Search

G-protein-coupled receptor-regulated cAMP production from endosomes can specify signaling to the nucleus by moving the source of cAMP without changing its overall amount. How this is possible remains unknown because cAMP gradients dissipate over the nanoscale, whereas endosomes typically localize micrometers from the nucleus. We show that the key location-dependent step for endosome-encoded transcriptional control is nuclear entry of cAMP-dependent protein kinase (PKA) catalytic subunits. These are sourced from punctate accumulations of PKA holoenzyme that are densely distributed in the cytoplasm and titrated by global cAMP into a discrete metastable state, in which catalytic subunits are bound but dynamically exchange. Mobile endosomes containing activated receptors collide with the metastable PKA puncta and pause in close contact. We propose that these properties enable cytoplasmic PKA to act collectively like a semiconductor, converting nanoscale cAMP gradients generated from endosomes into microscale elevations of free catalytic subunits to direct downstream signaling.

Introduction Indications for prophylactic antibiotic therapy in nonoperative, closed fractures of the orbit and zygoma remain controversial and are based on anecdotal data. The purpose of this study was to report the incidence of infectious sequelae among patients who presented to our institution with stated fractures and who were not administered prophylactic antibiotic therapy. The authors hypothesized that an increase in infectious complications would not be seen in these patients. Methods The authors conducted a prospective single site study from October 2015 to December 2019. Patients with closed, nonoperative fractures involving the orbit and/or zygoma were included. These patients did not receive prophylactic antibiotic therapy for their fracture patterns, and infectious complications including orbital cellulitis, meningitis, and bacterial sinusitis were noted if present during follow-up. Results Of 301 patients with closed, nonoperative orbital and/or zygomatic fractures, 268 were included in the study and 33 were excluded because of administration of antibiotics. The average age was 60 years, and the most common mechanism of injury was a traumatic fall. Fracture types included 100 orbital wall, 133 orbital floor, 15 orbital rim, 23 orbital roof, 48 zygoma, and 44 zygomaticomaxillary compound fractures. Infectious sequelae were not identified in any patient. Conclusions Of the 268 patients included in the study, no infectious complications were identified during the follow-up period. This outcome further supports that the use of prophylactic antibiotics is not indicated for closed, nonoperative fractures involving the orbital and/or zygoma. Prospective randomized control studies would be useful to support this outcome and better guide clinical antibiotic prescribing practices.

To test if and how chemotherapy-induced peripheral neurotoxicity (CIPN) is perceived differently by patients and physicians, making assessment and interpretation challenging. We performed a secondary analysis of the CI-PeriNomS study which included 281 patients with stable CIPN. We tested: (a) the association between patients' perception of activity limitation in performing eight common tasks and neurological impairment and (b) how the responses to questions related to these daily activities are interpreted by the treating oncologist. To achieve this, we compared patients' perception of their activity limitation with neurological assessment and the oncologists' blind interpretation. Distribution of the scores attributed by oncologists to each daily life maximum limitation ("impossible") generated three groups: Group 1 included limitations oncologists attributed mainly to motor impairment; Group 2 ones mainly attributed to sensory impairment and Group 3 ones with uncertain motor and sensory impairment. Only a subset of questions showed a significant trend between severity in subjective limitation, reported by patients, and neurological impairment. In Group 1, neurological examination confirmed motor impairment in only 51%-65% of patients; 76%-78% of them also had vibration perception impairment. In Group 2, sensory impairment ranged from 84% to 100%; some degree of motor impairment occurred in 43%-56% of them. In Group 3 strength reduction was observed in 49%-50% and sensory perception was altered in up to 82%. Interpretation provided by the panel of experienced oncologists was inconsistent with the neurological impairment. These observations highlight the need of a core set of outcome measures for future CIPN trials.

Simple Summary Cancer is one of the main causes of death worldwide and early detection is crucial for effective treatment. Scientists have therefore focused on the identification of circulating cancer-specific molecules, so-called markers, that can be detected with non- or less-invasive techniques. One attractive emerging marker is represented by circulating antibodies against molecules that are specific for the tumor cells and not produced by normal cells. Many such auto-antibodies have been discovered, but still not much is known about their significance and role in either promoting or inhibiting tumor growth. The aim of this review is to summarize the current knowledge on auto-antibodies in different cancer types and on their current or possible utilization for effective cancer management. Reference to autoimmune diseases will also be made, as they share with cancer the presence of such auto-antibodies. Abstract Auto-antibodies are classically associated with autoimmune diseases, where they are an integral part of diagnostic panels. However, recent evidence is accumulating on the presence of auto-antibodies against single or selected panels of auto-antigens in many types of cancer. Auto-antibodies might initially represent an epiphenomenon derived from the inflammatory environment induced by the tumor. However, their effect on tumor evolution can be crucial, as is discussed in this paper. It has been demonstrated that some of these auto-antibodies can be used for early detection and cancer staging, as well as for monitoring of cancer regression during treatment and follow up. Interestingly, certain auto-antibodies were found to promote cancer progression and metastasis, while others contribute to the body’s defense against it. Moreover, auto-antibodies are of a polyclonal nature, which means that often several antibodies are involved in the response to a single tumor antigen. Dissection of these antibody specificities is now possible, allowing their identification at the genetic, structural, and epitope levels. In this review, we report the evidence available on the presence of auto-antibodies in the main cancer types and discuss some of the open issues that still need to be addressed by the research community.

The use of virtual surgical planning and computer-aided design/computer-aided manufacturing has gained popularity in the surgical correction of craniosynostosis. This study expands the use of virtual surgical planning and computer-aided design/computer-aided manufacturing in cranial vault reconstruction by using these methods to reconstruct the anterior vault using a single endocortically-plated unit constructed from the posterior calvarium. This technique was designed to reduce the risk of undesirable contour deformities that can occur when multiple bone grafts are used to reconstruct the anterior vault and fronto-orbital rim. Six patients were included in this study, all of which had nonsyndromic craniosynostosis. Excellent aesthetic outcomes were obtained in all patients, without complication. Additionally, the placement of a single reconstructive unit constructed from the posterior calvarium was efficient, aesthetically pleasing, and minimized postoperative contour deformities secondary to bone gaps, resorption, and often palpable resorbable plates.

Large head and neck arteriovenous malformations are notoriously difficult to manage given their location and propensity for extreme hemorrhage. We propose a unique approach utilizing a Hyperform balloon and percutaneous Surgiflo sclerotherapy to provide intraoperative hemostatic stability during the excision of a left-sided scalp arteriovenous malformation. In a hybrid operating room a microcatheter balloon was fed into the left external carotid artery for occlusion of the malformation's main blood supply, and subsequently followed with digital subtraction angiography guided sclerotherapy of selective vessels. A split thickness graft was used to reconstruct the site of excision. This method offered optimal hemostatic control with a blood loss less than 120 cc. Our approach may offer safety advantages over traditional modalities and allow resection of head and neck lesions previously thought to be inoperable.

Spectral imaging is a powerful technique used to simultaneously study multiple fluorophore labels with overlapping emissions. Here, we present a computational spectral imaging method, which uses sample spatial fluorescence information as a reconstruction constraint. Our method addresses both the under-sampling issue of compressive spectral imaging and the low throughput issue of scanning spectral imaging. With simulated and experimental data, we have demonstrated the reconstruction precision of our method in two and three-color imaging. We have experimentally validated this method for differentiating cellular structures labeled with two red-colored fluorescent proteins, tdTomato and mCherry, which have highly overlapping emission spectra. Our method has the advantage of totally free wavelength choice and can also be combined with conventional filter-based sequential multi-color imaging to further improve multiplexing capability.

PURPOSE: To investigate the scale structure and psychometrics of the EORTC chemotherapy-induced peripheral neuropathy module (QLQ-CIPN20).METHODS: Using confirmatory factor analyses (CFA), we tested two hypothesized scale structure models of the QLQ-CIPN20 in 473 patients with non-small cell lung cancer, 281 patients with heterogeneous cancer diagnoses, and 500 patients with colorectal cancer. We also modeled the two hypothesized models as bi-factor models. These included a general factor, in addition to the specific domain factors. Additional models were investigated with exploratory factor analysis (EFA). Known groups validity was evaluated where justified.RESULTS: CFA could not confirm the two hypothesized models (Model 1: CFI < 0.926; TLI < 0.914; RMSEA > 0.077 and Model 2: CFI < 0.906; TLI < 0.887; RMSEA > 0.105) in any of the three samples. Including a general factor to these two hypothesized models to produce a bi-factor model also did not yield satisfactory results. Using EFA, we identified four different factor structures in the three samples that were unstable due to cross loadings of the items. When scoring the QLQ-CIPN20 as a simple, additive checklist evidence was found for known groups validity in the first two samples based on Common Toxicity Criteria (CTC-AE), and in the third sample based on exposure to CIPN-inducing chemotherapy.CONCLUSIONS: Neither CFA nor EFA yielded support for a stable subscale structure for the QLQ-CIPN20. Scoring the questionnaire as a simple additive checklist results in acceptable validity.

Background: asparagine-glycine-arginine-human tumour necrosis factor (NGR-hTNF), an agent selectively damaging the tumour vasculature, showed a biphasic dose–response curve in preclinical models. Previous phase I trials of NGR-hTNF indicated 0.8 and 45 μg/m2 as optimal biological and maximum-tolerated dose, respectively. Patients and methods: Two sequential cohorts of 12 colorectal cancer (CRC) patients who had failed standard therapies received NGR-hTNF 0.8 or 45 μg/m2 in combination with capecitabine–oxaliplatin (XELOX). Results: Median number of prior treatment lines was 3 in the low-dose and 2 in the high-dose cohort. Overall, 21 patients had been pretreated with oxaliplatin-based regimens. No grade 3–4 NGR-hTNF-related toxicities were observed. Grade 1–2 chills were reported in 43% and 40% of cycles in the low-dose and high-dose cohorts, respectively. In the low-dose cohort, one patient achieved a partial response and five had stable disease for a median of 4.6 months. In the high-dose cohort, six patients had stable disease for a median of 3.6 months. Three-month progression-free survival (PFS) rates were 50% and 33% in the low-dose and high-dose cohort, respectively. Three patients in low-dose cohort experienced PFS longer than PFS on last prior therapy. Conclusions: Both NGR-hTNF doses were safely combined with XELOX in pretreated CRC patients. Hint of activity was apparent only with low-dose NGR-hTNF.

Recent reports in model plant species have highlighted a role for DNA methylation pathways in the regulation of the somatic-to-reproductive transition in the ovule, suggesting that apomixis (asexual reproduction through seeds) likely relies on RdDM downregulation. Our aim was therefore to explore this hypothesis by characterizing genes involved in DNA methylation in the apomictic grass Eragrostis curvula. We explored floral transcriptomes to identify homologs of three candidate genes, for which mutations in Arabidopsis and maize mimic apomixis (AtAGO9/ZmAGO104, AtCMT3/ZmDMT102/ZmDMT105, and AtDDM1/ZmCHR106), and compared both their spatial and temporal expression patterns during reproduction in sexual and apomictic genotypes. Quantitative expression analyses revealed contrasting expression patterns for the three genes in apomictic vs sexual plants. In situ hybridization corroborated these results for two candidates, EcAGO104 and EcDMT102, and revealed an unexpected ectopic pattern for the AGO gene during germ line differentiation in apomicts. Although our data partially support previous results obtained in sexual plant models, they suggest that rather than an RdDM breakdown in the ovule, altered localization of AtAGO9/ZmAGO104 expression is required for achieving diplospory in E. curvula. The differences in the RdDM machinery acquired during plant evolution might have promoted the emergence of the numerous apomictic paths observed in plants. Fil: Selva, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Siena, Lorena Adelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentina Fil: Rodrigo, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; Argentina Fil: Garbus, Ingrid. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Ciencias de la Salud; Argentina Fil: Zappacosta, Diego Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; Argentina Fil: Romero, José Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina Fil: Ortiz, Juan Pablo Amelio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentina Fil: Pessino, Silvina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentina Fil: Leblanc, O.. Universidad de Montpellier; Alemania Fil: Echenique, Carmen Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; Argentina

Chemotherapy-induced peripheral neuropathy (CIPN) lacks standardized clinical measurement. The objective of the current secondary analysis was to examine data from the CIPN Outcomes Standardization (CI-PeriNomS) study for associations between clinical examinations and neurophysiological abnormalities. Logistic regression estimated the strength of associations of vibration, pin, and monofilament examinations with lower limb sensory and motor amplitudes. Examinations were classified as normal (0), moderately abnormal (1), or severely abnormal (2). Among 218 participants, those with class 1 upper extremity (UE) and classes 1 or 2 lower extremity (LE) monofilament abnormality were 2.79 (95% confidence interval [CI]: 1.28-6.07), 3.49 (95%CI: 1.61-7.55), and 4.42 (95%CI: 1.35-14.46) times more likely to have abnormal sural nerve amplitudes, respectively, compared to individuals with normal examinations. Likewise, those with class 2 UE and classes 1 or 2 LE vibration abnormality were 8.65 (95%CI: 1.81-41.42), 2.54 (95%CI: 1.19-5.41), and 7.47 (95%CI: 2.49-22.40) times more likely to have abnormal sural nerve amplitudes, respectively, compared to participants with normal examinations. Abnormalities in vibration and monofilament examinations are associated with abnormal sural nerve amplitudes and are useful in identifying CIPN.

We tested the hypothesis that mutant tau proteins that cause neurodegeneration and dementia differentially alter kinesin translocation along microtubules (MTs) relative to normal tau in vitro. We employed complementary in vitro motility assays using purified recombinant kinesin, purified recombinant tau, and purified bovine brain α:β tubulin to isolate interactions among these components without any contribution by cellular regulatory mechanisms. We found that kinesin translocates slower along MTs assembled by any of three independent tau mutants (4-repeat P301L tau, 4-repeat ΔN296 tau, and 4-repeat R406W tau) relative to its translocation rate along MTs assembled by normal, 4-repeat wild type (WT) tau. Moreover, the R406W mutation exhibited isoform specific effects; while kinesin translocation along 4-repeat R406W tau assembled MTs is slower than along MTs assembled by 4-repeat WT tau, the R406W mutation had no effect in the 3-repeat tau context. These data provide strong support for the notion that aberrant modulation of kinesin translocation is a component of tau-mediated neuronal cell death and dementia. Finally, we showed that assembling MTs with taxol before coating them with mutant tau obscured effects of the mutant tau that were readily apparent using more physiologically relevant MTs assembled with tau alone, raising important issues regarding the use of taxol as an experimental reagent and novel insights into therapeutic mechanisms of taxol action.

Background Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating and dose-limiting complication of cancer treatment. Thus far, the impact of CIPN has not been studied in a systematic clinimetric manner. The objective of the study was to select outcome measures for CIPN evaluation and to establish their validity and reproducibility in a cross-sectional multicenter study. Patients and methods After literature review and a consensus meeting among experts, face/content validity were obtained for the following selected scales: the National Cancer Institute-Common Toxicity Criteria (NCI-CTC), the Total Neuropathy Score clinical version (TNSc), the modified Inflammatory Neuropathy Cause and Treatment (INCAT) group sensory sumscore (mISS), the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30, and CIPN20 quality-of-life measures. A total of 281 patients with stable CIPN were examined. Validity (correlation) and reliability studies were carried out. Results Good inter-/intra-observer scores were obtained for the TNSc, mISS, and NCI-CTC sensory/motor subscales. Test–retest values were also good for the EORTC QLQ-C30 and CIPN20. Acceptable validity scores were obtained through the correlation among the measures. Conclusion Good validity and reliability scores were demonstrated for the set of selected impairment and quality-of-life outcome measures in CIPN. Future studies are planned to investigate the responsiveness aspects of these measures.

Quorum sensing (QS) is a bacterial communication mechanism in which secreted signaling molecules impact population function and gene expression. QS-like phenomena have been reported in eukaryotes with largely unknown contributing molecules, functions, and mechanisms. We identify Qsp1, a secreted peptide, as a central signaling molecule that regulates virulence in the fungal pathogen Cryptococcus neoformans. QSP1 is a direct target of three transcription factors required for virulence, and qsp1Δ mutants exhibit attenuated infection, slowed tissue accumulation, and greater control by primary macrophages. Qsp1 mediates autoregulatory signaling that modulates secreted protease activity and promotes cell wall function at high cell densities. Peptide production requires release from a secreted precursor, proQsp1, by a cell-associated protease, Pqp1. Qsp1 sensing requires an oligopeptide transporter, Opt1, and remarkably, cytoplasmic expression of mature Qsp1 complements multiple phenotypes of qsp1Δ. Thus, C.neoformans produces an autoregulatory peptide that matures extracellularly but functions intracellularly to regulate virulence.

Apomixis in plants consists of asexual reproduction by seeds. Here we characterized at structural and functional levels an apomixis-linked sequence of Paspalum simplex homologous to subunit 3 of the ORIGIN RECOGNITION COMPLEX (ORC3). ORC is a multiprotein complex which controls DNA replication and cell differentiation in eukaryotes. Three PsORC3 copies were identified, each one characterized by a specific expression profile. Of these, PsORC3a, specific for apomictic genotypes, is a pseudogene that was poorly and constitutively expressed in all developmental stages of apomictic flowers, whereas PsORC3b, the putative functional gene in sexual flowers, showed a precise time-related regulation. Sense transcripts of PsORC3 were expressed in the female cell lineage of both apomictic and sexual reproductive phenotypes, and in aposporous initials. Although strong expression was detected in sexual early endosperm, no expression was present in the apomictic endosperm. Antisense PsORC3 transcripts were revealed exclusively in apomictic germ cell lineages. Defective orc3 mutants of rice and Arabidopsis showed normal female gametophytes although the embryo and endosperm were arrested at early phases of development. We hypothesize that PsORC3a is associated with the down-regulation of its functional homolog and with the development of apomictic endosperm which deviates from the canonical 2(maternal):1(paternal) genome ratio. Fil: Siena, Lorena Adelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentina Fil: Ortiz, Juan Pablo Amelio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentina Fil: Calderini, Ornella. Istituto di Bioscienze e Biorisorse; Italia Fil: Paolocci, Francesco. Istituto di Bioscienze e Biorisorse; Italia Fil: Cáceres, Maria E.. Istituto di Bioscienze e Biorisorse; Italia Fil: Kaushal, Pankaj. Istituto di Bioscienze e Biorisorse; Italia Fil: Grisan, Simone. Istituto di Bioscienze e Biorisorse; Italia Fil: Pessino, Silvina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentina Fil: Pupilli, Fulvio. Istituto di Bioscienze e Biorisorse; Italia

Using a suite of biophysical tools, we assess the mechanical, structural, and functional properties of microtubules (MTs) stabilized by the chemotherapeutic compounds epothilone-A, epothilone-B, and taxol in vitro. We demonstrate that MTs stabilized by epothilone-A or epothilone-B are competent to bind tau proteins and support kinesin translocation. Kinesin speed is sensitive not only to the type of small molecule stabilizer used but also to the presence of the essential MT-associated protein tau. Epothilone-stabilized MTs are substantially less stiff than taxol-stabilized MTs. The addition of tau proteins to MTs stabilized by either epothilone compound or taxol further reduces stiffness. Taken together, these results suggest that small molecule stabilizers do not simply stabilize a “native” MT structure, but rather they modulate the structure, function, and mechanics of the MTs they bind. This may have important consequences to the therapeutic use of these agents in cancer chemotherapies. © 2012 Wiley Periodicals, Inc

Background NGR-hTNF consists of human tumour necrosis factor (hTNF) fused with the tumour-homing peptide Asp-Gly-Arg (NGR), which is able to selectively bind an aminopeptidase N overexpressed on tumour blood vessels. Preclinical antitumour activity was observed even at low doses. We evaluated the activity and safety of low-dose NGR-hTNF in colorectal cancer (CRC) patients failing standard therapies. Patients and methods Thirty-three patients with progressive disease at study entry received NGR-hTNF 0.8μg/m 2 given intravenously every 3weeks. The median number of prior treatment regimens was three (range, 2–5). One-quarter of patients had previously received four or more regimens and two-thirds targeted agents. Progression-free survival (PFS) was the primary study objective. Results NGR-hTNF was well tolerated. No treatment-related grade 3 to 4 toxicities were detected, most common grade 1 to 2 adverse events being short-lived, infusion-time related chills (50.0%). One partial response and 12 stable diseases were observed, yielding a disease control rate of 39.4% (95% CI, 22.9–57.8%). Median PFS and overall survival were 2.5months (95% CI, 2.1–2.8) and 13.1months (95% CI, 8.9–17.3), respectively; whereas in patients who achieved disease control the median PFS and overall survival were 3.8 and 15.4months, respectively. In an additional cohort of 13 patients treated at same dose with a weekly schedule, there was no increased toxicity and 2 patients experienced PFS longer than 10months. Conclusion Based on tolerability and preliminary evidence of disease control in heavily pretreated CRC patients, NGR-hTNF deserves further evaluation in combination with standard chemotherapy.

Background: The anti-epidermal growth factor receptor (EGFR) antibody cetuximab is active in heavily pretreated patients with metastatic colorectal cancer (mCRC) both in monotherapy and in combination with chemotherapy (CT). This study assesses the antitumor activity of single-agent cetuximab in CT-naive patients. Patients and methods: Phase II clinical trial was used. Patients were EGFR positive by immunohistochemistry and were not candidate for radical surgery, even in the case of substantial tumor shrinkage. Cetuximab was administered weekly. Results: Thirty-nine patients were treated and evaluated. The most common adverse event was skin toxicity (89% any grade; 48% grade 1; 31% grade 2; 10% grade 3). One patient had a complete response and three obtained partial responses (10% overall response rate). Thirteen patients had stable disease (34%). Twenty-two patients experienced progressive disease (56%). Overall median time to progression (TTP) was 2 months, and the responders individual TTP was 12, 9, 9, and 6 months. Conclusions: Even in chemo-naive patients, cetuximab as single agent is active only in a small fraction of mCRC, similarly to what has been reported for heavily pretreated patients. The extent of benefit when response occurs is, however, such that it is mandatory to intensify the search for the predictive markers of response to cetuximab therapy.

Composite scales such as the Total Neuropathy Score clinical version (TNSc(©) ) have been widely used to measure neurological impairment in a standardized manner but they have been criticized due to their ordinal setting having no fixed unit. This study aims to improve impairment assessment in patients with chemotherapy-induced peripheral neuropathy (CIPN) by subjecting TNSc(©) records to Rasch analyses. In particular, we wanted to investigate the influence of factors affecting the use of the TNSc(©) in clinical practice. TNSc(©) has 7 domains (sensory, motor, autonomic, pin-prick, vibration, strength, and deep tendon reflexes [DTR]) each being scored 0-4. Data obtained in 281 patients with stable CIPN were subjected to Rasch analyses to determine the fit to the model. The TNSc(©) did not meet Rasch model's expectations primarily because of misfit statistics in autonomic and DTR domains. Removing these two, acceptable model fit and uni-dimensionality were obtained. However, disordered thresholds (vibration and strength) and item bias (mainly cultural) were still seen, but these findings were kept to balance the assessment range of the Rasch-Transformed TNSc(©) (RT-TNSc(©) ). Acceptable reliability findings were also obtained. A 5-domains RT-TNSc(©) may be a more proper assessment tool in patients with CIPN. Future studies are needed to examine its responsive properties.

Apomixis gives rise to a novel prospect in agricultural production: the opportunity to establish genetically stable, seed-propagating clones of crops, which can perpetuate themselves across countless sporophytic generations. This asexual mode of reproduction, that naturally occurs in some angiosperms, may prove to be an unrivalled biotechnological tool to improve crop yields. Nevertheless, an exhaustive molecular characterisation of the phenomenon is still needed. This article presents the current state of knowledge on the molecular genetic basis of gametophytic apomixis, and discusses the perspectives profiled by an extensive use of the character in agriculture.

Apomixis is an asexual mode of reproduction by seed, which is found in a number of families of higher plants. During apomictic processes, both meiosis and egg cell fertilisation are omitted making offspring exact genetic replicas of the mother plant. To gain an insight into the molecular basis of apomictic reproduction, gene expression in mature ovaries containing reduced and unreduced embryo sacs was compared in Brachiaria, an apomictic tropical forage grass. Experimental systems for the molecular study of apomixis and sexuality have limitations mainly resulting from numerous differences in the genetic make-up of the plant material. When unrelated to the reproductive behaviour, these may dramatically affect the results if a conventional differential display strategy is applied (comparison made on a one-to-one plant basis). To overcome these limitations, we examined mRNA populations extracted from ovaries collected from pooled samples of various apomictic and sexual plants using the polymerase chain reaction (PCR) differential display and non radioactive cDNA detection procedures. Using this approach, two mRNAs whose expression appeared to be related to the late stages of apomictic reproduction were identified. These results indicate that gene expression in pooled mRNA analysis is a promising tool to elucidate the molecular basis of apomixis.

Background In cross-sectional studies, ambulatory blood pressure (ABP) correlates more closely than clinic BP with the organ damage of hypertension. Whether ABP predicts development or regression of organ damage over time better than clinic BP, however, is unknown. Methods and Results In 206 essential hypertensive subjects with left ventricular hypertrophy (LVH), we measured clinic supine BP, 24-hour ABP, and left ventricular mass index (LVMI, echocardiography) before and after 12 months of treatment with lisinopril (20 mg UID) without or with hydrochlorothiazide (12.5 or 25 mg UID). Measurements included random-zero, clinic orthostatic, and home BP. In all, 184 subjects completed the 12-month treatment period. Before treatment, clinic supine BP was 165±15/105±5 mm Hg (systolic/diastolic), 24-hour average BP was 149±16/95±11 mm Hg, and LVMI was 158±32 g/m 2 . At the end of treatment, they were 139±12/87±7 mm Hg, 131±12/83±10 mm Hg, and 133±26 g/m 2 , respectively ( P r =.34/.27, P r =.42/.38, P Conclusions In hypertensive subjects with LVH, regression of LVH was predicted much more closely by treatment-induced changes in ABP than in clinic BP. This provides the first longitudinally controlled evidence that ABP may be clinically superior to traditional BP measurements.

Ilex paraguariensis plants were subjected to progressive soil water deficit, and differential display (DD) was used to analyse gene expression in leaves to characterise physiological responses to mild and severe water deficits. A cDNA fragment showing strong homology with the flavoprotein subunit (SDH1) of succinate:ubiquinone oxidoreductase (succinate dehydrogenase, SDH, EC 1.3.5.1) was upregulated in plants exposed to drought. Quantitative real-time PCR revealed that the SDH1-like transcript level began to increase when the leaf relative water content (RWC) decreased to 78% and peaked when the RWC dropped to 57%. A correlation between abscisic acid (ABA) concentration and variations in transcript levels was assessed by GC-SIM. After rehydration, SDH1 mRNA and ABA returned to their initial levels. In stressed leaves sprayed with ABA SDH1 mRNA accumulated in greater levels compared to stressed leaves that did not receive ABA. Moreover, the enzymatic activity of succinate dehydrogenase increased 1.5-fold in the mature leaves of ABA-treated plants. This physiological response may be related to the tendency of this species to minimise water losses through stomatal closure in the early stages of dehydration to avoid tissue desiccation. As the leaf water potential diminished due to an increase in water restriction, I. paraguariensis leaf tissues reacted by making osmotic adjustments to sustain tissue metabolic activity, which enables the recovery of photosynthesis upon re-watering. These results provide new insights concerning the linkage between plant respiration and photosynthetic metabolism that could be potentially further used in breeding programs aiming water tolerant genotypes. Fil: Acevedo, Raúl Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina Fil: Maiale, Santiago Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Pessino, Silvina Claudia. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bottini, Ambrosio Ruben. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Ruiz, Oscar Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Sansberro, Pedro Alfonso. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina

On the basis of the amino acid sequence of isolated tryptic peptides, it has been established that the differentiation enhancing factor, produced and active on murine erythroleukemia (MEL)cells, possesses a unique sequence, with no similarity to that of known proteins. Accordingly, this factor can be defined as a novel biologically active peptide. An antisense oligodeoxynucleotide, deduced from the sequence of a nonadecapeptide (produced by tryptic digestion of the factor), decreases the rate and the extent of MEL cell differentiation, induced by hexamethylenebisacetamide. In these cells the amount of the factor is reduced to one third of that constitutively present in untreated cells. Exogenous addition of the factor restores cell inducibility to normal values. Taken together, these results demonstrate the presence in MEL cells of a new factor, structurally and firnctionally unrelated to any of the known biologically active peptides, and suggest its crucial role in the promotion of an initial signal, in chemically induced erythroid differentiation.

Paspalum notatum Flugge is a warm-season forage grass with mainly diploid (2n =20) and autotetraploid (2n = 40) representa-tives.Diploid races reproduce sexually andrequire crosspollination due to a self-incom-patible mating system, while autotetraploids reproduce by aposporous apomixis. The objec-tives of this work were to develop a genetic linkage map of Paspalum notatum Flugge at the tetraploid level, identify the linkage/s group/s associated with apomixis and carry out a general characterization of its mode of inheritance. A pseudo test-cross F1 family of 113 individuals segregating for the mode of reproduction was obtained by crossinga syn-thetic completely sexual tetraploid plant (Q4188) as female parent with a naturalaposporous individual (Q4117) as pollen donor.Map construction was based on single-dose markers (SDAFs) segregating from both par-ents. Two linkage maps (female and male) were constructed. Within each map, homolo-gous groups were assembled by detectingrepulsion-phase linked SDAFs. Putative Q4188 and Q4117 homolog groups were identified by mapping shared single dose mark-ers (BSDF). The Q4188 map consisted of 263 markers distributed on 26 co-segregation groups over a total genetic distance of 1.590.6 cM, while the Q4117 map contained 216 loci dispersed on 39 co-segregation groups along 2.265.7 cM, giving an estimated genome cov-erage of 88% and 83%, respectively. Seven and 12 putative homologous chromosomes were detected within Q4188 and Q4117 maps,respectively. Afterward, ten female and male homologous chromosomes were identified by mapping BSDFs. In the Q4117 map, a single linkage group was associated with apospory. It was characterized by restriction in recombina-tion and preferential chromosome pairing. A BPSD marker mapping within this group allowed the detection of the female homolog and the putative four male groups of the set carrying apospory. Fil: Stein, Juliana. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pessino, Silvina Claudia. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Martínez, Eric Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina Fil: Rodriguez Romero, María Pía. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Siena, Lorena Adelina. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Quarin, Camilo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina Fil: Ortiz, Juan Pablo Amelio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina

For over 40 years, fluorouracil has been the only drug registered for the treatment of metastatic colorectal cancer. During the past 5 years, combination chemotherapy regimens including either irinotecan or oxaliplatin have proven to be superior to fluorouracil monotherapy in randomized clinical trials, in terms of response rate, progression-free survival and overall survival. Both doublets demonstrated similar efficacy, therefore either combination can be considered standard first-line treatment for metastatic colorectal cancer. Recently, a new orally active fluorouracil analog, capecitabine, and two targeted biological agents, cetuximab and bevacizumab, have been added to the armamentarium of drugs active against metastatic colorectal cancer, thus making the scenario more complex. Moreover, ongoing clinical trials are currently testing new promising molecularly targeted agents. Thus, we are facing a new era in which the rapid clinical development of novel agents is outpacing the ability to perform Phase III clinical trials. At present, there is no single standard treatment suitable for all patients. However, general principles of management can be derived from the available clinical data in order to guide the therapeutic choice and individualize treatment.

Progeny tests employing molecular markers allow the identification of individuals originated by sexual means among the offspring of a facultative apomict. The objective of this work was to evaluate the effect of the pollination timing on the proportion of sexually formed individuals in progenies of a facultative apomictic Paspalum notatum genotype. Progeny families of approx. 30 plants each were generated at five different pollination times: 1–3 d pre‐anthesis; at anthesis; and 2, 4 and 6 d post‐anthesis. Cytoembryological analyses indicated that approx. 17 % of the ovules carried a meiotic cytologically reduced embryo sac in florets formed simultaneously with those used for crosses. The parental plants and the five F1 families were analysed using RAPD molecular markers. Ninety‐five oligonucleotides were assayed on the progenitors in order to search for male‐specific bands. Eight primers presenting clear polymorphic bands were selected for use in the progeny tests. The proportion of sexually produced progeny reached 3·4 % before anthesis and 20 % at anthesis, while pollination after anthesis generated only maternal plants. A second progeny of 97 plants obtained from pollination at anthesis produced 16 off‐type plants (16·5 %), of which only one was a BIII hybrid (2n + n). Our results indicate that pollination at anthesis allows the greatest potential for sexuality to be expressed in this facultative apomictic genotype. When pollination is delayed as soon as 2 d after anthesis, only the aposporous sacs develop endosperm through pseudogamy to set seed. Fil: Espinoza, Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina Fil: Pessino, Silvina Claudia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Quarin, Camilo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina Fil: Valle, Estela Marta. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The NKp46 molecule has been proposed to play the role of triggering receptor in the natural cytotoxicity mediated by human NK cells. In this study we have identified the gene encoding the murine NKp46 homologue that we termed MAR-1. The MAR-1 gene is localized on chromosome 7 that is synthenic to the human chromosome 19 where the NKp46 gene is located. MAR-1 encodes a type I transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily that, like human NKp46, is characterized by two C2-type Ig-like domains, a transmembrane portion containing a positively charged residue and a cytoplasmic tail lacking the immunoreceptor tyrosine-based activation motif (ITAM). The MAR-1 protein is expressed on the surface of cell transfectants and displays a molecular mass of approximately 46 kDa similar to that of its human counterpart. Semiquantitative RT-PCR analysis showed that MAR-1, similar to the human NKp46, is selectively expressed by NK cells. The MAR-1 protein displays 58 % identity with the human NKp46 receptor. This high homology together with the presence of a charged amino acid (Arg) in the transmembrane portion suggest that MAR-1 may associate at the cell membrane into a multimeric complex with ITAM containing polypeptides.

NK cells display several killer inhibitory receptors (KIRs) specific for different alleles of major histocompatibility complex (MHC) class I molecules. A family of KIRs are represented by type I transmembrane proteins belonging to the Immunoglobulin Superfamily (Ig-SF). In the present study we describe a cDNA, termed cl.15.212, that encodes for a type I transmembrane protein displaying approximately 50% sequence homology with other Ig-SF members. The protein encoded by cl.15.212 (termed p49 according to its apparent molecular weight of 49 kDa) is characterized by two extracellular Ig-like domains, a 115-amino acid cytoplasmic tail containing a single immuno-receptor tyrosine-based inhibitory motif (ITIM) typical of KIR. Different from the other KIRs, the cl.15.212 transcript is expressed by all NK cells and by a fraction of T-cell clones expressing KIR. To determine the specificity of the cl.15.212-encoded receptor, we generated a chimeric protein, formed by the ectodomain of p49 and the Fc portion of human IgG1 (p49-Fc). Soluble molecules bound efficiently to LCL721.221 (221) cells transfected with HLA-G1, -A3, -B46 alleles and weakly to the -B7 allele. On the other hand, they did not bind to 221 cells either untransfected or transfected with HLA-A2, -B51, -Cw3, or-Cw4.

The potential regulatory role of delta protein kinase C (delta PKC) in murine erythroleukaemia cell differentiation was studied by using antisense oligodeoxynucleotides targeting the translation initiation region of mouse delta PKC mRNA. Cell treatment with antisense oligonucleotides, at a concentration of 20 microM, followed by hexamethylenebisacetamide induction, produced a specific 2-fold increase in the differentiation rate of both slowly and rapidly differentiating murine erythroleukaemia cell clones. Cell permeabilization by a cationic lipid resulted in a decrease of one order of magnitude in the amounts of antisense oligonucleotides necessary to elicit the maximal response, and accelerated the kinetics of the stimulatory effect. These changes in murine erythroleukaemia cell differentiation rates, observed in both cell clones, were associated with 60% and 50% decreases, respectively, in delta PKC immunoreactive protein in slowly and rapidly differentiating cells. The present results indicate strongly that basal levels of delta PKC in murine erythroleukaemia cells are essential in regulating the initial differentiation rate of these cells in response to chemical induction, and provide further evidence that this PKC isoform plays a fundamental role in maintaining the undifferentiated phenotype of murine erythroleukaemia cells.

A 29 kD protein previously isolated from murine erythroleukemia (MEL) cells and shown to enhance the rate of differentiation of these cells has now been demonstrated to possess an amino acid sequence identical to that reported for the 30 kD heparin-binding protein from developing rat brain, named amphoterin after its highly dipolar structure. The identity between the two proteins has been established on the basis of a strong heparin binding affinity and a complete homology in the amino acid sequences of N-terminal region as well as of several tryptic peptides. Furthermore, the cDNA encoding this protein has been isolated from MEL cell mRNA, by means of reverse transcriptase-polymerase chain reaction, and its sequence was found to correspond to that of amphoterin. The MEL cell differentiation enhancing factor, previously abbreviated as DEF, is again confirmed to reduce the latent period preceding the appearance of hexamethylenebisacetamide induced cell commitment and to stimulate the catalytic activity of α-protein kinase C. Thus, here we demonstrate that a protein expressed in MEL cells, whose sequence is identical to that previously reported for amphoterin, plays an essential role in promoting cell differentiation, thereby indicating anew relevant function of amphoterin.

The presence of alpha, delta, epsilon, theta, and zeta protein kinase C isoforms in DS19 murine erythroleukemia cells has been established in this study. In addition, the mRNA levels of these isozymes have been measured by quantitative reverse transcriptase-polymerase chain reaction. Isoform delta has been found to be the most abundant isotype, whereas isoform zeta resulted to be present in only few copies. Furthermore, the expression levels of all five protein kinase C isozymes have been studied in three cell clones, derived from parental DS19 cells and characterized by different susceptibilities to differentiation. This comparative analysis indicated that the calcium-independent isozymes (delta, epsilon, zeta, and theta) display significantly higher expression levels in cells less prone to differentiation. On the other hand, the mRNA levels of the only calcium-dependent isoform present (alpha) fluctuate poorly from one cell clone to the other, but are the highest in the cell clone characterized by the fastest rate of differentiation. This study represents the first complete characterization of the basal levels of specific protein kinase C isotypes in different murine erythroleukemia cell clones and provides further evidence for the role of individual isozymes in the early events that trigger chemical induced murine erithroleukemia cell differentiation.

We compared final height to height at diagnosis (expressed as a standard deviation score, SDS), predicted adult height (according to the Bayley and Pinneau method) and target genetic height (expressed as mean parental height in cm, +6.5 for males and -6.5 for females) in 37 patients (15 males, 22 females) with insulin-dependent diabetes mellitus (IDDM), aged 20.6 +/- 3.3 years (16.6-27), with 11.8 +/- 3.7 years (5.2-19.2) mean duration of disease. In the 22 females, final height (162.4 +/- 5.7 cm; range, 150-174 cm) was higher than predicted (161.5 +/- 7.8 cm; range, 146-176.2 cm) and target genetic height (159.7 +/- 3.8 cm; range, 152.8-167.3 cm), although not significantly. Female patients showed a positive correlation between final height and both predicted (P < 0.05) and target genetic height (P < 0.005). No difference was observed in final height between patients diagnosed in the prepubertal or pubertal phase (162.2 +/- 4.6 cm vs. 163.4 +/- 6.2 cm; P-value n.s.). In the 15 males, final height (173.4 +/- 4.4 cm; range, 166.5-181 cm), lower than predicted (175.4 +/- 4.9 cm; range, 166-183 cm), was higher than target genetic height (169.9 +/- 4.8 cm; range, 162.4-177 cm) (P < 0.05). Male patients showed a positive correlation between final height and target genetic height (P < 0.05). No difference was found in final height between patients diagnosed in the prepubertal or pubertal phase (173.6 +/- 3.5 cm vs. 172.7 +/- 5.5 cm; P-value n.s.).(ABSTRACT TRUNCATED AT 250 WORDS)

In addition to alpha, delta and epsilon-protein kinase C, murine erythroleukemia cells contain zeta-PKC and also a c-PKC isoform, named alpha 1, which shows cross-reactivity with an anti-alpha-PKC antipeptide antibody. In a C44 MEL cell clone, characterized by a high rate of differentiation, both c-PKC forms are expressed at a level higher than that of the N23 MEL cell clone which differentiates at a low rate and contains higher levels of epsilon-PKC and particularly of the delta-PKC isozyme. In the course of MEL cell differentiation, delta-PKC in N23 cells and alpha 1-PKC in C44 cells are rapidly down-regulated and the overall process is almost completed before cell commitment. Of the other three PKC isozymes present in both clones, only alpha-PKC is down-regulated to a significant extent. It is proposed that modulation of the signal delivered by each PKC isozyme is one of the biochemical mechanisms involved in MEL cell differentiation.

In murine erythroleukemia (MEL) cells the length of the latent period before the onset of hexamethylenebisacetamide induced terminal erythroid differentiation is inversely correlated to the intracellular level of delta-PKC. This is supported by the following experimental evidence. V3.17[44] MEL cell line, characterized by a very high rate of differentiation, contains an amount of delta-PKC protein one third lower than that present in the N23 MEL cell line, characterized by a very low rate of differentiation. A similar difference in the amount of delta-PKC mRNA is present in the two cell lines. In N23 cells, following addition of HMBA, the amount of delta-PKC protein and delta-PKC mRNA is down-regulated to one third its original value, which now corresponds to that constitutively present in V3.17[44] cells. Furthermore, in these cells the levels of delta-PKC protein and of its specific mRNA are unaffected by treatment with HMBA. Following introduction of homologous purified delta-PKC both MEL cell variants display a longer latent period before the onset of differentiation from 50 to 75 hours in N23 cell line and from 20 to 40 hours in V3.17[44] cells, respectively. Taken together, these results suggest that a delta-PKC related signal plays a negative role in the early stages of MEL cell differentiation and that the level of the kinase is controlled through a down-regulation process upon exposure to the chemical inducer.

e14077 Background: NGR-hTNF consists of tumour necrosis factor (TNF) fused to the tumour homing peptide (NGR), which selectively binds an aminopeptidase N expressed on tumour blood vessels. Preclin...

Facilitated sugar entry into mammalian cells is catalysed by multiple isoforms of the glucose transporter and regulated by hormonal stimuli, nutritional status and oncogenesis. A large reserve of latent glucose transport capacity must be maintained by muscle and adipose cells that are sensitive to insulin, the primary activator of sugar uptake after feeding. Intracellular sequestration of sugar transporters accounts for a large part of this latent capacity, but new findings suggest that there is also reversible suppression of intrinsic catalytic activity of those glucose transporters residing at the cell surface. The mechanism of this suppression appears to be occlusion or disruption of the exofacial sugar-binding sites on the glucose-transporter proteins.

A sample of 30 alcoholic patients, selected according to a rigorous criterion, was compared with two samples of the general population with respect to the distribution of HLA antigens. The sole inclusion criterion was the presence of signs and symptoms of an alcohol withdrawal syndrome. The alcoholic sample did not differ from the general population sample with respect to ABO blood group distribution, and had minimal evidence of alcoholic liver disease. The alcoholic group showed highly significant differences from the two general population samples with respect to the HLA system, consisting of a much higher frequency of HLA B40 and DR4 antigens and a much lower frequency of DR3. The results are consistent with the view that alcoholism may have a precise genetic determination.

In order to investigate structure and function of beta-subunit extracellular portion, four polyclonal antibodies (AP1, AP2, AP3 and AP4) toward peptides comprised in this region were generated. None of them recognizes native human and rat insulin receptor both in vitro and in whole cells. Two antibodies, AP1 and AP2, immunoprecipitate isolated (DTT-reduced) human beta-subunits and bind to human IM-9 cell after alpha-subunit tryptic cleavage. Only AP1 recognizes rat beta-subunit both in vitro and in trypsin treated rat FAD cells. These findings suggest that: (i) the extracellular portion of the insulin receptor beta-subunit is partially covered by the alpha-subunit in human and rat native insulin receptors; (ii) human and rat beta-subunit extracellular domains are different, at least in the amino acid sequence corresponding to residues 785-796 of the human insulin receptor.

As indicated by direct evidence, obtained by altering the cell-membrane permeability for Ca2+ in murine erythroleukaemia (MEL) cells, calpain is the triggering factor which connects fluctuations of the intracellular Ca2+ concentrations to the decay of protein kinase C (PKC), as well as to the kinetics of cell differentiation induced by hexamethylenebisacetamide. Cell exposure to verapamil caused a profound decrease in the rate of PKC down-regulation and a slower initial rate of accumulation of mature erythroid cells, whereas addition of the Ca2+ ionophore A23187 produced opposite effects. The high susceptibility of PKC-delta to calpain degradation, at concentrations of Ca2+ much lower than those required for degradation of the other PKC isoforms, may be explained by the finding that this kinase isoform is predominantly associated with the cell membrane. The different cellular localizations, as well as the different susceptibilities to calpain digestion, further support the hypothesis that in MEL cells the various PKC isoforms play distinct biological functions that are critical for the maintenance of the undifferentiated state of the cell and for its commitment to terminal erythroid differentiation.

e15030 Background: Imatinib inhibits PDGFR interfering with pericytes, the structural support to newly formed tumor blood vessels. It may thus synergize with bevacizumab. Microenvironment and tumor targeted agents along with chemotherapy could be a promising add-on approach. Methods: cetuximab 500 mg/m2, bevacizumab 5 mg/kg and modified FOLFOX-6 were given i.v. on day 1 and repeated every 2 weeks. Imatinib 400 mg/day per os was given continuously. Due to the cost and potential toxicity of the combination, the endpoint for this phase II study was very ambitious: at least 25% of complete response (medically or surgically achieved), lasting a minimum of 12 months in advanced untreated colorectal cancer patients with clearly unresectable disease. Results: Of 26 patients (16 with 1 site of disease), 17 completed the first 4 months of treatment according to the protocol, while 9 had to discontinue one biologic drug due to side effects (5 cetuximab, 3 imatinib and 1 bevacizumab). Grade 3–4 toxicity: diarrhea 12%, neutropenia 24%, skin rash 24%, hypersensitivity reactions 16%, asthenia 8%, neuro 8%. All patients were evaluable for response. Eleven responses ( 1 CR and 10 PR), 13 SD and 2 PD were observed, corresponding to 42% RR ( 95% CI = 23–61). The minimum follow up is 12 months; median PFS is 10 months. One patient among responders underwent radiofrequency ablation and 17 patients underwent surgery: 8 R-0, 3 R-1, 5 R-2 and one exploratory laparotomy. Major post surgical complications occurred in 5/17 patients. No evidence of macroscopic disease after the entire treatment plan was obtained in 13/26 patients: 12 surgical and 1 medical CR. Seven/13 were disease free at 6 months, but only 3 were still disease free at 12 months. ERCC1, ERCC2/XDP, GSTP1,TS,EGF, COX2, CYCLIN D, FCgR polymorphisms and K-RAS mutations were evaluated on all 26 patients, but no correlations were found with clinical outcome. Conclusions: The triple combination of biologics with modified FOLFOX-6 is feasible and tolerable as initial aggressive treatment. The primary endpoint of the study was not met . In fact the activity, 42% RR, was not outstanding and the high resectability rate, 69%, is misleading in the light of the short duration of the surgically induced CR. [Table: see text]

3553 Background: The most relevant recent advance in the treatment of metastatic colorectal cancer is the fact that cure is still possible under very selected conditions. However the goal of chemotherapy remains palliative in the great majority of patients, justifying less toxic innovative approaches in so- called “window of opportunity trials”. We have pursued this idea in a phase II study of cetuximab monotherapy in chemo-naive patients with advanced colorectal cancer beyond any possibility of curative resection. Methods: Patients with non-resectable metastatic colorectal cancer (at least two metastatic sites and/or otherwise inoperable metastatic disease) were treated with cetuximab (400 mg/m2 week 1 and 250 mg/m2 weekly thereafter) until progressive disease (PD) or unacceptable toxicity. The primary end-point was objective response; secondary end-points were: stable disease, time to treatment failure and time to progression. According to Simon’s two-stage design, the number of responses/patients to stop the trial was 0/10 for stage 1 and 3/29 for stage 2. Results: Of the 44 patients screened, 42 (97%) had EGFR-expressing tumors. Thirty-nine patients (median age: 69) initiated the treatment. Two had grade 3 allergic reactions to cetuximab at the first administration leading to treatment discontinuation. The most common adverse event was skin toxicity, which occurred in 90% of the patients ( 31% grade 2, 10 % grade 3). We observed 1 complete response, 3 partial responses, 13 stable diseases (5 of which were minor responses), and 22 PD. The duration of the 4 responses were 12, 9, 9 and 6 months. Median time to progression was 2.0 months. Conclusions: The study is negative because the response rate is low. However, the duration of benefit in the few responding patients is such that it is imperative to find the molecular determinants of cetuximab activity in these cases. [Table: see text]