicted local control included patient age, previously untreated vs. recurrent lesions, presence of clinical symptoms, and extent of radiotherapy fields. Recurrence patterns were predominantly local; 26 of 31 patients (84%) who developed local recurrence after treatment had recurrent cancer limited to the primary site., Conclusions: Many patients with skin cancer and symptomatic perineural invasion have disease that is incompletely resectable. Approximately half these patients will be cured with aggressive irradiation alone or combined with surgery. Age, prior treatment, and clinical symptoms influence the likelihood of cure.

Purpose: To determine if follow-up computed tomography (CT) after definitive radiation therapy for laryngeal or hypopharyngeal (laryngopharyngeal) carcinoma allows the detection of local failure earlier than clinical examination alone., Materials and Methods: Pre- and post-radiation therapy follow-up CT scans in 66 patients were reviewed retrospectively. All patients underwent definitive hyperfractionated radiation therapy and were followed up clinically for at least 2 years after its completion. Post-radiation therapy CT scans (N = 153) were evaluated for posttreatment changes with a three-point score: A score of 1 represented expected posttreatment changes; 2, focal mass with a maximal diameter of less than 1 cm and/or asymmetric obliteration of laryngeal tissue planes; or 3, focal mass with a maximal diameter equal to or greater than 1 cm or estimated tumor volume reduction of less than 50%. All patients underwent the first posttreatment CT study 1-6 months after therapy. New or progressive laryngeal cartilage changes were noted. The clinical impression of the larynx at the time of each follow-up CT scan was also recorded., Results: In 12 of 29 (41%) patients with treatment failure at the primary site, follow-up CT scans were definite for local failure (score, 3) a mean of 5.5 months (median, 3.5 months; range, 1-17 months) before clinical examination results., Conclusion: In many patients, follow-up CT shows local failure earlier than does clinical examination alone.

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60(c-src) or p59(fyn) results in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60(c-src) or p59(fyn) to induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60(c-src) is hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60(c-src) from a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60(c-src) to focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.

Epidermal growth factor stimulates migration of a number of cell types, yet the signaling pathways that regulate epidermal growth factor-stimulated migration are poorly defined. In this report, we employ a transient transfection migration assay to assess the role of components of the Ras-mitogen-activated protein (MAP) kinase signaling pathway in epidermal growth factor-stimulated chemotaxis of rat embryo fibroblasts. Expression of dominant negative Ras blocks epidermal growth factor-mediated chemotaxis, while constitutively active Ras has no effect on chemokinesis or chemotaxis. PD98059 and U0126, inhibitors of MAP kinase kinase (MEK) activity, decreased epidermal growth factor-stimulated migration, while kinase-defective MEK1, an inhibitor of MAP kinase activation, enhanced migration. To understand the paradoxical effects of these molecules on epidermal growth factor-induced migration, we examined the role of c-Raf on migration. Expression of either wild type c-Raf or the catalytic domain of c-Raf effectively inhibited epidermal growth factor-stimulated cell migration. We suggest that, whereas Ras activity is necessary to promote epidermal growth factor-stimulated migration, sustained activation of c-Raf may be important in down-regulating migratory signaling pathways triggered by epidermal growth factor receptor activation. Further, activation of c-Raf upon inhibition of the MEK-MAP kinase pathway may contribute to the inhibition of cell migration observed with pharmacological MEK inhibitors.

Affinity chromatography, employing the extracellular domain of the Sea receptor, was used to enrich Sea-binding proteins from chicken serum. One isolated protein bound both a Sea-immunoglobulin fusion protein and an antisera raised against murine macrophage stimulating protein. Amino-terminal sequencing of the dual-reactive protein yielded sequences which were identical to the predicted alpha and beta subunits of chicken macrophage stimulating protein. The partially purified chicken macrophage stimulating protein caused autophosphorylation of the Sea receptor. Previous work showed that recombinant expression of fully activatible human or mouse macrophage stimulating protein required a specific Cys to Ala substitution (Wahl, R. C., Costigan, V. J., Batac, J. P., Chen, K., Cam, L., Courchesne, P. L., Patterson, S. D. Zhang, K., and Pacifici, R. E. (1997) J. Biol. Chem. 272, 1-4). Therefore, we expressed both the wild type and the specific Cys to Ala form of chicken macrophage stimulating protein as recombinant proteins. After proteolytic activation, only conditioned media from COS cells transfected with the C665A chicken macrophage stimulating protein, but not from wild type chicken macrophage-stimulating protein, or control vector, was detected by the Sea-immunoglobulin fusion protein in Western blotting experiments. Conditioned media containing the C665A chicken macrophage-stimulating protein readily caused Sea phosphorylation, while conditioned media containing the wild type chicken macrophage-stimulating protein was only effective at inducing receptor phosphorylation at high concentrations. In addition to receptor phosphorylation, the C665A chicken macrophage-stimulating protein induced phosphorylation of Shc, Erk1, and Erk 2. We conclude that macrophage-stimulating protein is a ligand of the Sea receptor protein-tyrosine kinase.

Focal adhesion kinase (FAK) has been implicated in cellular processes that control cell adhesion, migration, cell cycle progression, and apoptosis. FRNK (FAK-related nonkinase) is the autonomously expressed, noncatalytic C-terminal portion of FAK. When ectopically expressed in cells, FRNK has been shown to act as a negative regulator of FAK activity, inhibiting cell spreading, migration, and cell cycle progression. The mechanisms that regulate FRNK expression during embryonic development and the functional role of FRNK in normal cell homeostasis remain poorly understood. Herein we show that FRNK expression in chicken cells is directed by an alternative promoter residing within an intron of FAK, positioned 3' of the exon encoding sequences for the catalytic domain and 5' of the exon encoding sequences for the C-terminal domain of FAK (e.g., FRNK). Using probes specific for FRNK, we show that FRNK expression occurs early in chicken embryogenesis, being readily detected at day 3, 6, or 9. Late in embryogenesis, at day 18, FRNK is expressed in a tissue-specific manner, predominately in lung and intestine cells. Western blot analysis of mouse tissues with a FAK-specific antibody revealed the expression of FRNK in the mouse lung. Reverse transcriptase PCR analysis of mouse lung RNA revealed the presence of spliced FRNK mRNAs containing 5' untranslated sequences derived from a positionally conserved exon present in the mouse genome. FAK is the first example of a tyrosine kinase regulated by a domain under the control of an alternative intronic promoter. It is also the first example of a focal adhesion-associated protein regulated by such a mechanism and thus represents a novel means for the modulation of cell adhesion signaling.

Purpose: To determine if pre-radiotherapy (RT) and/or post-radiotherapy computed tomography (CT) can predict local failure in patients with laryngeal carcinoma treated with definitive RT., Methods and Materials: The pre- and post-RT CT examinations of 59 patients (T3 glottic carcinoma [n = 30] and T1-T4 supraglottic carcinoma [n = 29]) were reviewed. For each patient, the first post-RT CT study between 1 and 6 months after irradiation was used. All patients were treated with definitive hyperfractionated twice-daily continuous-course irradiation to a total dose of 6,720-7,920 cGy, and followed-up clinically for at least 2 years after completion of RT. Local control was defined as absence of primary tumor recurrence and a functioning larynx. On the pre-treatment CT study, each tumor was assigned a high-or low-risk profile for local failure after RT. The post-RT CT examinations were evaluated for post-treatment changes using a three-point post-RT CT-score: 1 = expected post-RT changes; 2 = focal mass with a maximal diameter of < 1 cm and/or asymmetric obliteration of laryngeal tissue planes; 3 = focal mass with a maximal diameter of > 1 cm, or < 50% estimated tumor volume reduction., Results: The local control rates at 2 years post-RT based on pre-treatment CT evaluation were 88% for low pre-treatment risk profile patients (95% CI: 66-96%) and 34% (95% CI: 19-50%) for high pre-treatment risk profile patients (risk ratio 6.583; 95% CI: 2.265-9.129;p = 0.0001). Based on post-treatment CT, the local control rates at 2 years post-RT were 94% for score 1, 67% for score 2, and 10% for score 3 (risk ratio 4.760; 95% CI: 2.278-9.950 p = 0.0001). Post-RT CT scores added significant information to the pre-treatment risk profiles on prognosis., Conclusions: Pre-treatment CT risk profiles, as well as post-RT CT evaluation can identify patients, irradiated for laryngeal carcinomas, at high risk for developing local failure. When the post-RT CT score is available, it proves to be an even better prognosticator than the pre-treatment CT-risk profile.

Ischemia is associated with a loss of cytosolic calcium homeostasis. Intracellular stores, particularly in endoplasmic reticulum, are critical for the maintenance of calcium homeostasis. Recent studies have shown that ischemia significantly inhibited microsomal calcium uptake mediated by Mg(2+)/Ca(2+) ATPase, the major mechanism of endoplasmic reticulum calcium sequestration. This study was initiated to determine whether the decreased calcium uptake caused by ischemia was the result of inhibition of Mg(2+)/Ca(2+) ATPase activity or an uncoupling of calcium uptake from ATP hydrolysis. The microsomal Mg(2+)/Ca(2+) ATPase specific inhibitor thapsigargin partially inhibited ATPase activity and completely inhibited calcium uptake. ATPase inhibited by thapsigargin was considered microsomal Mg(2+)/Ca(2+) ATPase. Ischemia from 5 to 60 min had no significant effect on thapsigargin sensitive ATPase activity. However, under identical conditions, increasing ischemia from 5 to 60 min significantly inhibited microsomal calcium uptake. Comparing calcium uptake to ATP hydrolysis as ischemia increased from 5 to 60 min revealed that the coupling ratio of calcium molecules sequestered to ATP molecules hydrolyzed became significantly decreased. The results demonstrated that the effect of ischemia on microsomal calcium uptake was mediated by an uncoupling of calcium transport from Mg(2+)/Ca(2+) ATPase activity., (Copyright 1999 Elsevier Science B.V.)

Bone resorption is initiated by osteoclast attachment to the mineralized matrix, cytoskeletal reorganization, cellular polarization, and the formation of the sealing zone. The present study examines the interaction between PYK2 and p130(Cas) (Crk-associated substrate), suggested to be part of the signaling pathway initiated by osteoclast adhesion. Using murine osteoclast-like cells (OCLs) and their mononuclear precursors (pOCs), generated in a co-culture of bone marrow and osteoblastic MB1.8 cells, we show that: 1) p130(Cas) is tyrosine-phosphorylated upon adhesion of pOCs to vitronectin or ligation of beta3 integrins; 2) p130(Cas) colocalizes with PYK2 and the cytoskeletal proteins F-actin, vinculin, and paxillin in the podosomal-rich ring-like structures of OCLs plated on glass and in the sealing zone in actively resorbing OCLs on bone; 3) p130(Cas) and PYK2 form a stable complex in pOCs, independent of tyrosine phosphorylation of either molecule, and this complex is present in Src (-/-) OCLs, in which neither protein is phosphorylated or associated with the osteoclast adhesion structure; 4) the association of p130(Cas) and PYK2 is mediated by the SH3 domain of p130(Cas) and the C-terminal domain of PYK2. These findings suggest that p130(Cas) and its association with PYK2 may play an important role in the adhesion-dependent signaling that leads to cytoskeletal reorganization and formation of the sealing zone during osteoclast activation.

Purpose: To address outcomes in clinically asymptomatic patients in whom the unexpected finding of microscopic perineural invasion is noted at the time of surgery., Methods and Materials: The 35 patients included in this study had skin cancers of the head and neck treated with curative intent between January 1965 and April 1995 at the University of Florida. All patients were without clinical or radiographic evidence of perineural invasion but, at the time of biopsy or surgical excision, had the incidental finding of microscopic perineural invasion. Definitive therapy consisted of radiotherapy alone after lesion biopsy (3 patients) or surgical excision preceded (2 patients) or followed (30 patients) by radiotherapy. All patients had follow-up for at least 1 year, 13 patients (37%) had follow-up for at least 5 years., Results: The 5-year local control rate was 78%. The 5-year local control rate for the few patients treated with radiotherapy alone was statistically similar to that for patients treated with surgery and radiotherapy (100% vs. 77%, p = 0.4). Multivariate analysis for factors affecting local control included sex, histology, age, treatment group, clinical T stage, initial histologic differentiation, and previously untreated vs. recurrent tumors, none of which was found to be significant., Conclusions: Both surgery plus radiotherapy and radiotherapy alone provide a relatively high rate of local control for patients with incidentally discovered perineural invasion secondary to skin cancer.

Between 1964 and 1996, 123 patients were treated for T4 oropharyngeal carcinoma; 93 were treated with radiation therapy alone; 30 were treated with induction chemotherapy and radiation therapy. Patients who received induction chemotherapy and radiation therapy were treated between 1985 and 1996; during this time 39 patients were treated with radiation therapy alone. Five-year local control rates for patients undergoing chemotherapy and radiation therapy, radiation therapy alone (all patients), and radiation therapy alone (patients treated since September 1985) were 63%, 38%, and 48%, respectively. The five-year rates of freedom from distant metastasis were 87%, 73%, and 76%, respectively. The five-year actuarial cause-specific survival rates were 58%, 27%, and 37%, respectively, while the five-year absolute survival rates were 42%, 17%, and 23%, respectively. Improvements in local control and freedom from distant metastasis in those receiving chemotherapy were not statistically significant, while the improvements in cause-specific survival and absolute survival were significant at the P < or = 0.05 level. Induction chemotherapy may improve the cure rate for patients with T4 oropharyngeal carcinoma. Although encouraging, these data are nonrandomized and should be interpreted with caution.

Graf, the GTPase regulator associated with focal adhesion kinase was previously shown to have GAP activity for &Rgr; A and Cdc42 in vitro (Hildebrand et al 1996 Mol. Cell Biol. 16: 3169-3178). In this study we sought to determine whether Graf acted at the level of Cdc42, Rho, or both in vivo and whether Graf was a signal terminator or transducer for these proteins. Microinjection of Graf cDNA into subconfluent Swiss 3T3 cells (in the presence of serum) has marked effects on cell shape and actin localization. Graf expression causes clearing of stress fibers followed by formation of long actin based filopodial-like extensions. Similar phenotypes were observed following injection of the Rho-inhibitor, C3 into these cells. The Graf response was dependent on GAP activity, since injection of Graf cDNA containing point mutations in the GAP domain (R236Q or N351V) which block enzymatic activity, does not confer this phenotype. Injection of Graf into Swiss 3T3 cells in which Rho has been down-regulated by serum starvation has no effect on cell morphology. Using this system, we demonstrate that Graf blocks sphingosine-1-phosphate (SPP) stimulated (Rho-mediated) stress fiber formation. Conversely, Graf expression does not inhibit bradykinin stimulated (Cdc42-mediated) filopodial extensions. These data indicate that Graf is a GAP for Rho in vivo. To further substantiate these results we examined the effect of Graf over-expression on Rho-mediated neurite retraction in nerve growth factor (NGF)-differentiated PC12 cells. In PC12 cells, which express relatively high levels of endogenous Graf, overexpression of Graf (but not Graf containing the R236Q mutation) enhances SPP-induced neurite retraction. These data indicate the possibility that Graf may be an effector for Rho in certain cell types.