Harvesting 48V at the National Superconducting Cyclotron Laboratory.

As part of an effort to develop aqueous isotope harvesting techniques at radioactive beam facilities, 48V and a cocktail of primary- and secondary-beam ions created by the fragmentation reaction of a 160 MeV/nucleon 58Ni beam were stopped in an aqueous target cell. After collection, 48V was separated from the mixture of beam ions using cation-exchange chromatography. The extraction efficiency from the aqueous solution was (47.0 ± 2.5)%, and the isolated 48V had a radiochemical purity of 95.8%. This proof-of-concept work shows that aqueous isotope harvesting could provide significant quantities of rare isotopes which are currently unavailable at conventional facilities.

A novel approach for BCR-ABL1 standardization to improve International Scale estimation.

INTRODUCTION: Standardization of BCR-ABL1 messenger RNA quantification by real-time PCR on the International Scale (IS) is critical for monitoring therapy response in chronic myelogenous leukaemia. Since 2006, BCR-ABL1 IS standardization is propagated along reference laboratories by calculating a laboratory-specific conversion factor (CF), co-ordinated in Europe through the European Treatment and Outcome Study project. Although this process has proven successful to some extent, it has not been achievable for all laboratories due to the complexity of the process and the stringent requirements in terms of numbers of samples to be exchanged. In addition, several BCR-ABL1 IS quantification methods and secondary reference materials became commercially available. However, it was observed that different IS methods generate consistently different results. METHODS: To overcome these difficulties, we have developed an alternative and simple approach of CF calculation, based on the retrospective analysis of existing external quality assessment (EQA) data. Our approach does not depend on the exchange of samples and is solely based on the mathematical CF calculation using EQA results. RESULTS AND CONCLUSION: We have demonstrated by thorough statistical validation that this approach performs well in converting BCR-ABL1 measurements to improve IS estimation. In expectation of a true golden standard method for BCR-ABL1 IS quantification, the proposed method is a valuable alternative.

EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.

PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

Fish response to the temporal hierarchy of the natural flow regime in the Daly River, northern Australia.

In this study, relationships between flow variation across multiple temporal scales and the distribution and abundance of three fish species, western rainbowfish Melanotaenia australis, sooty grunter Hephaestus fuliginosus and barramundi Lates calcarifer were examined at eight sampling reaches in the Daly River, Northern Territory, Australia. Discharge was highly seasonal during the study period of 2006-2010 with a distinct wet-dry discharge pattern. Significant catchment-wide correlations were identified between species abundance and hydrologic variables across several scales describing the magnitude and variability of flow. A Bayesian hierarchical model which accounted for >80% of variation in abundances for all species and age classes (i.e. juvenile and adult), identified the extent to which the influence of short-term flow variation was dependent upon the historical flow regime. There were distinct ontogenetic differences in these relationships for H. fuliginosus, with variability of recent flows having a negative effect on juveniles which was stronger at locations with higher historical mean daily flow. Lates calcarifer also displayed ontogenetic differences in relationships to flow variation with adults showing a positive association with increase in recent flows and juveniles showing a negative one. The effect of increased magnitude of wet-season flows on M. australis was negative in locations with lower historical mean daily flow but positive in locations with higher historical mean daily flow. The results highlighted how interactions between multiple scales of flow variability influence the abundance of fish species according to their life-history requirements.

Reflections and proposals to assure quality in molecular diagnostics.

Molecular diagnostic testing has become an important tool in clinical laboratories. Accreditation according to the international quality standard ISO15189:2007 for medical laboratories is required for reimbursement of several molecular diagnostic tests in Belgium. Since the ISO15189:2007 standard applies to medical laboratories in general, the particular requirements for quality and competence are mentioned in general terms, not taking into account the specificities of molecular biology testing. Therefore, the working group "MolecularDiagnostics.be" described a consensus interpretation of chapter 5, Technical requirements, of the ISO standard for application in molecular diagnostic laboratories. The manuscript can be used as an instrument to prepare internal and external audits that meet the 15015189:2007 (chapter 5) criteria.

Guidelines for an integrated diagnostic approach of chronic lymphoproliferative disorders in the routine laboratory of haematology in Belgium.

This paper summarizes the minimal workout of chronic lymphoproliferative disorders in a routine laboratory of haematology as recommended by a team of experienced laboratory supervisors in Belgium, taking into account the specific organisation of healthcare in Belgium, the innovations in the field of molecular analyses and related reimbursement. The starting point was essentially based upon clinical and/or haematological indications and it is emphasized that conclusions should be drawn in close dialogue with the clinician and experts in cytogenetics and histopathology. Reports made in the laboratory should be based upon an integration of cytomorphological, immunophenotypical and molecular data. These guidelines are not intended to be used as universal 'diagnostic pathways', but should be useful in developing local diagnostic pathways. It is well understood that this consensus, being valid anno 2009, may rapidly change with new technologies being introduced and new targets discovered.

Non-randomized, prospective, multi-centre evaluation of the ABSOLUTE .035 peripheral self-expanding stent system for occluded or stenotic superficial femoral or proximal popliteal arteries (ASSESS Trial): acute and 30-day results.

AIM: The aim of the paper was to investigate the performance of the ABSOLUTE .035 Peripheral Self-Expanding Stent System in preventing restenosis of superficial femoral or proximal popliteal arteries. Due to a lack of large controlled trials proving its long-term durability femoropopliteal artery stenting is still a matter of debate. In this paper we report the study design, the acute and short-term results of a prospective European registry on the treatment of TASC B and C femoropopliteal lesions with the use of the ABSOLUTE stent. METHODS: This prospective, non-randomized, multi-centre study enrolled 122 patients with symptomatic peripheral occlusive disease at 14 sites in Europe. Patients were included with obstructed femoropopliteal arteries. Key inclusion criteria were de novo lesions > or = 4.0 mm and < or = 7.0 mm in diameter, and > or = 40 mm and < or = 200 mm in length. Single target vessel treatment had to be performed with a maximum of three stents. RESULTS: Mean target lesion length was 108 +/- 44 mm (range 22.2 to 200 mm) and mean reference vessel diameter 4.6 +/- 0.8 mm by quantitative angiography; 71% of the lesions analyzable by quantitative angiography (QA) had total occlusions. A total of 227 stents were implanted, 224 of which were deployed successfully (98.7%). Mean percentage of diameter stenosis was reduced from 90.9 +/- 15.5 % (range 41.3 to 100) to 19.0 +/- 8.4% (range 2.3 to 41.5). Device and procedural success were 83.6% each whereas technical success reached 100%. Sixteen lesions had a > or = 30% residual stenosis post-procedure, 6 of them (37.5%) rated as being calcified. Eleven patients experienced major complications (9.1%) and 6 patients experienced minor complications (5%) within 30 days. Duplex ultrasound based 1-month restenosis rate was 9.3%. Target lesion revascularization (TLR) and target vessel revascularization (TVR) rates were 0.8% and 1.7%, respectively and amputation rate was 0.8%. Mean ankle-brachial index (ABI) at rest and after exercise increased significantly from baseline to 30 days follow-up by 0.63 +/- 0.20 to 0.94 +/- 0.17 and from 0.44 +/- 0.23 to 0.85 +/- 0.21, respectively (P<0.001 each). CONCLUSION: The treatment of TASC B and C femoro-popliteal lesions with use of the ABSOLUTE stent is safe and feasible. Short-term follow-up documents persistent improvement of hemodynamics. The 6- and 12-month data have to be awaited for further conclusions:

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Expression in Escherichia coli of the death domain of the human p55 tumor necrosis factor receptor.

The p55 tumor necrosis factor receptor (TNF-RI) is the main receptor by which TNF exerts its effects. The signaling capacity largely depends on the presence of an intact C-terminal protein-protein interaction domain, a so-called death domain (DD). Here we report the expression and purification of the human TNF-RI DD as a fusion with the Escherichia coli thioredoxin A (TRX) protein. When expressed under control of the bacteriophage T7 promoter, TRX-DD accumulates as a soluble protein in the cytoplasm of E. coli. The TRX-DD protein was released from the cells into the periplasmic fraction after osmotic shock. Due to self-association of the DD, a large part of the material appeared as multimers; it could be removed by selective precipitation and a combination of ion-exchange and size-exclusion chromatography. This purification protocol yielded 30 mg of purified, monomeric protein from 1 liter of shake-flask culture. The purified TRX-DD was found to be functional as it still bound to the TNF-RI-associated DD protein and the intracellular part of TNF-RI. We conclude that TRX-DD is correctly folded and can be used for further structure/function analysis.

Structure-activity relationship of the p55 TNF receptor death domain and its lymphoproliferation mutants.

Upon stimulation with tumor necrosis factor (TNF), the TNF receptor (TNFR55) mediates a multitude of effects both in normal and in tumor cells. Clustering of the intracellular domain of the receptor, the so-called death domain (DD), is responsible for both the initiation of cell killing and the activation of gene expression. To characterize this domain further, TNFR55 DD was expressed and purified as a thioredoxin fusion protein in Escherichia coli. Circular dichroism, steady-state and time-resolved fluorescence spectroscopy were used to compare TNFR55 DD with DDs of the Fas antigen (Fas), the Fas-associating protein with DD (FADD) and p75 nerve growth factor receptor, for which the 3-dimensional structure are already known. The structural information derived from the measurements strongly suggests that TNFR55 DD adopts a similar fold in solution. This prompted a homology modeling of the TNFR DD 3-D structure using FADD as a template. In vivo studies revealed a difference between the two lymphoproliferation (lpr) mutations. Biophysical techniques were used to analyze the effect of changing Leu351 to Ala and Leu351 to Asn on the global structure and its impact on the overall stability of TNFR55 DD. The results obtained from these experiments in combination with the modeled structure offer an explanation for the in vivo observed difference.

Reduction and temporary stabilization of acetabular fractures using ASIF mandibular reduction forceps: technique and results using plate fixation in 25 dogs.

OBJECTIVE: To report the results of acetabular fracture fixation in 25 dogs in which a specialized forceps (ASIF mandibular reduction forceps, Synthes USA, Paoli, PA) was used to obtain fracture reduction and stabilization. STUDY DESIGN: A retrospective clinical study. ANIMAL POPULATION: Twenty-five client-owned dogs with traumatic acetabular fractures. METHODS: The mandibular reduction forceps (MRF) use a screw on each side of the fracture to attach the clamp directly to the bone and permit direct manipulation of the fragments. Medical records from 25 dogs with acetabular fractures were reviewed to determine the effectiveness of this technique in obtaining, and then maintaining, fracture reduction while a plate was being applied. RESULTS: Clinical results were considered successful in 24 of 25 dogs; the small size of 1 dog prevented application of the MRF. The final reduction and fixation of the fractures was evaluated as anatomic in 17 dogs, near-anatomic in 6 dogs, and nonanatomic in 1 dog. CONCLUSIONS AND CLINICAL RELEVANCE: Application of the MRF is an effective technique for aiding the reduction of acetabular fractures in dogs. It maintains reduction while simultaneously permitting unimpeded access to the dorsal acetabular rim, thus facilitating accurate contouring of a plate. Accurate reduction and rigid fixation of articular fractures is essential to prevent secondary osteoarthritis.

Signal transduction by tumor necrosis factor and gene regulation of the inflammatory cytokine interleukin-6.

Interleukin (IL)-6 is a multifunctional cytokine that can be induced by a plethora of chemical or physiological compounds, including the inflammatory cytokines tumor necrosis factor (TNF) and IL-1. The molecule TNF has a trimeric configuration and thus binds to membrane-bound, cellular receptors to initiate cell death mechanisms and signaling pathways leading to gene induction. Previously, we showed that induced clustering of the intracellular domains of the p55 TNF receptor, or of their respective 'death domains' only, is sufficient to activate the nuclear factor kappa B (NF-kappa B) and several mitogen-activated protein kinase (MAPK) pathways. NF-kappa B is the exclusive transcription factor for induction of the IL-6 gene in response to TNF and functions as the final trigger to activate a multiprotein complex, a so-called 'enhanceosome', at the level of the IL-6 promoter. Furthermore, the enhanceosome displays histone acetylation activity, which turned out to be essential for IL-6 gene activation via NF-kappa B. However, activation of NF-kappa B alone is not sufficient for IL-6 gene induction in response to TNF, as inhibition of the coactivated extracellular signal-regulated kinase and p38 MAPK pathways blocks TNF-mediated gene expression. Nevertheless, the transactivating NF-kappa B subunit p65 is not a direct target of MAPK phosphorylation. Thus, we postulated that other components of the enhanceosome complex are sensitive to MAPK cascades and found that MAPK activity is unequivocally linked to the histone acetylation capacity of the enhanceosome to stimulate gene expression in response to TNF. In contrast, glucocorticoid repression of TNF-driven IL-6 gene expression does not depend on abrogation of histone acetyltransferase activity, but originates from interference of the liganded glucocorticoid receptor with the contacts between NF-kappa B p65 and the promoter configuration around the TATA box.

Structure/Function analysis of p55 tumor necrosis factor receptor and fas-associated death domain. Effect on necrosis in L929sA cells.

Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). In contrast, triggering of the fibrosarcoma cell line L929sA gives rise to characteristic cellular changes resulting in necrosis. The intracellular domain of TNF-R55 can be subdivided into two parts: a membrane-proximal domain (amino acids 202-325) and a C-terminal death domain (DD) (amino acids 326-413), which has been shown to be necessary and sufficient for apoptosis. Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defined by swelling of the cells, rapid membrane permeabilization, absence of nuclear condensation, absence of DNA hypoploidy, and generation of mitochondrial reactive oxygen intermediates, is also confined to the DD. The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone on TNF-induced necrosis was also observed with receptors solely containing the DD. TNF-R55-mediated necrosis is not affected by the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80-205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80-205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1-111) lacking the DD is prevented by CrmA. These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway.

Glucocorticoids repress NF-kappaB-driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell.

Glucocorticoids (GCs) are used to combat inflammatory diseases. Their beneficial effect relies mainly on the inhibition of NF-kappaB- and/or AP-1-driven proinflammatory gene expression. Previously, we have shown that GCs repress tumor necrosis factor-induced IL-6 gene expression by an NF-kappaB-dependent nuclear mechanism without changing the DNA-binding capacity of NF-kappaB or the expression levels of the cytoplasmic inhibitor of NF-kappaB (IkappaB-alpha). In the present work, we investigate the effect of GC repression on different natural and/or recombinant NF-kappaB-driven reporter gene constructs in the presence of increasing amounts of various coactivator molecules, such as CREB-binding protein (CBP), p300, and SRC-1. We found that GCs maintain their repressive capacities, irrespective of the amount of cofactor present in the cell. Similar results were obtained for the reciprocal transrepression of a GC receptor (GR) element-driven reporter gene by p65. We demonstrate that neither the expression levels of p65 and CBP nor their physical association are affected by activated GR. Using Gal4 chimeras, we show that repression by GCs is specific for p65-mediated transactivation, ruling out competition for limiting nuclear factors as the major underlying mechanism of gene repression. In addition, the transactivation potential of a point-mutated Gal4-p65 variant with a decreased CBP interaction capability is still repressed by GR. Finally, we present evidence that the specificity of GC repression on p65-driven gene expression is codetermined by the TATA box context.

The nuclear factor-kappaB engages CBP/p300 and histone acetyltransferase activity for transcriptional activation of the interleukin-6 gene promoter.

Expression of the pleiotropic cytokine interleukin (IL)-6 can be stimulated by the proinflammatory cytokine tumor necrosis factor (TNF) and the microbial alkaloid staurosporine (STS). In this report, the transcriptional mechanisms were thoroughly investigated. Whereas transcription factors binding to the activator protein-1-, cAMP-responsive element-, and CAAT enhancer-binding protein-responsive sequences are necessary for gene activation by STS, nuclear factor (NF)-kappaB alone is responsible and sufficient for inducibility by TNF, which reveals distinct signaling pathways for both compounds. At the cofactor level, cAMP-responsive element-binding protein-binding protein (CBP) or p300 potentiate basal and induced IL-6 promoter activation via multiple protein-protein interactions with all transcription factors bound to the promoter DNA. However, the strongest promoter activation relies on the p65 NF-kappaB subunit, which specifically engages CBP/p300 for maximal transcriptional stimulation by its histone acetyltransferase activity. Moreover, treatment of chromatin-integrated promoter constructions with the histone deacetylase inhibitor trichostatin A exclusively potentiates TNF-dependent (i.e. NF-kappaB-mediated) gene activation, while basal or STS-stimulated IL-6 promoter activity remains completely unchanged. Similar observations were recorded with other natural NF-kappaB-driven promoters, namely IL-8 and endothelial leukocyte adhesion molecule (ELAM). We conclude that, within an "enhanceosome-like" structure, NF-kappaB is the central mediator of TNF-induced IL-6 gene expression, involving CBP/p300 and requiring histone acetyltransferase activity.

Multiple signal transduction pathways regulate TNF-induced actin reorganization in macrophages: inhibition of Cdc42-mediated filopodium formation by TNF.

TNF is known to regulate macrophage (Mphi) migration, but the signaling pathways mediating this response have not been established. Here we report that stimulation of the 55-kDa TNF receptor (TNFR-1) induced an overall decrease in filamentous actin (F-actin), inhibited CSF-1- and Cdc42-dependent filopodium formation, and stimulated macropinocytosis. Using a panel of TNFR-1 mutants, the regions of the receptor required for each of these responses were mapped. The decrease in F-actin required both the death domain and the membrane proximal part of the receptor, whereas inhibition of filopodium formation and increased pinocytosis were only dependent upon a functional death domain. When the TNF-induced decrease in F-actin was inhibited using either receptor mutants or the compound D609, TNF-stimulated actin reorganization at the cell cortex became apparent. This activity was dependent upon the FAN-binding region of TNFR-1. We conclude that different domains of TNFR-1 mediate distinct changes in the Mphi cytoskeleton, and that the ability of TNF to inhibit Mphi chemotaxis may be due to decreased filopodium formation downstream of Cdc42.

Live birth of a bear cub following nonsurgical embryo collection.

In the near future, 6 of 8 bear species will face extinction mainly because of loss of their natural habitat. This loss of habitat will ultimately require some of these bears to be maintained in zoos and wildlife preserves in the hope of conserving genetic diversity. If the giant panda is representative of other bear species, reproductive performance will be inhibited in such an environment. In this study, we used the nonendangered American black bear (Ursus americanus) as the model for developing appropriate embryo transfer procedures. The donor bear mated numerous times between late May and early June. In late July we anesthetized her and used a series of telescoping sheaths to gain access to the uterus Then we passed a catheter through the largest sheath, inflated the balloon, and, using a 20-mL syringe, repeatedly infused into and then aspirated from the uterus PBS + BSA. We emptied the syringe into Petri dishes and observed 2 embryos. We rinsed the embryos, placed them in human tubal fluid + HSA + HEPES and then held them at 35 degrees C for 5 h. The recipient mated during mid-June; in late July we anesthetized her and, with the aid of laparoscopy, transferred an embryo into the cranial portion of the uterine horn ipsilateral to the ovary containing a CL. The recipient delivered 2 cubs in January. Necropsy results indicated that the neonates lived for 6 to 8 wk before succumbing to flooding in the den. The DNA from hair samples belonging to the neonates indicated that the male cub belonged to the donor, the female cub to the recipient. The delayed implantation mechanism in bears probably allowed for the successful development of the embryo in the presence of a substantial asynchrony between the donor and the recipient (13 d). We conclude that embryo transfer is possible in the American black bear and can lead to the birth of live cubs.

The 55-kDa tumor necrosis factor receptor induces clustering of mitochondria through its membrane-proximal region.

The cytokine tumor necrosis factor (TNF) activates diverse signaling molecules resulting in gene expression, differentiation, and/or cell death. Here we report a novel feature induced by TNF, namely translocation of mitochondria from a dispersed distribution to a perinuclear cluster. Mitochondrial translocation correlated with sensitivity to the cell death-inducing activity of TNF and was mediated by the 55-kDa TNF receptor (TNF-R55), but not by Fas, indicating that the signaling pathway requires a TNF-R55-specific but death domain-independent signal. Indeed, using L929 cells that express mutant TNF-R55, we showed that the membrane-proximal region of TNF-R55 was essential for signaling to mitochondrial translocation. In the absence of translocation, the cell death response was markedly delayed, pointing to a cooperative effect on cell death. Translocation of mitochondria, although dependent on the microtubules, was not imposed by the latter and was equally induced by TNF-independent immunoinhibition of the motor protein kinesin. Additionally, immunoinhibition with antibody directed against the tail domain of kinesin synergized with TNF-induced cell death. Based on this functional mimicry, we propose that a TNF-R55 membrane-proximal region-dependent signal impedes mitochondria-associated kinesin, resulting in cooperation with the TNF-R55 death domain-induced cytotoxic response and causing the observed clustering of mitochondria.

Ethanol modulates cocaine-induced behavioral change in inbred mice.

We recently conducted a study of the behavioral effects of combined cocaine and ethanol in genetically defined mice. Male and female C57BL/6 (B6) and DBA/2 (D2) were tested in an automated activity monitor on 2 consecutive days. On day 1, all animals received an IP injection of sterile saline and were placed into the activity monitor for 30 min. Behaviors measured were total distance traveled, stereotypy, nosepokes, and wall-seeking. On day 2, all animals were tested again for 15 min following injection of one of the following: saline, 10% v/v ethanol at 2.0 g kg(-1) or 2.0 g kg(-1) ethanol plus 5, 15, or 30 mg kg(-1) cocaine. Cocaine alone at the same doses was injected into separate groups of animals. For the B6 strain, the overall effect of ethanol was to reduce cocaine-induced locomotor stimulation; no consistent effect of ethanol on cocaine-induced locomotion was observed in D2 mice. Cocaine-induced inhibition of nosepokes in both strains and sexes was partially reversed by ethanol. Ethanol also partially reversed cocaine-elevated stereotypy in both strains and both sexes. In B6 mice, cocaine-increased wall seeking tended to be reversed by coadministration of ethanol, whereas no consistent pattern was observed in the D2s. Results from this study suggest that the several measures affected by cocaine (locomotor activity, stereotypy, exploration, thigmotaxis) were, in turn, differentially affected by concurrent treatment with ethanol. Furthermore, our results point to genetic-based differences in ethanol's effects on cocaine-related behaviors. We address the implications for combined ethanol and cocaine use in humans.

Induction of unresponsiveness to tumor necrosis factor (TNF) after autocrine TNF expression requires TNF membrane retention.

Tumor necrosis factor (TNF) has a specific gene-inducing activity on many cell types and exerts a cytotoxic effect on a number of tumor cell lines. However, several tumor cell types are resistant to TNF-induced effects, and some of these produce TNF. We previously demonstrated that introduction of an exogenous TNF gene in the TNF-sensitive cell line L929sA induced autocrine TNF production and unresponsiveness to the cytotoxic activity of TNF. This resistance required biologically active TNF and was correlated with complete down-modulation of the TNF receptors on the cell surface. We have now characterized this process in more detail. The role of expression of the membrane-bound TNF proform and its subsequent proteolytic processing in the induction of TNF unresponsiveness was investigated. Exchange of the TNF presequence for the signal sequence of interleukin-6 resulted in production of secreted TNF, but not in induction of TNF resistance. On the other hand, expression of non-secretable, membrane-bound TNF generated complete TNF unresponsiveness. To explore whether the requirement for anchoring reflected a specific functional role of the TNF presequence, the latter was replaced by the membrane anchor of trimeric chicken hepatic lectin. Expression of this construct induced complete TNF unresponsiveness. Hence, the role of the TNF presequence in the induction of TNF unresponsiveness only involves its function as a membrane anchor, which permits oligomerization of the TNF molecule into a biologically active homotrimer.