Graft versus neuroblastoma reaction is efficiently elicited by allogeneic bone marrow transplantation through cytolytic activity in the absence of GVHD

Continuous efforts are dedicated to develop immunotherapeutic approaches to neuroblastoma (NB), a tumor that relapses at high rates following high-dose conventional cytotoxic therapy and autologous bone marrow cell (BMC) reconstitution. This study presents a series of transplant experiments aiming to evaluate the efficacy of allogeneic BMC transplantation. Neuro-2a cells were found to express low levels of class I major histocompatibility complex (MHC) antigens. While radiation and syngeneic bone marrow transplantation (BMT) reduced tumor growth (P < 0.001), allogeneic BMT further impaired subcutaneous development of Neuro-2a cells (P < 0.001). Allogeneic donor-derived T cells displayed direct cytotoxic activity against Neuro-2a in vitro, a mechanism of immune-mediated suppression of tumor growth. The proliferation of lymphocytes from congenic mice bearing subcutaneous tumors was inhibited by tumor lysate, suggesting that a soluble factor suppresses cytotoxic activity of syngeneic lymphocytes. However, the growth of Neuro-2a cells was impaired when implanted into chimeric mice at various times after syngeneic and allogeneic BMT. F1 (donor-host) splenocytes were infused attempting to foster immune reconstitution, however they engrafted transiently and had no effect on tumor growth. Taken together, these data indicate: (1) Neuro-2a cells express MHC antigens and immunogenic tumor associated antigens. (2) Allogeneic BMT is a significantly better platform to develop graft versus tumor (GVT) immunotherapy to NB as compared to syngeneic (autologous) immuno-hematopoietic reconstitution. (3) An effective GVT reaction in tumor bearing mice is primed by MHC disparity and targets tumor associated antigens.     

Therapeutic antibodies: Discovery and development using the ProteOn XPR36 biosensor interaction array system

Therapeutic monoclonal antibodies are becoming a significant and rapidly growing class of therapeutic pharmaceuticals. Their discovery and development requires fast and high-throughput methodologies for screening and selecting appropriate candidate antibodies having high affinity for the target as well as high specificity and low cross-reactivity. This study demonstrates the use of the ProteOn XPR36 protein interaction array system and its novel approach, termed One-Shot Kinetics, for the rapid screening and selection of high-affinity antibodies. This approach allows multiple quantitative protein binding analyses in parallel, providing association, dissociation, and affinity constants for several antibodies or supernatants simultaneously in one experiment. We show that the ProteOn XPR36 system is a valuable tool for use across multiple stages of the therapeutic antibody discovery and development process, enabling efficient and rapid screening after panning, affinity maturation, assay validation, and clone selection.     

Long-term obesity levels in female OLETF rats following time-specific post-weaning food restriction

Obesity and the metabolic syndrome represent serious health threats affecting increasing numbers of individuals, with females being more affected than males and with growing incidence among children and adolescents. In the present study, we used the OLETF rat model of early-onset obesity to examine the influence of different timing of food restriction on long-term obesity levels in females. Food restriction took place at different time windows: from weaning until postnatal day (PND) 45 (early); from weaning until PND90 (chronic); or from PND45 until PND70 (late). Follow-up continued until PND90. During and after the termination of the diet-restriction period, we focused on peripheral adiposity-related measures such as fat pad weight (brown, retroperitoneal and inguinal); inguinal adipocyte size and number; and leptin, oxytocin and glucose levels. We also examined body weight, feeding efficiency, spontaneous intake after release from diet-restriction, and plasma creatinine levels and estrous cycle characteristics as a result of the chronic diet. The results suggest that while food restriction produced significant weight and adiposity loss, OLETF females presented poor weight loss retention after the early and late short-term diets. The estrous cycle structure and time of first estrous of the OLETF rats were normalized by chronic food restriction. Females responded to early food restriction in a different manner than males did in previous studies, further emphasizing the importance of sex-appropriate approaches in the investigation and treatment of the pathologies related to obesity and the metabolic syndrome.     

Lithium-calcium exchange is mediated by a distinct potassium-independent sodium-calcium exchanger

Sodium-calcium exchangers have long been considered inert with respect to monovalent cations such as lithium, choline, and N-methyl-d-glucamine. A key question that has remained unsolved is how despite this, Li(+) catalyzes calcium exchange in mammalian tissues. Here we report that a Na(+)/Ca(2+) exchanger, NCLX cloned from human cells (known as FLJ22233), is distinct from both known forms of the exchanger, NCX and NCKX in structure and kinetics. Surprisingly, NCLX catalyzes active Li(+)/Ca(2+) exchange, thereby explaining the exchange of these ions in mammalian tissues. The NCLX protein, detected as both 70- and 55-KDa polypeptides, is highly expressed in rat pancreas, skeletal muscle, and stomach. We demonstrate, moreover, that NCLX is a K(+)-independent exchanger that catalyzes Ca(2+) flux at a rate comparable with NCX1 but without promoting Na(+)/Ba(2+) exchange. The activity of NCLX is strongly inhibited by zinc, although it does not transport this cation. NCLX activity is only partially inhibited by the NCX inhibitor, KB-R7943. Our results provide a cogent explanation for a fundamental question. How can Li(+) promote Ca(2+) exchange whereas the known exchangers are inert to Li(+) ions? Identification of this novel member of the Na(+)/Ca(2+) superfamily, with distinct characteristics, including the ability to transport Li(+), may provide an explanation for this phenomenon.     

A Novel, Multifunctional c-Cbl Binding Protein in Insulin Receptor Signaling in 3T3-L1 Adipocytes

The protein product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with endogenous c-Crk and Fyn. These results suggest a role for tyrosine-phosphorylated c-Cbl in 3T3-L1 adipocyte activation by insulin. A yeast two-hybrid cDNA library prepared from fully differentiated 3T3-L1 adipocytes was screened with full-length c-Cbl as the target protein in an attempt to identify adipose-specific signaling proteins that interact with c-Cbl and potentially are involved in its tyrosine phosphorylation in 3T3-L1 adipocytes. Here we describe the isolation and the characterization of a novel protein that we termed CAP for c-Cbl-associated protein. CAP contains a unique structure with three adjacent Src homology 3 (SH3) domains in the C terminus and a region showing significant sequence similarity with the peptide hormone sorbin. Both CAP mRNA and proteins are expressed predominately in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. CAP associates with c-Cbl in 3T3-L1 adipocytes independently of insulin stimulation in vivo and in vitro in an SH3-domain-mediated manner. Furthermore, we detected the association of CAP with the insulin receptor. Insulin stimulation resulted in the dissociation of CAP from the insulin receptor. Taken together, these data suggest that CAP represents a novel c-Cbl binding protein in 3T3-L1 adipocytes likely to participate in insulin signaling.     

The pituitary of Aphanius dispar (Rüppell) from hypersaline marshes and freshwater

Summary An ultrastructural study of the rostral pars distalis of the pituitary of Aphanius dispar specimens taken from freshwater or hypersaline marshes revealed significant structural differences which indicate higher activity of the prolactin cells in the hypotonic medium. Prolactin cells from freshwater specimens had larger secretory granules, a higher amount of endoplasmic reticulum, and expanded intercellular spaces with many secretory lakes. These cells contained an unusual cytoplasmic structure, consisting of twisted canals with vesicular lumina, connected to the endoplasmic reticulum of the cell. This structure is about 1–2 m in diameter.Stellate cells are characterized by extracellular spacing junctions which are particularly noticeable at the confluence of the interstellate cell canaliculi and the pericapillary space.Abbreviations FW freshwater - HS hypersaline - NS neurosecretory - PCB paracrystalline body - PNH proximal neurohypophysis - RPD rostral pars distalis - SG secretory granule - SW seawater This paper is dedicated with affectionate respect to Professor Berta Scharrer on the occasion of her 70th birthdayThe assistance of Cynthia Bellon in editing this paper is gratefully acknowledged     

Biodegradation of wheat straw lignocarbohydrate complexes (LCC)

Summary In laboratory and semi-industrial scale experiments the influence of the substrate water content, temperature, and incubation time on the progress of solid state fermentation of straw colonized by white rot fungi was investigated. The parameters used to evaluate the fermentation process were degradation of total organic matter and lignin, in vitro digestibility, the content of water soluble substances in the substrate and the pH.The degradation of total organic matter was species specific. Only Trametes hirsuta enhanced the degradation at elevated temperature (30 °C). With Abortiporus biennis, Ganoderma applanatum, and Pleurotus serotinus, elevated temperature had and adverse effect. Prolonged incubation only improved degradation of straw by the relatively slowgrowing fungi Ganoderma applanatum, Lenzites betulina, and Pleurotus sajor caju.Elevated temperature and prolonged incubation shifted the relative degradation rates in favour of total organic matter degradation. With Ganoderma applanatum, Pleurotus ostreatus, and Pleurotus serotinus lignin degradation, even on an absolute scale, was less at 30 °C than at 22 °C.In general, the in vitro digestibility also decreased, when the incubation time and temperature were raised. With Ganoderma applanatum the in vitro digestibility dropped below the value of the sterile straw control.Solid state fermentation of straw was at an optimum at a medium water content of 75 ml/25 g of substrate. However, most of the fungi tested could digest straw over a wide range of water content. At higher water contents (125–150 ml/25 g of substrate) an increased production of aerial mycelium was observed.In semi-industrial batch experiments (40 kg) with Abortiporus biennis the in vitro digestibility dropped below the reference value for sterile straw during the first 19 days of incubation. Later, the in vitro digestibility again rose and reached its optimum after about 60 days. The in vitro digestibility in the semi-industrial experiments was always lower than in the laboratory experiments (+9% and +25%, respectively).In long term experiments (2.5 kg batches, 8 months of incubation) very different values for the in vitro digestibility were found, and these depended on the fungus used (Abortiporus biennis, +16%; Pleurotus ostreatus, +4%; and Ganoderma applanatum, –27%).     

Discovery and SAR of substituted 3-oxoisoindoline-4-carboxamides as potent inhibitors of poly(ADP-ribose) polymerase (PARP) for the treatment of cancer

Through conformational restriction of a benzamide by formation of a seven-membered hydrogen-bond with an oxindole carbonyl group, a series of PARP inhibitors was designed for appropriate orientation for binding to the PARP surface. This series of compounds with a 3-oxoisoindoline-4-carboxamide core structure, displayed modest to good activity against PARP-1 in both intrinsic and cellular assays. SAR studies at the lactam nitrogen of the pharmacophore have suggested that a secondary or tertiary amine is important for cellular potency. An X-ray structure of compound 1e bound to the protein confirmed the formation of a seven-membered intramolecular hydrogen bond. Though revealed previously in peptides, this type of seven-membered intramolecular hydrogen bond is rarely observed in small molecules. Largely due to the formation of the intramolecular hydrogen bond, the 3-oxoisoindoline-4-carboxamide core structure appears to be planar in the X-ray structure. An additional hydrogen bond interaction of the piperidine nitrogen to Gly-888 also contributes to the binding affinity of 1e to PARP-1.