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Addressing the ongoing decline of Pacific Lamprey Entosphenus tridentatus across its range along the west coast of North America requires an understanding of all life history phases. Currently, spawning surveys (redd counts) are a common tool used to monitor returning adult salmonids, but the methods are in their infancy for Pacific Lamprey. To better understand the spawning phase, our objective was to assess temporal spawning trends, redd abundance, habitat use, and spatial patterns of spawning at multiple spatial scales for Pacific Lamprey in the Willamette River basin, Oregon. Although redd density varied considerably across surveyed reaches, the observed temporal patterns of spawning were related to physical habitat and hydrologic conditions. As has been documented in studies in other basins in the Pacific Northwest, we found that redds were often constructed in pool tailouts dominated by gravel, similar to habitat used by spawning salmonids. Across the entire Willamette Basin, Pacific Lamprey...

Extensive phase II metabolism of an advanced PKCε inhibitor resulted in sub-optimal pharmacokinetics in rat marked by elevated clearance. Synthesis of the O-glucuronide metabolite as a standard was followed by three distinct strategies to specifically temper phase II metabolic degradation of the parent molecule. In this study, it was determined that the introduction of proximal polarity to the primary alcohol generally curbed O-glucuronidation and improved PK and physical chemical properties while maintaining potency against the target. Utilization of a Jacobsen hydrolytic kinetic resolution to obtain optically enriched final compounds is also discussed.

Background. Emergency departments (EDs) are commonly used by pediatric patients for nonemergent reasons. There is little information regarding how parents perceive their use of the ED and primary care availability during evening hours. Methods. We conducted a survey of parental perspectives of ED use during evening hours. Participants were parents of pediatric patients (age 0-18 years) at a large quaternary medical center’s ED presenting between 17:00 and 22:00 hours from January 15, 2013 to March 12, 2013. Results. Most patients had a primary care pediatrician (98/102, 96.1%); 80% of their pediatricians (78/98) did not have evening hour availability. Overall, 46.7% of parents would have preferred to go to their primary care pediatrician. Conclusions. Many parents who expect outpatient treatment prefer to take their child to a primary care pediatrician during evening hours, but present to the ED because of lack of primary care access.

Lampreys (Petromyzontiformes) have persisted over millennia and now suffer a recent decline in abundance. Complex life histories may have factored in their persistence; anthropogenic perturbations in their demise. The complexity of life histories of lampreys is not understood, particularly for the anadromous Pacific lamprey, Entosphenus tridentatus Gairdner, 1836. Our goals were to describe the maturation timing and associated characteristics of adult Pacific lamprey, and to test the null hypothesis that different life histories do not exist. Females exhibited early vitellogenesis – early maturation stages; males exhibited spermatogonia – spermatozoa. Cluster analyses revealed an “immature” group and a “maturing–mature” group for each sex. We found statistically significant differences between these groups in the relationships between (i) body mass and total length in males; (ii) Fulton’s condition factor and liver lipids in males; (iii) the gonadosomatic index (GSI) and liver lipids in females; (iv) GSI and total length in females; (v) mean oocyte diameter and liver lipids; and (vi) mean oocyte diameter and GSI. We found no significant difference between the groups in the relationship of muscle lipids and body mass. Our analyses support rejection of the hypothesis of a single life history. We found evidence for an “ocean-maturing” life history that would likely spawn within several weeks of entering fresh water, in addition to the formerly recognized life history of spending 1 year in fresh water prior to spawning—the “stream-maturing” life history. Late maturity, semelparity, and high fecundity suggest that Pacific lamprey capitalize on infrequent opportunities for reproduction in highly variable environments.

Background Information. Tumour cells can be induced to undergo apoptosis after treatment with the tumour necrosis factor -related death-inducing ligand (TRAIL). Although human pancreatic cancer cells show varying degrees of response they can be sensitised to the pro-apoptotic effects of TRAIL in the presence of celastrol, a natural compound extracted from the plant Tripterygium wilfordii Hook F. One important aspect of the cellular response to TRAIL is the control of protein synthesis, a key regulator of which is the eukaryotic initiation factor 4E-binding protein, 4E-BP1. Results. We examined the effects of celastrol and TRAIL in several pancreatic cancer cell lines. In cells that are normally resistant to TRAIL, synergistic effects of TRAIL plus celastrol on commitment to apoptosis and inhibition of protein synthesis were observed. These were associated with a strong up-regulation and dephosphorylation of 4E-BP1. The enhancement of 4E-BP1 expression, which correlated with a threefold increase in the level of the 4E-BP1 transcript, was blocked by inhibitors of reactive oxygen species and the JNK protein kinase. When the expression of 4E-BP1 was reduced by an inducible micro-RNA, TRAIL-mediated apoptosis was inhibited. Conclusion. These results suggest that 4E-BP1 plays a critical role in the mechanism by which TRAIL and celastrol together cause apoptotic cell death in human pancreatic tumour cells.

Mephedrone (MMC) is a relatively new recreational drug that has rapidly increased in popularity in recent years. This study explored the characteristics of intravenous MMC self-administration in the rat, with methamphetamine (METH) used as a comparator drug. Male Sprague-Dawley rats were trained to nose poke for intravenous MMC or METH in daily 2 h sessions over a 10 d acquisition period. Dose-response functions were then established under fixed- and progressive-ratio (FR and PR) schedules over three subsequent weeks of testing. Brains were analyzed ex vivo for striatal serotonin (5-HT) and dopamine (DA) levels and metabolites, while autoradiography assessed changes in the regional density of 5-HT and serotonin transporter (SERT) and DA transporter (DAT) and induction of the inflammation marker translocator protein (TSPO). Results showed that MMC was readily and vigorously self-administered via the intravenous route. Under a FR1 schedule, peak responding for MMC was obtained at 0.1 mg/kg/infusion, versus 0.01 mg/kg/infusion for METH. Break points under a PR schedule peaked at 1 mg/kg/infusion MMC versus 0.3 mg/kg/infusion for METH. Final intakes of MMC were 31.3 mg/kg/d compared to 4 mg/kg/d for METH. Rats self-administering MMC, but not METH, gained weight at a slower rate than control rats. METH, but not MMC, self-administration elevated TSPO receptor density in the nucleus accumbens and hippocampus, while MMC, but not METH, self-administration decreased striatal 5-hydroxyindolacetic acid (5-HIAA) concentrations. In summary, MMC supported high levels of self-administration, matching or exceeding those previously reported with other drugs of abuse.

We evaluated the impact of clinical and molecular characteristics on overall survival (OS) in 108 patients with indolent (n=41) and advanced SM (advSM, n=67). Organomegaly was measured by magnetic resonance imaging (MRI)-based volumetry of liver and spleen. In multivariate analysis of all patients, an increased spleen volume greater than or equal to450?ml (hazard ratio [HR], 5.2; 95% confidence interval [CI], [2.1–13.0]; P=0.003) and an elevated alkaline phosphatase (AP; HR 5.0 [1.1–22.2]; P=0.02) were associated with adverse OS. The 3-year OS was 100, 77, and 39%, respectively (P

The orexin/hypocretin system is important for appetitive motivation towards multiple drugs of abuse, including nicotine. Both OX1 and OX2 receptors individually have been shown to influence nicotine self-administration and reinstatement. Due to the increasing clinical use of dual orexin receptor antagonists in the treatment of disorders such as insomnia, we examined whether a dual orexin receptor antagonist may also be effective in reducing nicotine seeking. We tested the effect of intracerebroventricular (i.c.v.) administration of the potent and selective dual orexin receptor antagonist TCS1102 on orexin-A-induced food self-administration, nicotine self-administration and reinstatement of nicotine-seeking in rats. Our results show that 30 μg of TCS1102 i.c.v. abolishes orexin-A-induced increases in food self-administration but does not reduce nicotine self-administration. Neither i.c.v. 10 μg nor 30 μg of TCS1102 reduced compound reinstatement after short-term (15 days) self-administration nicotine, but 30 μg transiently reduced cue/nicotine compound reinstatement after chronic self-administration (29 days). These results indicate that TCS1102 has no substantial effect on motivation for nicotine seeking following chronic self-administration and no effect after shorter periods of intake. Orexin receptor antagonists may therefore have little clinical utility against nicotine addiction.

Background HER2 is one of the predominant therapeutic targets in breast cancer. The metastatic selection process may lead to discrepancies between the HER2 status of the primary tumor and circulating tumor cells (CTCs). This study analyzed the HER2 status of CTCs in patients with HER2-positive primary breast cancer at the time of diagnosis. Aim of the study was to assess potential discordance of HER2 status between primary tumor and CTCs, as this may have important implications for the use of HER2-targeted therapy. Methods The number and HER2 status of CTCs out of 30ml peripheral blood were assessed in 642 patients using the CellSearch System (Janssen Diagnostics, USA). The cutoff for CTC positivity was the presence of at least 1 CTC, and the cutoff for HER2 positivity of CTCs was the presence of at least 1 CTC with a strong HER2 staining. Results 258 (40.2%) of the 642 patients were positive for CTCs (median 2; range 1–1,689). 149 (57.8%) of these 258 patients had at least 1 CTC with strong HER2 staining. The presence of HER2-positive CTCs was not associated with tumor size (p = 0.335), histopathological grading (p = 0.976), hormone receptor status (ER: p = 0.626, PR: p = 0.263) or axillary lymph node involvement (p = 0.430). Overall, 83 (32.2%) of the CTC-positive patients exclusively had CTCs with strong HER2 staining, whereas 31 (12.0%) had only CTCs with negative HER2 staining. Within-sample variation in the HER2 status of CTCs was found in 86 (57.8%) of the 149 patients with more than 1 CTC. Conclusion This study demonstrated that discordance between the HER2 expression of CTCs and that of the primary tumor frequently occurs in early breast cancer. Future follow-up evaluation will assess whether this discrepancy may contribute to trastuzumab resistance.

Purpose The 21-gene Recurrence Score (RS) assay is a validated prognostic/predictive tool in early hormone receptor–positive breast cancer (BC); however, only a few prospective outcome results have been available so far. In the phase III PlanB trial, RS was prospectively used to define a subset of patients who received only endocrine therapy. We present 3-year outcome data and concordance analysis (among biomarkers/RS). Patients and Methods Central tumor bank was established prospectively from PlanB (intermediate and high-risk, locally human epidermal growth factor receptor 2–negative BC). After an early amendment, HR-positive, pN0-1 patients with RS ≤ 11 were recommended to omit chemotherapy. Results From 2009 to 2011, PlanB enrolled 3,198 patients with a median age of 56 years; 41.1% had node-positive and 32.5% grade 3 disease. In 348 patients (15.3%), chemotherapy was omitted based on RS ≤ 11. After 35 months median follow-up, 3-year disease-free survival in patients with RS ≤ 11 and endocrine therapy alone was 98% versus 92% and 98% in RS > 25 and RS 12 to 25 in chemotherapy-treated patients, respectively. Nodal status, central and local grade, the Ki-67 protein encoded by the MKI67 gene, estrogen receptor, progesterone receptor, tumor size, and RS were univariate prognostic factors for disease-free survival; only nodal status, both central and local grade, and RS were independent multivariate factors. Histologic grade was discordant between central and local laboratories in 44%. RS was positively but moderately correlated with the Ki-67 protein encoded by the MKI67 gene and grade and negatively correlated with progesterone receptor and estrogen receptor. Conclusion In this prospective trial, patients with enhanced clinical risk and omitted chemotherapy on the basis of RS ≤ 11 had excellent 3-year survival. The substantial discordance observed between traditional prognostic markers and RS emphasizes the need for standardized assessment and supports the potential integration of standardized, well-validated genomic assays such as RS with clinicopathologic prognostic factors for chemotherapy indication in early hormone receptor–positive BC.

Background: Routine use of multigene RT-PCR based assays as Recurrence Score (RS) vs. single markers (grade, uPA/PAI-1, HR, HER2, Ki-67) is controversially discussed in early BC. Several definitions of luminal A/B subtypes have been proposed by St. Gallen guidelines and individual researchers (grade 1/2 vs. 3, Ki-67 cut-offs 14 or 20%). Recently, integration of PR>20% was proposed as an immunhistochemical luminal A subtype definition (Ki-67 Methods: planB trial (04/09 to 11/11: n=2,449 randomized for 6xTC vs. 4xEC-4xDOC in locally HER2− BC; n=3197 registered; n=3072 available for central tumor bank). RS was used as selection criterion for chemotherapy (CTx) omission in HR+ BC (if RS Results: RS distribution in 2569 HR+ tumors: 0–11 (18%), 12–25 (60%), >25 (22%) (RS1853%/34%/13%). Luminal A subtype based on Ki-67 cut-off of 20% was included in the luminal A/Ki-67 Luminal A/B based on local/central grade: 79%/21%; 67.9%/32.1%. In 354 pN0-1 patients, CTx was omitted based on low RS (88% compliance). In this group, 62% were high-risk by clinical-pathological criteria. Allocation to luminal A/B subtypes based on local/central grade varied substantially: overall concordance was 72%, but 40% of locally luminal B (HR+/G3) were luminal A (HR+/G1/2) by central and 60% of centrally luminal B were luminal A locally. Moderate correlations were found between RS and central grade (Spearman correlation rs=0.317; p < 0.001), local grade (0.321; p < 0.001) and Ki-67 (rs = 0.374; p < 0.001), particularly due to poor correlations in the RS group Inclusion of PR>20% moderately improved the correlation between luminal subtypes and RS to rs=0.34; p < 0.001. Data on allocation to luminal subtypes by local/central Ki-67 will be presented at the meeting. Conclusions: Our results represent the first prospective correlation of different luminal A/B definitions vs. a guideline mentioned gene signature (RS). High RS usually implies high grade and luminal B classification, the converse is not true. There is substantial heterogeneity in risk assessment by luminal A/B classification and grade in low/intermediate RS groups. Concordance for central/local assessment of grade and luminal A/B status was limited. There is strong need for standardization of molecular subtype's immunhistochemical/clinical definition prior to implementation into daily routine. Clinical significance of these findings will be addressed by further follow up of the WSG-planB trial. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-08.

We report the incidence of male intersex in adult Pacific lamprey (Entosphenus tridentatus (Richardson, 1836)) during their pre-spawning migration in fresh water. Although "hermaphrodites" have been suggested in other adult lampreys, this is the first detailed description and discussion of this phenomenon. A total of 0.5% of our adult Pacific lamprey from Willamette Falls (2 out of 427 adults) were intersex, with oocytes in the testes. This phenomenon was identifiable only by histological examination. The testes of the intersex males were immature, in the beginning stages of meiosis. One intersex male possessed primary growth or perinucleolar stage oocytes loosely interspersed throughout the testes, and the other pos- sessed at least 6 mid-vitellogenic oocytes (0.6 mm, mean long diameter) separate from the testes. Because premetamorphic lamprey can possess both female and male gonial cells, we hypothesize that intersex is a remnant larval trait and that these fish failed to fully develop into males during metamorphosis. Resume : Nous rendons compte de l'incidence d'intersexualite chez des mâles adultes de lamproie du Pacifique (Entosphe- nus tridentatus (Richardson, 1836)) durant leur migration en eau douce precedant le frai. Bien que la presence « d'herma- phrodites » ait ete suggeree chez d'autres lamproies adultes, il s'agit des premieres description et discussion detaillees de ce phenomene. Au total, 0,5 % des lamproies du Pacifique adultes de Willamette Falls recensees (2 individus sur 427 adultes) etaient intersexuelles, presentant des oocytes dans les testicules. Ce phenomene n'est observable que par un examen histolo- gique. Les testicules des mâles intersexuels etaient immatures, aux stades initiaux de la meiose. Un mâle intersexuel posse- dait des oocytes au stade de croissance primaire ou perinucleolaire dissemines ca et la dans les testicules et l'autre possedait au moins six oocytes a des stades intermediaires de la vitellogenese (diametre long moyen de 0,6 mm) separes des testicu- les. Etant donne que la lamproie premetamorphique peut presenter des cellules goniales femelles et mâles, nous avons emis l'hypothese que l'intersexualite est un caractere larvaire residuel et que ces poissons ne se sont pas developpes complete- ment en individus mâles durant la metamorphose. Mots-cles : intersexualite, gonochoriste, poisson primitif, reproduction, maturation, cyclostomes. (Traduit par la Redaction)

The Pacific lamprey (Entosphenus tridentatus) is in decline in the Columbia River Basin, and translocating adult lamprey to bypass difficult migration corridors has been implemented since 2000. We describe and report results from two current translocation programs, provide context for use of translocation, and discuss potential benefits, risks, and uncertainties. Both translocation programs appear to have increased the number of spawning adults and the presence of larvae and juveniles; however, any subsequent increase in naturally spawning adults will require at least one, and likely more, generations to be realized. It was seen that the number of adults entering the Umatilla River increased beginning four years after the first translocations. Potential benefits of translocation programs are increased pheromone production by ammocoetes to attract adults, increased lamprey distribution and abundance in target areas, increased marine-derived nutrients, and promotion of tribal culture. Potential ris...

OBJECTIVE: To determine whether the activation of innate immune responses, which can be elicited by pathogenic and endogenous triggers, is associated with the presence of Epstein-Barr virus (EBV) infection in the multiple sclerosis (MS) brain. METHODS: White matter postmortem MS (n = 10) and control tissue (n = 11) was analyzed for the expression of the proinflammatory cytokine interferon (IFN) by immunohistochemistry and for EBV by using the highly sensitive method of EBV-encoded RNA (EBER) in situ hybridization. RESULTS: We detected overexpression of IFN in active areas of white matter MS lesions but not in inactive MS lesions, normal-appearing white matter, or normal brains. The presence of IFN in macrophages and microglia (expressing human leukocyte antigen class II) is suggestive of local production as part of an acute inflammatory process. Interestingly, EBERs were also specifically detected in areas where IFN was overexpressed in these preselected active MS lesions. EBER+ cells were also found in CNS lymphoma and stroke cases, but were absent in other control brains. We next addressed a potential mechanism, e.g., the role of EBERs in eliciting IFN production, and transfected EBERs into human embryonic kidney (HEK) cells. We used HEK cells that stably expressed Toll-like receptor-3, which recognizes double-stranded RNAs, associated with many viral infections. EBERs elicited IFN production in vitro. CONCLUSION: These findings suggest that latent EBV infection may contribute to the inflammatory milieu in active MS lesions by activating innate immune responses, e.g., IFN production. Unraveling the underlying mechanisms may help in uncovering causal pathways and developing better treatment strategies for MS and other neuroinflammatory diseases.

We describe the migration distances and timing of the adult Pacific lamprey, Entosphenus tridentatus, in the Willamette River Basin (Oregon, U.S.A.). We conducted aerial surveys to track radio-tagged fish upstream of a major waterfall and hydropower complex en route to spawning areas. We detected 24 out of the 43 fish that passed the waterfall-hydropower complex. Of the detected fish, 17 were detected multiple times. Their maximum migration distance upstream in the mainstem Willamette approximated a normal distribution. The maximum distance migrated upstream did not significantly correlate with total body length (r = −0.186, P = 0.385) or date that the fish passed Willamette Falls (r = −0.118, P = 0.582). Fish migrated primarily during the spring to early summer period before stopping during the summer, when peak river temperatures (≥20°C). However, at least three fish continued to migrate upstream after September. Behavior ranged from relatively slow migration, followed by holding; to rapid migration, followed by slow migration further up in the basin. This study provides a basis for informing more detailed research on Pacific lamprey in the future.

Sea lampreys, Petromyzon marinus, are invasive to the Laurentian Great Lakes where they have decimated native fishes. Great Lakes sea lampreys have been subjected to control measures for several decades, and the drive to control them has led to major advances in understanding their biology and in informing management. In contrast, anadromous sea and Pacific (Entosphenus tridentatus) lampreys have co-evolved with their oceanic prey. Both of these anadromous lampreys are in decline, and a limited amount of information on their biology has stymied conservation. The tendency has been to make biological inferences about anadromous lampreys based on the Great Lakes sea lamprey without justifiable evidence. We identify areas in which key information is missing for the juvenile (parasitic feeding) phase and adult freshwater spawning migrations, and compare and contrast information for these lampreys. Our comparisons reveal major differences, some intriguing similarities, and key unknowns that will requir...

Background: Despite progress in adjuvant breast cancer (BC) treatment, overtreatment due to decisions based on conventional prognostic factors (lymph nodes (LN), grade, tumor size) alone remains an important clinical problem. Recurrence Score®, RS is validated using pre-specified analyses, genes, cutt-offs in several randomized patient collectives as a potent prognostic and predictive marker in HR positive BC. Here, we present the first WSG-Plan B trial preplanned correlation analysis of central grade, nodal status, RS, and luminal A/B subtypes. Material and Methods: The West German Study Group (WSG) Plan B trial (planned n=2,448) is a randomized Phase-III-trial evaluating anthracyline-free adjuvant chemotherapy (Cht) (6x TC) vs. standard 4xEC-4xDOC in high-risk N0 and N+ HER2-negative BC. In HR+ BC with 0-3 LN, RS is the decision criterion for (>11) or against (≥11) Cht randomization. Tumor blocks are prospectively evaluated by a central pathologist for grade, histology and Ki-67. In HR+ disease, molecular subtypes (luminal A vs. B) are grouped using a Ki-67 cut-off of 13.25% (Cheang et al, JNCI 2009) and 20% (Penault-Llorca et al JCO 2009). Exploratory analysis with Elston-Ellis central grade and Ki-67 based (mitosis measured by Ki-67) grade will be performed. Results: As of June 2010, 1375 patients in 96 centers have been registered and 1067 randomized into Plan B. In 298 patients, RS results and clinical-pathological characteristics were available. In 266 patients complete information on all factors was available (132 N0, 134 N+). RS groups using the pre-specified trial cut-offs of 0-11, 12-25 and 30) were 48%, 38%, 14%. Central grade 1, 2, or 3 was observed in 4%, 50%, and 46% of cases. We observed a modest (Spearman) correlation between central grade and RS (e.g 83.8% of G3 tumors with RS >25, rs=0.331, P25), and guideline-recommended 30 (see table 1).Discussion: For the first time, we demonstrate a modest but significant correlation between centrally assed tumor grade, molecular classes by IHC Ki-67 cutt offs, and RS, particularly in the high RS group. However, our data also indicate that there is substantial discordance between the methods and thus an urgent need for optimzing risk-stratification classifiers. Table 1. Association of RS and molecular subtypes Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-12.

Chantier qualité GA; International audience; Intake of sweet-alcoholic drinks during adolescence is believed to favor alcohol abuse and dependence in adulthood. This study examined the influence of early exposure to ethanol with or without sucrose on the consumption of sweet or alcoholic solutions in adulthood. Adolescent rats (from post-natal day 30–46) were given continuous free access to tap water and either 5% sucrose, 5% ethanol or mixed 5% sucrose–5% ethanol. The control group was given access to water only. Upon reaching adulthood (post-natal day 60), rats were tested for saccharin (sweet), quinine (bitter) and ethanol consumption using a two-bottle free-choice paradigm. The results indicated that pre-exposure to ethanol did not alter the intake of sweet or ethanol solutions in adulthood. However, rats exposed to sucrose during adolescence showed a decreased consumption of both sweet and ethanol solutions. Because alcohol has a sweet taste component, an additional group of rats, pre-exposed to either 5% sucrose or water during adolescence, was tested for intravenous ethanol self-administration (preventing oral sensory stimulation) and in a new model of simultaneous access to oral saccharin and intravenous ethanol that results in higher total ethanol intake. Relative to controls, sucrose-exposed rats showed reduced operant self-administration of saccharin, yet no differences were found for intravenous ethanol self-administration. Altogether, these findings indicate that sucrose exposure during adolescence persistently affected the perception of sweet taste reward and thereby alcohol’s acceptance in adulthood.

522Background: Recent studies draw different conclusions concerning whether omission of anthracyclines (A) in adjuvant chemotherapy for HER2-negative early breast cancer (EBC) may reduce toxicity w...

The feasibility of using preformed calcium sulfate (CS) space-making devices (SMDs) for bone regeneration was explored using a rabbit calvarial model. Twenty-four CS devices were fabricated. Twelve of these were SMDs, which consisted of a domed head that served as the actual space-maker, and a stalk or "tail" portion used to affix the device to the bone. A second set of control devices (CDs) was fabricated that consisted of only the tail portion. CDs were made of medical-grade CS, as were 9 of the SMDs. Six of the CS SMDs were loaded with high or low concentrations of simvastatin. The remaining 3 SMDs were made of a CS/bioactive glass composite. One SMD and 1 CD were implanted bilaterally in the parietal bones of 12 New Zealand White rabbits, which were euthanized 8 weeks following surgery. All implants were well tolerated. In all animals, the side receiving the SMD exhibited greater thickness than did the control sites. The addition of simvastatin resulted in a statistically significant difference in calvarial thickness. The CS/bioactive glass composite also yielded encouraging results. The CS resorbable SMDs are worthy of further investigation.

Translationally controlled tumour protein (TCTP) is a highly conserved protein present in all eukaryotic organisms. Various cellular functions and molecular interactions have been ascribed to this protein, many related to its growth-promoting and antiapoptotic properties. TCTP levels are highly regulated in response to various cellular stimuli and stresses. We have shown recently that the double-stranded RNA-dependent protein kinase, PKR, is involved in translational regulation of TCTP. Here we extend these studies by demonstrating that TCTP is downregulated in response to various proapoptotic treatments, in particular agents that induce Ca(++) stress, in a PKR-dependent manner. This regulation requires phosphorylation of protein synthesis factor eIF2alpha. Since TCTP has been characterized as an antiapoptotic and Ca(++)-binding protein, we asked whether it is involved in protecting cells from Ca(++)-stress-induced apoptosis. Overexpression of TCTP partially protects cells against thapsigargin-induced apoptosis, as measured using caspase-3 activation assays, a nuclear fragmentation assay, using fluorescence-activated cell sorting analysis, and time-lapse video microscopy. TCTP also protects cells against the proapoptotic effects of tunicamycin and etoposide, but not against those of arsenite. Our results imply that cellular TCTP levels influence sensitivity to apoptosis and that PKR may exert its proapoptotic effects at least in part through downregulation of TCTP via eIF2alpha phosphorylation.

In Gram-positive bacteria, sortase enzymes assemble surface proteins and pili in the cell wall envelope. Sortases catalyze a transpeptidation reaction that joins a highly conserved LPXTG sorting signal within their polypeptide substrate to the cell wall or to other pilin subunits. The molecular basis of transpeptidation and sorting signal recognition are not well understood, because the intermediates of catalysis are short lived. We have overcome this problem by synthesizing an analog of the LPXTG signal whose stable covalent complex with the enzyme mimics a key thioacyl catalytic intermediate. Here we report the solution structure and dynamics of its covalent complex with the Staphylococcus aureus SrtA sortase. In marked contrast to a previously reported crystal structure, we show that SrtA adaptively recognizes the LPXTG sorting signal by closing and immobilizing an active site loop. We have also used chemical shift mapping experiments to localize the binding site for the triglycine portion of lipid II, the second substrate to which surface proteins are attached. We propose a unified model of the transpeptidation reaction that explains the functions of key active site residues. Since the sortase-catalyzed anchoring reaction is required for the virulence of a number of bacterial pathogens, the results presented here may facilitate the development of new anti-infective agents.

IntroductionManagement practices within the commercial forest,agriculture and power generation industries in thePacific Northwest (USA) have resulted in warming ofstream temperatures to >20 C during the summertime(Beschta et al. 1987; NRC 1996; EPA 2003; USACE2007). The effects of these warm temperatures havebeen most studied in salmonids (Oncorhynchus spp.),where warm temperatures have been linked to elevatedmetabolic rates and energetic expenditures (Brett1995; Lee et al. 2003; Rand et al. 2006), use of coolwater refugia (Goniea et al. 2006; High et al. 2006),altered run times (Quinn & Adams 1996; Hodgson Q Keefer et al. 2007). Pacificlamprey (Entosphenus tridentatus) co-occur withPacific salmon in these warming watersheds, and theyhave a similar life cycle to Pacific salmon (anadromyand semelparity). However, little is known about theeffects of summer temperatures on maturing adultPacific lamprey.After ceasing their parasitic stage in the ocean,Pacific lamprey return to freshwater during the spring(April–June; Beamish 1980), and then begin theirinitial upstream migration during the summer (July–September; Scott & Crossman 1973), before overwin-tering during October–March (Scott & Crossman1973). Like other anadromous lampreys (Kott 1971;Beamish et al. 1979; Larsen 1980), Pacific lamprey donot feed during this prolonged freshwater residency(Beamish 1980; Whyte et al. 1993) and somaticenergy reserves fuel sexual maturation (Kott 1971;Beamish et al. 1979; Larsen 1980). As a result, Pacificlamprey shrink in body size (Beamish 1980; Whyteet al. 1993) prior to maturing, spawning, and thendying the following spring (April–July; Pletcher 1963,cited in Scott & Crossman 1973; Beamish 1980). Mostspawning activity in Western Oregon rivers occurs

Keywords Agnatha.Sarcopterygii.Polypteriformes.Acipenseriformes.Holosteans.PrimitiveAnyone who has seen a lamprey, sturgeon, bowfin, orany other ‘primitive fish’ can attest to the mystique ofthese animals. Primitive fishes can (in my case) evokethe wonder experienced as children over dinosaurs, agroup that is younger in geologic time than manyprimitive fishes (Kardong 1995). The ancient lineageand persistence of primitive fishes through time makesthem amenable to studies of phylogeny and evolution.Primitive fishes are especially interesting from ananthropomorphic view, as some, lungfishes and coela-canths, may be more closely related to humans than toother fishes (Maisey 1996;Janvier,Figure1.3inChapter 1; Youson, Chapter 9). In my case, the ancientlineage of primitive fishes also makes them interestingto study with respect to their physiological adaptationsto their present and past environments. Many of theprimitive fishes have changed very little in appearanceover time and it is a wonder that they have persisted forso long.The authors of the book chapters occasionallyspeculate about how the physiology of primitive fishesadapted them to past environments (Chapter 1) or hasenabledthemtopersistincurrentenvironments(Chapter10). For the most part, however, care is taken by allauthors(especiallyJanvierinChapter1)toacknowledgethat the physiology we see now may not indicateadaptation to past environments (witnessed by themarine origin of lungfishes, which now only exist infreshwater; Janvier, Chapter 1). Fossils typically captureonlythecalcifiedskeletalremainsoforganismsandomitthesoftanatomyinwhichmanyphysiologicalprocessesoccur. As a result, fishes lacking a rigid, calcifiedskeleton have relatively poor fossil records (e.g.,lampreys; Janvier, Chapter 1). Skeletons have theirlimitations for fishes with bony elements, as functioncannot always be deduced from morphology (e.g., seeWainwright 2007). ‘In sum, very few fossil data aredirectly informative to primitive vertebrate physiolo-gists’ (Janvier, Chapter 1, pg. 40).‘Primitive Fishes’ is a well-bound book that staysopen on your desktop for easy access and reference. Ipreferred the ‘old-style’ covers of past books in theFish Physiology series (Volumes 1–13). However thecover of ‘Primitive Fishes’ presents an attractive colorscheme with a whimsical and arresting line drawing ofprimitive fishes by the author of Chapter 1, Janvier.My only complaint about how the book is ‘puttogether’ is that the color inserts for figures are often,but not always, inserted several pages away from the

Deepwater ciscoes (genus Coregonus, subgenus Leucichthys) radiated into six phenotypes that occupy different depths in the Great Lakes, based upon body size and lipid content. Large, lipid-dense ciscoes occupy greater depths than small, lean ciscoes. This relationship is observed between adults and juveniles of the most prevalent deepwater cisco, the bloater (C. hoyi Gill). Lipid-dense adult bloaters are restricted to the hypolimnion, whereas lean juveniles are found primarily in the epilimnion. This article critically reviews, synthesizes, and provides hypotheses from the literature on the ecology of body size and depth use of bloater. Factors influencing depth use in bloater are categorized by Fry's (1971) environmental factors. The case is made for two parsimonious hypotheses to explain the depth distribution by body size of bloater: (1) the optimal foraging-antipredation (OFA) hypothesis, and (2) the mass-specific metabolism hypothesis. The OFA hypothesis relates abundance of piscivores with abundance...

We estimated the survival of juvenile salmonids out-migrating through the lower Columbia River to the Pacific Ocean. We tested the null hypotheses that no association exists between survival and transportation type (including barge transportation and in-river migration with no transportation), release date, river flow, mean body weight, and tag type. During 2002-2004, spring-summer (SS) and fall Chinook salmon Oncorhynchus tshawytscha and steelhead O. mykiss were implanted with uniquely coded radio or acoustic transmitter tags and released during the early, middle, and late out-migration periods. A series of receiver lines were used to detect these fish and estimate survival. Estimated survival varied considerably with transportation type and release date for fall Chinook salmon and steelhead, but the results were dependent upon year. Estimated survival between the lowermost dam (Bonneville Dam) and the upper estuary (river kilometer 46) was relatively high for SS Chinook salmon and steelhead, wh...

Signaling through mammalian target of rapamycin complex 1 (mTORC1) is stimulated by amino acids and insulin. Insulin inactivates TSC1/2, the GTPase-activator complex for Rheb, and Rheb·GTP activates mTORC1. It is not clear how amino acids regulate mTORC1. FKBP38 (immunophilin FK506-binding protein, 38 kDa), was recently reported to exert a negative effect on mTORC1 function that is relieved by its binding to Rheb·GTP. We confirm that Rheb binds wild type FKBP38, but inactive Rheb mutants showed contrasting abilities to bind FKBP38. We were unable to observe any regulation of FKBP38/mTOR binding by amino acids or insulin. Furthermore, FKBP38 did not inhibit mTORC1 signaling. The translationally controlled tumor protein (TCTP) in Drosophila was recently reported to act as the guanine nucleotide-exchange factor for Rheb. We have studied the role of TCTP in mammalian TORC1 signaling and its control by amino acids. Reducing TCTP levels did not reproducibly affect mTORC1 signaling in amino acid-replete/insulin-stimulated cells. Moreover, overexpressing TCTP did not rescue mTORC1 signaling in amino acid-starved cells. In addition, we were unable to see any stable interaction between TCTP and Rheb or mTORC1. Accumulation of uncharged tRNA has been previously proposed to be involved in the inhibition of mTORC1 signaling during amino acid starvation. To test this hypothesis, we used a Chinese hamster ovary cell line containing a temperature-sensitive mutation in leucyl-tRNA synthetase. Leucine deprivation markedly inhibited mTORC1 signaling in these cells, but shifting the cells to the nonpermissive temperature for the synthetase did not. These data indicate that uncharged tRNALeu does not switch off mTORC1 signaling and suggest that mTORC1 is controlled by a distinct pathway that senses the availability of amino acids. Our data also indicate that, in the mammalian cell lines tested here, neither TCTP nor FKBP38 regulates mTORC1 signaling.

Background information. The translational inhibitor protein 4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] regulates the availability of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds the 5′ cap structure present on all cellular mRNAs. Its ability to associate with initiation factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive manner by 4E-BP1. Phosphorylation of 4E-BP1 decreases the affinity of this protein for eIF4E, thus favouring the binding of eIF4G and enhancing translation. We have previously shown that induction or activation of the tumour suppressor protein p53 rapidly leads to 4E-BP1 dephosphorylation, resulting in sequestration of eIF4E, decreased formation of the eIF4F complex and inhibition of protein synthesis. Results. We now report that activation of p53 also results in modification of 4E-BP1 to a truncated form. Unlike full-length 4E-BP1, which is reversibly phosphorylated at multiple sites, the truncated protein is almost completely unphosphorylated. Moreover, the latter interacts with eIF4E in preference to full-length 4E-BP1. Inhibitor studies indicate that the p53-induced cleavage of 4E-BP1 is mediated by the proteasome and is blocked by conditions that inhibit the dephosphorylation of full-length 4E-BP1. Measurements of the turnover of 4E-BP1 indicate that the truncated form is much more stable than the full-length protein. Conclusions. The results suggest a model in which proteasome activity gives rise to a stable, hypophosphorylated and truncated form of 4E-BP1, which may exert a long-term inhibitory effect on the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the growth-inhibitory and pro-apoptotic effects of p53.

Rats were trained to self-administer cocaine in a distinctive context (context A). They were then extinguished in a second context (context B) prior to test for cocaine-seeking in the original training context, context A (group ABA), context B (group ABB) or no test (group AB0). Group ABA showed renewal of extinguished cocaine-seeking associated with c-Fos induction in basolateral amygdala, lateral hypothalamus, and infralimbic prefrontal cortex. Groups ABA and ABB showed test-associated c-Fos induction in prelimbic prefrontal cortex, nucleus accumbens (core, shell, rostral pole), striatum, lateral amygdala, perifornical hypothalamus, and ventral tegmental area. Double immunofluorescence revealed that renewal-associated c-Fos was expressed in orexin-negative lateral hypothalamic neurons whereas test-associated c-Fos was expressed in orexin-positive perifornical hypothalamic neurons. Retrograde tracing from lateral hypothalamus with cholera toxin revealed only sparse dual-labeled neurons in basolateral amygdala and infralimbic prefrontal cortex, suggesting that these regions contribute to renewal of cocaine-seeking independently of their projections to lateral hypothalamus. Retrograde tracing from the ventral tegmental area suggested that hypothalamic contributions to cocaine-seeking are likewise independent of projections to the midbrain. These results suggest that renewal of cocaine-seeking depends critically on basolateral amygdala, lateral hypothalamus, and infralimbic prefrontal cortex. Whereas basolateral amygdala and lateral hypothalamus contributions may be common to renewal of extinguished cocaine-, alcohol-, and sucrose-seeking, infralimbic prefrontal cortex contributions appear unique to renewal of cocaine-seeking and may reflect the habitual nature of relapse to cocaine.

The availability of the eukaryotic polypeptide chain initiation factor 4E (eIF4E) for protein synthesis is regulated by the 4E-binding proteins (4E-BPs), which act as inhibitors of cap-dependent mRNA translation. The ability of the 4E-BPs to sequester eIF4E is regulated by reversible phosphorylation at multiple sites. We show here that, in addition, 4E-BP1 is a substrate for polyubiquitination and that some forms of 4E-BP1 are simultaneously polyubiquitinated and phosphorylated. In Jurkat cells inhibition of proteasomal activity by MG132 enhances the level of hypophosphorylated, unmodified 4E-BP1 but only modestly increases the accumulation of high-molecular-weight, phosphorylated forms of 4E-BP1. In contrast, inhibition of protein phosphatase activity with calyculin A reduces the level of unmodified 4E-BP1 but strongly enhances the amount of phosphorylated, high-molecular-weight 4E-BP1. Turnover measurements in the presence of cycloheximide show that, whereas 4E-BP1 is normally a very stable protein, calyculin A decreases the apparent half-life of the normal-sized protein. Affinity chromatography on m(7)GTP-Sepharose indicates that the larger forms of 4E-BP1 bind very poorly to eIF4E. We suggest that the phosphorylation of 4E-BP1 may play a dual role in the regulation of protein synthesis, both reducing the affinity of 4E-BP1 for eIF4E and promoting the conversion of 4E-BP1 to alternative, polyubiquitinated forms.

To understand the functional-physiological influences on the depth distribution of deepwater cisco (bloater), Coregonus hoyi, in the Laurentian Great Lakes, we compared their gas bladder anatomy with the putative ancestor, shallowwater cisco, C. artedi, from a shallow inland lake. There were no general differences in the gas bladder anatomy—both coregonines possessed simple micro retia (counter-current gas multipliers) and an open pneumatic duct connection between the bladder and esophagus. Bloater possessed a significantly smaller duct diameter relative to body size in comparison with shallowwater cisco, yet the estimated resistance to air flow through the duct of bloater was ∼7.2 times lower than that of cisco. In light of these findings, we discuss evidence for two modes of gas bladder inflation in bloater: 1) gas deposition via micro retia at depth, and 2) gas acquisition via aquatic surface gulping (ASG). Because the hypolimnetic distribution of adult bloater precludes them from using ASG, we infer that these fish utilize micro retia to deposit gas into their bladders. We hypothesize that bloater compensate for relatively modest rates of gas deposition by not voiding excess gas. This “gas bladder conservation (GBC) hypothesis” for bloater is supported by four pieces of evidence: 1) deepwater habitation with no access to the surface to perform ASG, 2) a lack of extensive retia and modest gas deposition rates in coregonines, 3) a tendency to bloat when brought from depth to the surface, and 4) previous findings of a confined depth range, centered around the depth of neutral buoyancy.

Ultra wide-band frequency-modulated continuous-wave imaging systems operating in the lower terahertz regime have proven to be highly desirable for non-destructive testing applications. However, either quasi-optical configurations or the characteristics of typically used antennas as well as temperature-dependent nonlinear frequency sweeps restrict the achievable spatial resolution and reliability of these systems. In this contribution we report on the implementation of a dielectric rectangular rod antenna and a software-assisted feedback control loop to overcome these limitations.

Drug use may be exacerbated in environments which lack alternative means of engaging in rewarding behaviour. When alternative rewards are available, drug use may decrease-an effect that can be harnessed for therapeutic benefit. This idea is particularly well-supported by recent preclinical evidence demonstrating that a majority of rats will readily choose a potent non-drug reward over cocaine or heroin. Here we examine whether the same holds true for nicotine, a drug considered to have one of the highest addiction liabilities amongst drugs of abuse. Rats were trained to nose-poke separately for saccharin or nicotine on alternate days. Using a discrete-trial, forced-choice procedure, rats were then allowed to choose between nicotine and saccharin. This was followed by choice testing after a decrease in saccharin concentration (0.2-0%), omission of the fluid reward, an increase in nicotine concentration and following an extended nicotine self-administration history. All rats demonstrated a clear and immediate preference for saccharin at all times. This was despite variations in reward concentrations, or after an extensive nicotine history. Notably, rats preferred to nose-poke for water over nicotine and would omit responses when no fluid was delivered, rather than resume responding for nicotine. Overall, this study confirms and extends to nicotine previous research on other drugs of abuse, including cocaine and heroin. The ease with which rats quit nicotine in the present study contrasts with the well-known difficulty of humans to quit tobacco smoking. Possible factors that could explain this apparent discrepancy are discussed.

The prevalence of hedonic foods and associated advertising slogans has contributed to the rise of the obesity epidemic in the modern world. Research has shown that intake of these foods disrupt dopaminergic systems. It may be that a disruption of these circuits produces aberrant learning about food–cue relationships. We found that rodents given 28 days of intermittent access to sucrose exhibited a deficit in the ability to block learning about a stimulus when it is paired in compound with food and another stimulus that has already been established as predictive of the food outcome. This deficit was characterized by an approach to a cue signaling food delivery that is usually blocked by prior learning, an effect dependent on dopaminergic prediction-error signaling in the midbrain. Administering the D2 agonist quinpirole during learning restored blocking in animals with a prior history of sucrose exposure. Further, repeated central infusions of ghrelin produced a deficit in blocking in the same manner as sucrose exposure. We argue that changes in dopaminergic systems resulting from sucrose exposure are mediated by a disruption of ghrelin signaling as rodents come to anticipate delivery of the highly palatable sucrose outside of normal feeding schedules. This suggestion is supported by our finding that both sucrose and ghrelin treatments resulted in increases in amphetamine-induced locomotor responding. Thus, for the first time, we have provided evidence of a potential link between alterations in D2 receptors caused by the intake of hedonic foods and aberrant learning about cue–food relationships capable of promoting inappropriate feeding habits. In addition, we have found preliminary evidence to suggest that this is mediated by changes in ghrelin signaling, a finding that should stimulate further research into modulation of ghrelin activity to treat obesity.

The combined use of 3,4-methylenedioxymethamphetamine (MDMA, 'Ecstasy') with methamphetamine (METH) by recreational drug users is of particular concern due to their similar pharmacological and toxic profiles. In the current study we sought to elucidate why combining these particular drugs is such a popular choice among party-drug users. This was investigated through characterisation of the possible interactive effects of MDMA on METH intravenous self-administration. The first experiment involved characterisation of the METH dose-response curve for intravenous self-administration. Male Hooded-Wistar rats were trained to self-administer intravenous METH (0.01-0.3 mg/kg/infusion) and an inverted-U dose-response curve was obtained. In Experiment 2, a second squad of rats self-administered 0.01, 0.03 or 0.1 mg/kg/infusion METH and had small amounts of MDMA (0.001-0.03 mg/kg) then introduced into the infusion solution. Addition of MDMA to the METH infusion solution resulted in a dose independent reduction in responding. In Experiment 3, a third squad of rats was treated 20 min pre-session with an intraperitoneal injection of saline, 1.25 or 2.5 mg/kg of MDMA or METH to evaluate whether the reduction in responding evident in Experiment 2 was due to an MDMA-induced decrease in locomotor activity. Pre-treatment with intraperitoneal MDMA or METH had no effect on METH self-administration nor activity. We hypothesise that the reduction in METH self-administration caused by MDMA may reflect inhibitory effects of MDMA-induced 5-HT release on dopaminergic mechanisms.

Recovery of Endangered Species Act—listed salmonids in the Columbia River basin has relied upon the efficacy of the U.S. Army Corps of Engineer's juvenile salmon transportation program to move fish past Snake and Columbia River hydropower dams. The effectiveness of this program has been assessed by the indirect method of comparing smolt-to-adult returns. We present some of the first data and mortality estimates of barged and run-of-river (ROR) radio-tagged juvenile spring–summer Chinook salmon Oncorhynchus tshawytscha after release in the lower Columbia River, representing years of study. Our data suggest that smolt mortality (1) is very low for ROR and barged fish between Bonneville Dam and the estuary proper, a migratory distance of 180 river kilometers (rkm); (2) occurs in the lower estuary (rkm 0–46); (3) varies more across dates within a year than between years or between passage types (barged or ROR); (4) increases with time within a season and increasing numbers of avian predators, includi...

Epstein-Barr virus is a potent mitogen for human B lymphocytes and is associated with a large number of human malignancies. This large virus expresses several genes that may contribute to the transformed phenotype of infected cells and it possesses multiple strategies for the inhibition of apoptosis. The interferon-inducible protein kinase PKR is an important target for the anti-apoptotic actions of the virus and its activity is regulated by the small untranslated RNA, EBER-1. This review summarizes the mechanisms of action of EBER-1 and other Epstein-Barr virus gene products in the regulation of apoptosis, and presents a model of how EBER-1 exerts its effects on PKR activity.

Many virulence factors in Gram-positive bacteria are covalently anchored to the cell-wall peptidoglycan by sortase enzymes, a group of widely distributed cysteine transpeptidases. The Staphylococcus aureus Sortase A protein (SrtA) is the archetypal member of the Sortase family and is activated by Ca2+, an adaptation that may facilitate host colonization as elevated concentrations of this ion are encountered in human tissue. Here we show that a single Ca2+ ion bound to an ordered pocket on SrtA allosterically activates catalysis by modulating both the structure and dynamics of a large active site loop. Detailed nitrogen-15 relaxation measurements indicate that Ca2+ may facilitate the adaptive recognition of the substrate by inducing slow micro- to millisecond time-scale dynamics in the active site. Interestingly, relaxation compensated Carr-Purcell-Meiboom-Gill experiments suggest that the time scale of these motions is directly correlated with ion binding. The results of site-directed mutagenesis indicate that this motional coupling is mediated by the side chain of Glu-171, which is positioned within the β6/β7 loop and shown to contribute to Ca2+ binding. The available structural and dynamics data are compatible with a loop closure model of Ca2+ activation, in which the β6/β7 loop fluctuates between a binding competent closed form that is stabilized by Ca2+, and an open, highly flexible state that removes key substrate contacting residues from the active site.

l -Threonine 2 was converted in seven steps into the protected aminomercaptoalcohol 8, a threonine mimic. This compound 8 was coupled to various oligopeptides to produce two different tetrapeptide analogues, for example, 11 and 17, which were shown to inhibit the Sortase enzymes (SrtA and SrtB) via covalent attachment of the thiol groups of 11 and 17 to the catalytically active cysteine residue of the Sortase enzymes.

Phosphothioates may provide metabolic stability when compared to their phosphate counterparts, while retaining the potency and efficacy as agonists at sphingosine-1-phosphate (S1P) G-protein coupled receptors. Unlike their phosphate precursors, phosphothioate compounds with S1P-receptor profiles similar to that of FTY720, an emerging immunomodulator, were shown to evoke prolonged lymphopenia in vivo. Analysis of mouse plasma concentrations for a series of related alcohol/phosphate/phosphothioate compounds showed the conversion of the phosphate to alcohol. These preliminary data highlight the importance of metabolic regulation of S1P receptor ligands.

The rate of initiation of protein synthesis in mammalian cells in culture is regulated by the availability of essential amino acids. Previous reports have suggested that this effect is mediated by changes in the ratio of uncharged to charged tRNA species in these cells. We have investigated this possibility by examining the formation of 40-S subunit. Met-tRNAf ribosomal initiation complexes in extracts of Ehrlich ascites tumour cells; this is the stage in initiation previously shown to be regulated in response to lysine starvation of the intact cells. 1 Exposure of cells to a low concentration of cycloheximide makes polypeptide chain elongation the ratelimiting step, allows reformation of polysomes in starved cells and abolishes the effect of lysine deprivation on overall protein synthesis. Treatment with a high concentration of cycloheximide inhibits all protein synthesis and ‘freezes’ the polysomes in the state of aggregation which exists in the cell at that moment. The latter conditions also permit the tRNA pool to become fully charged in both fed and lysine-starved cells. In cell-free extracts prepared from cells treated with either low or high concentrations of cyclolieximide 40-S initiation complex formation nevertheless remains defective after lysine starvation of the cells. 2 Addition of deacylated tRNA, oxidized with periodate to prevent its recharging, to initiating cell-free extracts inhibits neither 40-S initiation complex formation nor overall protein synthesis in vitro. 3 Addition of lysine to an extract from lysine-starved cells, under conditions where this amino acid is charged on to endogenous tRNA, fails to stimulate either 40-S initiation complex formation or protein synthesis in vitro. These results suggest that amino acid starvation regulates the formation of 40-S polypeptide chain initiation complexes in mammalian cells by a mechanism which is not influenced by the rate of protein synthesis and does not directly involve uncharged tRNA.

1 Addition of haem to cell-free extracts from Ehrlich ascites tumour cells stimulates protein synthesis only in extracts from cells previously incubated in nutritionally complete conditions. Extracts from amino-acid-deprived cells do not respond to haem. The stimulation of protein synthesis in fed cell extracts is due to increased initiation on endogenous mRNA mediated by an increase in the levels of 40-S-subunit · Met-tRNA initiation complexes. Extracts from starved cells exhibit a defect in 40-S initiation complex formation which cannot be overcome by haem. 2 Experiments to test for the presence of an inhibitor of initiation in Ehrlich cell extracts by monitoring effects on translation in haem-supplemented reticulocyte lysates have revealed that extracts from both fed and starved cells contain one or more inhibitory activities which shut off protein synthesis, dissagregate polysomes and reduce the level of 40-S initiation complexes in the lysate. Extracts from starved cells are more inhibitory for protein synthesis than those from fed cells. 3 Initiation actor eIF-2 is phosphorylated by an endogenous Ehrlich cell protein kinase in vitro, but this occurs to the same extent in extracts from fed and starved cells. 4 We propose a possible model for the role of elF-2 in the control of protein synthesis by amino acid supply in Ehrlich cells.

The combined administration of heroin and cocaine ('speedball') is common among intravenous drug users. Dopamine receptors in the nucleus accumbens play a key role in cocaine self-administration; however, their role in speedball self-administration is unknown, as is the role of opiate receptors in this region.The effect of blocking dopamine D1, D2, mu-opiate or delta-opiate receptors in the nucleus accumbens on the intravenous self-administration of combined heroin and cocaine was examined in rats.Rats with bilateral cannulae implanted into the nucleus accumbens were trained to self-administer intravenous speedball (ratio of cocaine/heroin, 17:1) under a progressive ratio (PR) schedule. Prior to their self-administration session, rats were then microinjected with the dopamine D1 receptor antagonist SCH 23390 (1 and 6 nmol side(-1)), the D2 receptor antagonist raclopride (3 and 10 nmol side(-1)), the mu-opiate receptor antagonist CTOP (0.1, 0.3 and 1.0 nmol side(-1)), the delta-opiate receptor antagonist naltrindole (1.0, 3.0 and 10 nmol side(-1)) or a cocktail of SCH 23390 (1 nmol side(-1)) and CTOP (0.1 nmol side(-1)) into the nucleus accumbens.Microinjection of SCH 23390, raclopride or CTOP into the nucleus accumbens produced dose-dependent decreases in breakpoints under the PR schedule, while naltrindole was without effect. The highest dose of SCH 23390 also significantly reduced locomotor activity measured during speedball self-administration. The combination of SCH 23390 and CTOP significantly reduced breakpoints, while not affecting locomotor activity.These results indicate that dopamine and mu-opiate receptors, but not delta-opiate receptors, in the nucleus accumbens are involved in the reinforcing effects of speedball. Combined administration of D1 and mu-opiate receptor antagonists may be more selective at reducing the reinforcing effects of speedball self-administration than either drug alone.

Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid with the capacity to induce a broad range of cellular responses via its interaction with the S1P family of G-protein coupled receptors. A member of this receptor family, S1P(4), is highly and almost exclusively expressed in the lymphoid system and has been implicated in regulation of cell shape and motility. This report describes the synthesis of several potent benzimidazole based S1P(4) receptor selective agonists. For instance, compound 9b displayed an EC(50)=36 nM at the S1P(4) receptor using a [gamma-(35)S]GTP binding assay as compared to an EC(50)=37 nM for the endogenous ligand. We also report the effects of altering stereochemistry at the C2 position, methylation at the C1 and C2 position, and activity differences between the alcohol and phosphate head groups of the analogues.

There is increasing evidence that deregulation of gene expression at the level of mRNA translation can contribute to cell transformation and the malignant phenotype. Two steps in the pathway of polypeptide chain initiation, viz. the assembly of the 43S initiation complex catalysed by polypeptide chain initiation factor eIF2 and the binding of eIF4E to eIF4G during the recruitment of mRNA to the ribosome, have been shown to be likely targets for changes associated with tumorigenesis. The activity of eIF2 is controlled by changes in phosphorylation of the alpha subunit of this factor. The availability of eIF4E for binding to eIF4G is regulated by the phosphorylation of a small family of eIF4E-binding proteins (the 4E-BPs). The activities of the protein kinases and/or phosphatases responsible for the (de)phosphorylation of these substrates may in turn be controlled by cellular and viral oncogenes and tumour-suppressor genes. This review will describe recent aspects of the mechanisms involved, with particular emphasis on the regulation of the eIF2 alpha kinase PKR and the control of 4E-BP phosphorylation by viral gene products, growth-inhibitory cytokines and the tumour-suppressor protein p53.