Precision medicine for suicidality: from universality to subtypes and personalization.

Suicide remains a clear, present and increasing public health problem, despite being a potentially preventable tragedy. Its incidence is particularly high in people with overt or un(der)diagnosed psychiatric disorders. Objective and precise identification of individuals at risk, ways of monitoring response to treatments and novel preventive therapeutics need to be discovered, employed and widely deployed. We sought to investigate whether blood gene expression biomarkers for suicide (that is, a 'liquid biopsy' approach) can be identified that are more universal in nature, working across psychiatric diagnoses and genders, using larger cohorts than in previous studies. Such markers may reflect and/or be a proxy for the core biology of suicide. We were successful in this endeavor, using a comprehensive stepwise approach, leading to a wealth of findings. Steps 1, 2 and 3 were discovery, prioritization and validation for tracking suicidality, resulting in a Top Dozen list of candidate biomarkers comprising the top biomarkers from each step, as well as a larger list of 148 candidate biomarkers that survived Bonferroni correction in the validation step. Step 4 was testing the Top Dozen list and Bonferroni biomarker list for predictive ability for suicidal ideation (SI) and for future hospitalizations for suicidality in independent cohorts, leading to the identification of completely novel predictive biomarkers (such as CLN5 and AK2), as well as reinforcement of ours and others previous findings in the field (such as SLC4A4 and SKA2). Additionally, we examined whether subtypes of suicidality can be identified based on mental state at the time of high SI and identified four potential subtypes: high anxiety, low mood, combined and non-affective (psychotic). Such subtypes may delineate groups of individuals that are more homogenous in terms of suicidality biology and behavior. We also studied a more personalized approach, by psychiatric diagnosis and gender, with a focus on bipolar males, the highest risk group. Such a personalized approach may be more sensitive to gender differences and to the impact of psychiatric co-morbidities and medications. We compared testing the universal biomarkers in everybody versus testing by subtypes versus personalized by gender and diagnosis, and show that the subtype and personalized approaches permit enhanced precision of predictions for different universal biomarkers. In particular, LHFP appears to be a strong predictor for suicidality in males with depression. We also directly examined whether biomarkers discovered using male bipolars only are better predictors in a male bipolar independent cohort than universal biomarkers and show evidence for a possible advantage of personalization. We identified completely novel biomarkers (such as SPTBN1 and C7orf73), and reinforced previously known biomarkers (such as PTEN and SAT1). For diagnostic ability testing purposes, we also examined as predictors phenotypic measures as apps (for suicide risk (CFI-S, Convergent Functional Information for Suicidality) and for anxiety and mood (SASS, Simplified Affective State Scale)) by themselves, as well as in combination with the top biomarkers (the combination being our a priori primary endpoint), to provide context and enhance precision of predictions. We obtained area under the curves of 90% for SI and 77% for future hospitalizations in independent cohorts. Step 5 was to look for mechanistic understanding, starting with examining evidence for the Top Dozen and Bonferroni biomarkers for involvement in other psychiatric and non-psychiatric disorders, as a mechanism for biological predisposition and vulnerability. The biomarkers we identified also provide a window towards understanding the biology of suicide, implicating biological pathways related to neurogenesis, programmed cell death and insulin signaling from the universal biomarkers, as well as mTOR signaling from the male bipolar biomarkers. In particular, HTR2A increase coupled with ARRB1 and GSK3B decreases in expression in suicidality may provide a synergistic mechanistical corrective target, as do SLC4A4 increase coupled with AHCYL1 and AHCYL2 decrease. Step 6 was to move beyond diagnostics and mechanistical risk assessment, towards providing a foundation for personalized therapeutics. Items scored positive in the CFI-S and subtypes identified by SASS in different individuals provide targets for personalized (psycho)therapy. Some individual biomarkers are targets of existing drugs used to treat mood disorders and suicidality (lithium, clozapine and omega-3 fatty acids), providing a means toward pharmacogenomics stratification of patients and monitoring of response to treatment. Such biomarkers merit evaluation in clinical trials. Bioinformatics drug repurposing analyses with the gene expression biosignatures of the Top Dozen and Bonferroni-validated universal biomarkers identified novel potential therapeutics for suicidality, such as ebselen (a lithium mimetic), piracetam (a nootropic), chlorogenic acid (a polyphenol) and metformin (an antidiabetic and possible longevity promoting drug). Finally, based on the totality of our data and of the evidence in the field to date, a convergent functional evidence score prioritizing biomarkers that have all around evidence (track suicidality, predict it, are reflective of biological predisposition and are potential drug targets) brought to the fore APOE and IL6 from among the universal biomarkers, suggesting an inflammatory/accelerated aging component that may be a targetable common denominator.

Towards understanding and predicting suicidality in women: biomarkers and clinical risk assessment.

Women are under-represented in research on suicidality to date. Although women have a lower rate of suicide completion than men, due in part to the less-violent methods used, they have a higher rate of suicide attempts. Our group has previously identified genomic (blood gene expression biomarkers) and clinical information (apps) predictors for suicidality in men. We now describe pilot studies in women. We used a powerful within-participant discovery approach to identify genes that change in expression between no suicidal ideation (no SI) and high suicidal ideation (high SI) states (n=12 participants out of a cohort of 51 women psychiatric participants followed longitudinally, with diagnoses of bipolar disorder, depression, schizoaffective disorder and schizophrenia). We then used a Convergent Functional Genomics (CFG) approach to prioritize the candidate biomarkers identified in the discovery step by using all the prior evidence in the field. Next, we validated for suicidal behavior the top-ranked biomarkers for SI, in a demographically matched cohort of women suicide completers from the coroner's office (n=6), by assessing which markers were stepwise changed from no SI to high SI to suicide completers. We then tested the 50 biomarkers that survived Bonferroni correction in the validation step, as well as top increased and decreased biomarkers from the discovery and prioritization steps, in a completely independent test cohort of women psychiatric disorder participants for prediction of SI (n=33) and in a future follow-up cohort of psychiatric disorder participants for prediction of psychiatric hospitalizations due to suicidality (n=24). Additionally, we examined how two clinical instruments in the form of apps, Convergent Functional Information for Suicidality (CFI-S) and Simplified Affective State Scale (SASS), previously tested in men, perform in women. The top CFI-S item distinguishing high SI from no SI states was the chronic stress of social isolation. We then showed how the clinical information apps combined with the 50 validated biomarkers into a broad predictor (UP-Suicide), our apriori primary end point, predicts suicidality in women. UP-Suicide had a receiver-operating characteristic (ROC) area under the curve (AUC) of 82% for predicting SI and an AUC of 78% for predicting future hospitalizations for suicidality. Some of the individual components of the UP-Suicide showed even better results. SASS had an AUC of 81% for predicting SI, CFI-S had an AUC of 84% and the combination of the two apps had an AUC of 87%. The top biomarker from our sequential discovery, prioritization and validation steps, BCL2, predicted future hospitalizations due to suicidality with an AUC of 89%, and the panel of 50 validated biomarkers (BioM-50) predicted future hospitalizations due to suicidality with an AUC of 94%. The best overall single blood biomarker for predictions was PIK3C3 with an AUC of 65% for SI and an AUC of 90% for future hospitalizations. Finally, we sought to understand the biology of the biomarkers. BCL2 and GSK3B, the top CFG scoring validated biomarkers, as well as PIK3C3, have anti-apoptotic and neurotrophic effects, are decreased in expression in suicidality and are known targets of the anti-suicidal mood stabilizer drug lithium, which increases their expression and/or activity. Circadian clock genes were overrepresented among the top markers. Notably, PER1, increased in expression in suicidality, had an AUC of 84% for predicting future hospitalizations, and CSNK1A1, decreased in expression, had an AUC of 96% for predicting future hospitalizations. Circadian clock abnormalities are related to mood disorder, and sleep abnormalities have been implicated in suicide. Docosahexaenoic acid signaling was one of the top biological pathways overrepresented in validated biomarkers, which is of interest given the potential therapeutic and prophylactic benefits of omega-3 fatty acids. Some of the top biomarkers from the current work in women showed co-directionality of change in expression with our previous work in men, whereas others had changes in opposite directions, underlying the issue of biological context and differences in suicidality between the two genders. With this study, we begin to shed much needed light in the area of female suicidality, identify useful objective predictors and help understand gender commonalities and differences. During the conduct of the study, one participant committed suicide. In retrospect, when the analyses were completed, her UP-Suicide risk prediction score was at the 100 percentile of all participants tested.

Understanding and predicting suicidality using a combined genomic and clinical risk assessment approach.

Worldwide, one person dies every 40 seconds by suicide, a potentially preventable tragedy. A limiting step in our ability to intervene is the lack of objective, reliable predictors. We have previously provided proof of principle for the use of blood gene expression biomarkers to predict future hospitalizations due to suicidality, in male bipolar disorder participants. We now generalize the discovery, prioritization, validation, and testing of such markers across major psychiatric disorders (bipolar disorder, major depressive disorder, schizoaffective disorder, and schizophrenia) in male participants, to understand commonalities and differences. We used a powerful within-participant discovery approach to identify genes that change in expression between no suicidal ideation and high suicidal ideation states (n=37 participants out of a cohort of 217 psychiatric participants followed longitudinally). We then used a convergent functional genomics (CFG) approach with existing prior evidence in the field to prioritize the candidate biomarkers identified in the discovery step. Next, we validated the top biomarkers from the prioritization step for relevance to suicidal behavior, in a demographically matched cohort of suicide completers from the coroner's office (n=26). The biomarkers for suicidal ideation only are enriched for genes involved in neuronal connectivity and schizophrenia, the biomarkers also validated for suicidal behavior are enriched for genes involved in neuronal activity and mood. The 76 biomarkers that survived Bonferroni correction after validation for suicidal behavior map to biological pathways involved in immune and inflammatory response, mTOR signaling and growth factor regulation. mTOR signaling is necessary for the effects of the rapid-acting antidepressant agent ketamine, providing a novel biological rationale for its possible use in treating acute suicidality. Similarly, MAOB, a target of antidepressant inhibitors, was one of the increased biomarkers for suicidality. We also identified other potential therapeutic targets or biomarkers for drugs known to mitigate suicidality, such as omega-3 fatty acids, lithium and clozapine. Overall, 14% of the top candidate biomarkers also had evidence for involvement in psychological stress response, and 19% for involvement in programmed cell death/cellular suicide (apoptosis). It may be that in the face of adversity (stress), death mechanisms are turned on at a cellular (apoptosis) and organismal level. Finally, we tested the top increased and decreased biomarkers from the discovery for suicidal ideation (CADM1, CLIP4, DTNA, KIF2C), prioritization with CFG for prior evidence (SAT1, SKA2, SLC4A4), and validation for behavior in suicide completers (IL6, MBP, JUN, KLHDC3) steps in a completely independent test cohort of psychiatric participants for prediction of suicidal ideation (n=108), and in a future follow-up cohort of psychiatric participants (n=157) for prediction of psychiatric hospitalizations due to suicidality. The best individual biomarker across psychiatric diagnoses for predicting suicidal ideation was SLC4A4, with a receiver operating characteristic (ROC) area under the curve (AUC) of 72%. For bipolar disorder in particular, SLC4A4 predicted suicidal ideation with an AUC of 93%, and future hospitalizations with an AUC of 70%. SLC4A4 is involved in brain extracellular space pH regulation. Brain pH has been implicated in the pathophysiology of acute panic attacks. We also describe two new clinical information apps, one for affective state (simplified affective state scale, SASS) and one for suicide risk factors (Convergent Functional Information for Suicide, CFI-S), and how well they predict suicidal ideation across psychiatric diagnoses (AUC of 85% for SASS, AUC of 89% for CFI-S). We hypothesized a priori, based on our previous work, that the integration of the top biomarkers and the clinical information into a universal predictive measure (UP-Suicide) would show broad-spectrum predictive ability across psychiatric diagnoses. Indeed, the UP-Suicide was able to predict suicidal ideation across psychiatric diagnoses with an AUC of 92%. For bipolar disorder, it predicted suicidal ideation with an AUC of 98%, and future hospitalizations with an AUC of 94%. Of note, both types of tests we developed (blood biomarkers and clinical information apps) do not require asking the individual assessed if they have thoughts of suicide, as individuals who are truly suicidal often do not share that information with clinicians. We propose that the widespread use of such risk prediction tests as part of routine or targeted healthcare assessments will lead to early disease interception followed by preventive lifestyle modifications and proactive treatment.

Evidence against dopamine D1/D2 receptor heteromers.

Hetero-oligomers of G-protein-coupled receptors have become the subject of intense investigation, because their purported potential to manifest signaling and pharmacological properties that differ from the component receptors makes them highly attractive for the development of more selective pharmacological treatments. In particular, dopamine D1 and D2 receptors have been proposed to form hetero-oligomers that couple to Gαq proteins, and SKF83959 has been proposed to act as a biased agonist that selectively engages these receptor complexes to activate Gαq and thus phospholipase C. D1/D2 heteromers have been proposed as relevant to the pathophysiology and treatment of depression and schizophrenia. We used in vitro bioluminescence resonance energy transfer, ex vivo analyses of receptor localization and proximity in brain slices, and behavioral assays in mice to characterize signaling from these putative dimers/oligomers. We were unable to detect Gαq or Gα11 protein coupling to homomers or heteromers of D1 or D2 receptors using a variety of biosensors. SKF83959-induced locomotor and grooming behaviors were eliminated in D1 receptor knockout (KO) mice, verifying a key role for D1-like receptor activation. In contrast, SKF83959-induced motor responses were intact in D2 receptor and Gαq KO mice, as well as in knock-in mice expressing a mutant Ala(286)-CaMKIIα that cannot autophosphorylate to become active. Moreover, we found that, in the shell of the nucleus accumbens, even in neurons in which D1 and D2 receptor promoters are both active, the receptor proteins are segregated and do not form complexes. These data are not compatible with SKF83959 signaling through Gαq or through a D1/D2 heteromer and challenge the existence of such a signaling complex in the adult animals that we used for our studies.

Cigarette smoke and human plasma lycopene depletion.

It is known that smokers have a higher risk of developing cardiovascular disease and lung cancer. Plasma carotenoid concentrations in smokers are generally lower than in non-smokers and this may be due to modifications in diet or a direct or indirect action of cigarette smoke on carotenoids in the plasma. Recently it was reported that reactive nitrogen species derived from cigarette smoke could diffuse across the lung alveolar cell wall into the plasma. Such species may modify circulating low density lipoprotein (LDL) and in the process reduce circulating carotenoid concentrations. In an effort to address this rational we have treated lycopene solutions, human plasma and isolated LDL with cigarette smoke and monitored all-(E)-lycopene, 5(Z)-lycopene and beta-carotene depletion. In plasma, the depletion of all-(E)-lycopene (15.0+/-11.0%, n=10) was greater than 5(Z)-lycopene (10.4+/-9.6%) or beta-carotene (12.4+/-10.5%). In LDL, both all-(E)- and 5(Z)-lycopene were more susceptible than beta-carotene (20.8+/-11.8%, 15.4+/-11.5% and 11.5+/-12.5%, n=3 respectively). The effects have been compared with Sin-1 reactions and isomerization of all-(E) lycopene is common to both treatments. The results clearly indicate that low plasma lycopene may be a direct consequence of smoke inhalation.

North Central Cancer Treatment Group (NCCTG) N0432: phase II trial of docetaxel with capecitabine and bevacizumab as first-line chemotherapy for patients with metastatic breast cancer.

BACKGROUND: Docetaxel (T; Taxotere) with capecitabine (X) is active against metastatic breast cancer (MBC); bevacizumab (BV) has demonstrated efficacy with taxanes in the first-line setting. This study was conducted to assess the safety and efficacy of TX-BV in patients with MBC. PATIENTS AND METHODS: In this single-arm, multicenter phase II study, patients received first-line bevacizumab 15 mg/kg and docetaxel 75 mg/m(2) on day 1 and capecitabine 825 mg/m(2) twice per day on days 1-14 every 21 days. Primary and secondary end points were tumor response rate (RR), overall survival (OS), progression-free survival (PFS), and toxicity. RESULTS: A total of 45 assessable patients received TX-BV for a median of seven cycles. Two complete and 20 partial responses were observed (overall RR 49%); nine patients had stable disease >6 months, for a clinical benefit rate of 69%. Median response duration was 11.8 months. Median OS and PFS were 28.4 and 11.1 months, respectively. Grade 3/4 adverse events included hand-foot syndrome (29%), fatigue (20%), febrile neutropenia (18%), and diarrhea (18%). In cycles 3-10, median dose levels of docetaxel and capecitabine were 60 mg/m(2) and 660 mg/m(2), respectively. CONCLUSION: TX-BV demonstrated significant activity; dose modifications were required to manage drug-related toxic effects.

Antisense-induced reduction in nucleus accumbens cyclic AMP response element binding protein attenuates cocaine reinforcement.

Repeated cocaine exposure up-regulates cyclic AMP signaling and increases the transcriptional activity of cyclic AMP response element binding protein (CREB) in the nucleus accumbens. To study the possibility that nucleus accumbens CREB activity regulates self-administration behavior, we tested the effects of a single, bilateral infusion of CREB antisense oligonucleotide into nucleus accumbens core and shell sub-regions on cocaine self-administration in rats. Nucleus accumbens core infusions of CREB antisense reduced CREB and the CREB-regulated immediate early gene brain-derived neurotrophic factor by 31 and 27%, respectively, but failed to alter levels of the homologous CREB family proteins cyclic AMP response element modulator and activating transcription factor 1, and had no effect on CREB levels in adjacent nucleus accumbens shell tissue. Similar infusions of CREB antisense in either core or shell produced a transient downward shift in cocaine self-administration dose-response curves on a fixed ratio 5 (five responses/injection) reinforcement schedule, indicating a reduction in cocaine reinforcement that fully recovered 3 days after treatment. CREB antisense also increased the threshold dose of cocaine required for reinstating cocaine self-administration, indicating that nucleus accumbens CREB levels regulate the incentive properties of cocaine. When access to cocaine was less restricted on a fixed ratio 1 schedule, infusion of CREB antisense in the core, but not shell, caused a transient (1-2 days) reduction in stabilized cocaine self-administration, but had no effect on responding maintained by sucrose pellets, indicating that basal CREB levels in the nucleus accumbens core regulate drug intake. None of these effects were produced by nucleus accumbens infusions of complementary sense oligonucleotide. These results suggest a necessary role for nucleus accumbens CREB activity in cocaine reinforcement, and, by converse analogy, up-regulation in CREB activity after chronic cocaine use could contribute to addiction-related increases in cocaine self-administration.

High sensitivity detection of molecular recognition using magnetically labelled biomolecules and magnetoresistive sensors.

Small magnetoresistive spin valve sensors (2 x 6 microm(2)) were used to detect the binding of single streptavidin functionalized 2 microm magnetic microspheres to a biotinylated sensor surface. The sensor signals, using 8 mA sense current, were in the order of 150-400 microV for a single microsphere depending on sensor sensitivity and the thickness of the passivation layer over the sensor surface. Sensor saturation signals were 1-2 mV representing an estimated 6-20 microspheres, with a noise level of approximately 10 microV. The detection of biomolecular recognition for the streptavidin-biotin model was shown using both single and differential sensor architectures. The signal data compares favourably with previously reported signals for high numbers of magnetic microspheres detected using larger multilayered giant magnetoresistance sensors. A wide range of applications is foreseen for this system in the development of biochips, high sensitivity biosensors and the detection of single molecules and single molecule interactions.

Effect of IGFBP-3 on IGF- and IGF-analogue-induced insulin-like growth factor-I receptor (IGFIR) signalling.

Insulin-like growth factor binding protein-3 (IGFBP-3) binds IGF-I and IGF-II with high affinity, at least an order of magnitude higher than the affiniy of the IGFs for the IGFIR. It has been hypothesized that IGFBP-3 inhibits IGF binding to the IGFIR via a mechanism independent of its ability to sequester IGFs. In the present study, we examined the effects of IGFBP-3 and its proteolytic fragments on the initial events of the IGFIR signalling pathway. IGFBP-3 inhibited IGF-I-, IGF-II-, Des(1-3)IGF-I- and Long(R3)IGF-I-induced IGFIR phosphorylation in a dose-dependent manner at similar concentration range but not QAYL-induced IGFIR-P. The((1-97))IGFBP-3 fragment was able to inhibit only IGF-I-induced IGFIR-P. The((1-97))IGFBP-3 fragment but not intact IGFBP-3 inhibited insulin-induced IGFIR-P. Monolayer cross-linking with [(125)I]IGFBP-3 indicated that there is no direct interaction of IGFBP-3 with the IGFIR. This study demonstrates that the effect on the initial step of IGFIR signalling by IGFBP-3 is largely due to its ability to sequester IGF and the IGF analogues in the extracellular milieu and not the result of any interaction of IGFBP-3 with the IGFIR or a mechanism independent of its ability to bind IGFs.

A method to measure the interaction of Rac/Cdc42 with their binding partners using fluorescence resonance energy transfer between mutants of green fluorescent protein.

Enhanced blue fluorescent protein (EBFP) and enhanced green fluorescent protein (EGFP) mutants of GFP in close proximity to one another can act as a fluorescence resonance energy transfer (FRET) pair. Unstructured amino acid linkers of varying length were inserted between EBFP and EGFP, revealing that linkers even as long as 50 amino acids can be accommodated and still allow FRET to occur. This led to the development of a novel biosensor for Rac/Cdc42 binding to their effector proteins based on the insertion of amino acids 75-118 of p21-activated kinase (PAK) between the GFP mutants. We demonstrate that this protein construct allows significant FRET between EBFP and EGFP and retains the ability to bind to Rac in its GTP-bound form with a binding affinity similar to the uncomplexed PAK fragment, and furthermore, on binding to Rac or Cdc42 a marked change in FRET takes place. This forms the basis for a simple, sensitive, and rapid method to measure binding of Rac/Cdc42 to their effector proteins. Since the signal is dependent upon the interaction with active GTP-bound forms it acts as a biosensor for the activation of Rac/Cdc42. It has the potential for use in live cells and for identifying localization of Rac/Cdc42 within subcellular compartments.

Application of beta-galactosidase enzyme complementation technology as a high throughput screening format for antagonists of the epidermal growth factor receptor.

We have applied enzyme complementation technology to develop a screen for antagonists of the epidermal growth factor (EGF) receptor. Chimeric proteins containing two weakly complementing deletion mutants of Escherichia coli beta-galactosidase (beta-gal), each fused to the EGF receptor extracellular and transmembrane domains, have been stably expressed in C2C12 cells. In this cell line, formation of active beta-gal is dependent on agonist-stimulated dimerization of the EGF receptor. We have developed a homogenous 384-well assay protocol and have applied this to characterize the pharmacology of the receptor and to develop a high throughput screen (HTS) for EGF receptor antagonists. The assay is tolerant to DMSO concentrations of up to 2% and, across 21 passages in culture, exhibits an EC(50) for EGF of 5.4 +/- 3.6 ng/ml (n = 11) and a Z' of 0.55 +/- 0.13 (n = 11). A random set of 1,280 compounds was screened in duplicate at 11 microM to examine the robustness of enzyme complementation technology and to characterize the false-positive hit rate in the assay. Using a cutoff of 40% inhibition of EGF-promoted beta-gal activity, the hit rate on day 1 was 2.5% and on day 2 was 1.9%. After retesting the active compounds, the hit rate was reduced to 0.4%, of which one of the compounds was identified as a beta-gal inhibitor and the remainder appeared to be nonspecific inhibitors in the assay. This technology is amenable to automated screen workstations, there are highly sensitive chemiluminescent and fluorescent beta-gal assay reagents amenable to detection in miniaturized plate formats, and the assay benefits from a low false-positive hit rate. Enzyme complementation technology may have wide application within the HTS environment for the detection of modulators of receptor activation or inhibitors of protein-protein interactions in mammalian cells.

Patterns of movement and seed dispersal of a tropical frugivore.

Movement is a fundamental feature of vertebrate behavior and can modify processes within populations and communities. Because tropical avian frugivores disperse seeds of many plant species, the temporal and spatial patterning of their movement will influence seed distribution within a habitat. To date, little is known about movement patterns of these birds. Here we consider the movement of an understory frugivore, Mionectes oleagineus. Movements of 16 non-breeding females were monitored using continuous radio-telemetry to provide a general description of movement patterns and to examine the fractal geometry of the spatial component of movement. Most movements were of short distance and duration, with the frequency distributions of both measures strongly skewed to the left. Over the range of measurement scales considered, the fractal dimension of M. oleagineus's movement increased with increasing measurement scale up to ca.100 m, whereafter it appeared to flatten out. We combined movement data with M. oleagineus gut-passage rates for seeds of six plant species to predict seed shadows. Estimated seed shadows were leptokurtic for four of the six plant species, with median dispersal distances for all species from 42 to 56 m. Dispersal distances were of the order of reported pollen dispersal distances, suggesting that even small seed dispersers like M. oleagineus can provide significant dispersal for plant genotypes. Gut-passage rate appears to determine the shape of the seed shadow, while movement determines dispersal scale.

Magnesium fluoride-dependent binding of small G proteins to their GTPase-activating proteins.

GTPase-activating proteins (GAPs) enhance the intrinsic GTPase activity of small G proteins, such as Ras and Rho, by contributing a catalytic arginine to the active site. An intramolecular arginine plays a similar role in heterotrimeric G proteins. Aluminum fluoride activates the GDP form of heterotrimeric G proteins, and enhances binding of the GDP form of small G proteins to their GAPs. The resultant complexes have been interpreted as analogues of the transition state of the hydrolytic reaction. Here, equilibrium binding has been measured using scintillation proximity assays to provide quantitative information on the fluoride-mediated interaction of Ras and Rho proteins with their respective GAPs, neurofibromin (NF1) and RhoGAP. High-affinity fluoride-mediated complex formation between Rho.GDP and RhoGAP occurred in the absence of aluminum; however, under these conditions, magnesium was required. Additionally, the novel observation was made of magnesium-dependent, fluoride-mediated binding of Ras.GDP to NF1 in the absence of aluminum. Aluminum was required for complex formation when the concentration of magnesium was low. Thus, either aluminum fluoride or magnesium fluoride can mediate the high-affinity binding of Rho. GDP or Ras.GDP to GAPs. It has been reported that magnesium fluoride can activate heterotrimeric G proteins. Thus, magnesium-dependent fluoride effects might be a general phenomenon with G proteins. Moreover, these data suggest that some protein.nucleotide complexes previously reported to contain aluminum fluoride may in fact contain magnesium fluoride.

Oxacillin-induced tissue necrosis.

OBJECTIVE: To report a case of oxacillin-induced tissue necrosis in which recommended concentration guidelines for dilution and administration were used. Oxacillin concentration data, potential risk factors, and treatment options for extravasation injuries are also briefly reviewed. CASE SUMMARY: Oxacillin was infused peripherally by infusion pump in a 79-year-old white woman as prophylactic antibiotic coverage for permanent pacemaker placement. Oxacillin extravasation occurred after the second postoperative dose. A dime-sized area of necrosis was noted at the heparin-lock insertion site. DISCUSSION: Only one case of oxacillin-induced necrosis has been reported. The degree of damage and concentration of drug used were not specifically described. Concentration may play a role in the appearance or absence of tissue damage after an antibiotic extravasation and should be taken into consideration when evaluating a drug's tissue toxicity potential. CONCLUSIONS: The potential exists for oxacillin 50 mg/mL to cause tissue damage in humans if an extravasation occurs. This reaction may be avoided with use of a less-concentrated preparation, avoidance of infusion pump administration, and identification of high-risk patients.

Genetic diversity in twenty variants of the avian polyomavirus.

To determine if different pathotypes of the avian polyomavirus (APV) exist and to compare the genomes of APVs originating from different geographic areas, dates, and species of birds, the partial sequences of 18 APVs were determined. New viral sequences were compared with three published APV sequences. Two of the new viruses had identical sequences. Forty point mutations were found at 31 loci. A 27-bp deletion was found in the VP2 and VP3 open reading frames of one virus. A duplication of the putative origin of replication and adjacent enhancer region was previously reported in one APV. Smaller duplications involving the origin in one APV and a second enhancer region in another were discovered. All duplications were in tissue culture-adapted viruses, suggesting they occurred during the isolation process. Excluding duplications and the deletion, maximum variation between viruses was small (11 bp). A maximum parsimony tree was constructed that contained three major branches. The three earliest isolates were on separate branches. The European viruses were confined to branch I, but APVs from the United States were on all three branches. Lovebird, budgerigar, and macaw APVs were also on each of the three branches, suggesting that species-specific pathotypes have not developed. Most nonsynonymous mutations occurred in a small portion of the VP2 and VP3 open reading frames, demonstrating a selection for these mutations. That a glycine at VP2 221 will inhibit virus replication in chicken embryo fibroblasts (CEFs) has been previously reported. In contrast, six of seven of the new APVs isolated in CEFs had a glycine at VP2 221.

The conserved arginine in rho-GTPase-activating protein is essential for efficient catalysis but not for complex formation with Rho.GDP and aluminum fluoride.

The Rho family of small GTP-binding proteins are downregulated by an intrinsic GTPase, which is enhanced by GTPase-activating proteins (GAPs). RhoGAPs contain a single conserved arginine residue that has been proposed to be involved in catalysis. Here, the role of this arginine has been elucidated by mutagenesis followed by determination of catalytic and equilibrium binding constants using single-turnover kinetics, isothermal titration calorimetry, and scintillation proximity assays. The turnover numbers for wild-type, R282A, and R282K RhoGAPs were 5.4, 0.023, and 0.010 s-1, respectively. Thus, the function of this arginine could not be replaced by lysine or alanine. Nevertheless, the R282A mutation had a minimal effect on the binding affinity of RhoGAP for either Rho. GTP or Rho.GMPPNP, which confirms the importance of the arginine residue for catalysis as opposed to formation of the protein-protein complex. The R282A mutant RhoGAP still increased the hydrolysis rate of Rho.GTP by 160-fold, whereas the wild-type enzyme increased it by 38000-fold. We conclude that this arginine contributes half of the total reduction of activation energy of catalysis. In the presence of aluminum fluoride, the R282A mutant RhoGAP binds almost as well as the wild type to Rho.GDP, demonstrating that the conserved arginine is not required for this interaction. The affinity of wild-type RhoGAP for the triphosphate form of Rho is similar to that for Rho.GDP with aluminum fluoride. These last two observations show that this complex is not associated with the free energy changes expected for the transition state, although the Rho.GDP.AlF4-.RhoGAP complex might well be a close structural approximation.

Reticuloendotheliosis in captive greater and Attwater's prairie chickens.

Reticuloendotheliosis in captive greater (Tympanuchus cupido pinnatus) and Attwater's (T. cupido attwateri) prairie chickens is reported for the first time. Between September 1993 and August 1994, two adult female wild-caught greater prairie chickens housed at Texas A&M University (College Station, Texas, USA) were observed with multiple subcutaneous nodules. Both birds were euthanatized. Complete necropsy examinations revealed lesions limited to the skin of each bird. Histopathologic examination of lesions revealed pleomorphic lymphoreticular cells suggestive of reticuloendotheliosis and reticuloendotheliosis virus (REV) was demonstrated in tumor tissue by polymerase chain reaction and virus isolation. Between September 1994 and June 1995, five additional greater prairie chickens and two Attwater's prairie chickens were euthanatized or found dead with evidence of lymphoreticular neoplasia in multiple organ systems. Initial testing of the captive flock in December 1994 for evidence of viremia and antibody to reticuloendotheliosis virus revealed over 50% of the tested birds were viremic, but none developed antibodies. Subsequent testing between January 1995 and January 1996 indicated that once infected with reticuloendotheliosis virus, Attwater's prairie chickens tended to remain outwardly healthy despite persistent viremia compared to infected greater prairie chickens which had higher morbidity and mortality rates within 60 to 90 days after initial detection of viremia and did not usually develop persistent viremia. Antibodies to REV were detected in only three captive greater prairie chickens and only in 1995. Six of the nine birds that were euthanatized or found dead due to reticuloendotheliosis developed viremia prior to death; three birds were not tested prior to death. Testing of free-ranging greater and Attwater's prairie chickens for reticuloendotheliosis is recommended prior to translocation or release.

A serologic survey for avian polyomavirus and Pacheco's disease virus in Australian cockatoos.

Sera collected from wild and captive Australian cockatoos and other psittacine species (n = 411) were tested for antibodies to avian polyomavirus (APV) and Pacheco's disease virus (PDV). Of 144 wild sulphur-crested cockatoos (Cacatua galerita) sampled at three regions in New South Wales (NSW) 96 (64.4%) birds had positive (>/= 1:32) neutralizing antibody titres to avian polyomavirus (range 1:32-1:2048). Two of 17 wild long-billed corellas (Cacatua tenuirostris) were also APV-antibody positive. However, no samples from 107 wild galahs (Eolophus roseicapillus) were positive for neutralizing antibody to APV. Sera were also collected from captive psittacine bird flocks from NSW, Tasmania, Victoria, and Western Australia. In a mixed aviary of cockatoos and lorikeets, APV antibody was detected in sera from sulphur-crested cockatoos, Major Mitchell's cockatoos (Cacatua leadbeateri), a white-tailed black cockatoo (Calyptorhynchus baudinii latirostris), a red-tailed black cockatoo (Calyptorhynchus magnificus) a single galah, a rainbow lorikeet (Trichoglossus haematodus), and a scaley-breasted lorikeet (Trichoglossus chlorolepidotus). All 411 wild and captive birds were negative for the presence of neutralizing antibody to PDV. These results indicate that wild sulphur-crested cockatoos in NSW are enzootically infected with avian polyomavirus and that the sampled populations are free of Pacheco's disease.