Enhancement of cytotoxic T-lymphocyte responses in patients with gastrointestinal malignancies following vaccination with CEA peptide–pulsed dendritic cells

Carcinoembryonic antigen (CEA) is strongly expressed in a vast majority of gastrointestinal carcinomas. Recently, epitope peptides of CEA were identified. We have demonstrated HLA-A24–restricted peptide, CEA652[9] (TYACFVSNL), was capable of eliciting specific cytotoxic T lymphocytes (CTLs) which could lyse tumor cells expressing HLA-A24 and CEA. HLA-A24 is the most applicable MHC class I allele in the Japanese population. In this pilot study, we have used the peptide-pulsed dendritic cells (DCs) generated from peripheral blood mononuclear cells (PBMCs) supplemented with GM-CSF and IL-4 as the source of the vaccine. Eight patients with advanced CEA-expressing gastrointestinal malignancies received subcutaneous injections every 2 or 3 weeks. Immunomonitoring was performed by ELISpot (enzyme-linked immunosorbent spot) assay to measure the precursor frequency of CTLs and their capacity to elicit antitumor CTLs in vitro. Four of seven patients have developed their CTL response after vaccinations. DTH reaction was observed in one of eight patients at the DC-injected site. Skin biopsy at the injected site showed the infiltration of the lymphocytes. Furthermore, A24/CEA peptide tetramer assay revealed an increase in peptide-specific T-cell precursor frequency in vaccinated patients. No significant toxic adverse effects were observed, except for mild diarrhea in one case after three vaccinations. Three patients have shown stabilization of the disease after vaccinations. In conclusion, our results clearly demonstrated that our vaccination protocol was safe and might develop a CEA-specific CTL response in cancer patients.     

A large-scale protein protein interaction analysis in Synechocystis sp. PCC6803.

Protein-protein interactions (PPIs) play crucial roles in protein function for a variety of biological processes. Data from large-scale PPI screening has contributed to understanding the function of a large number of predicted genes from fully sequenced genomes. Here, we report the systematic identification of protein interactions for the unicellular cyanobacterium Synechocystis sp. strain PCC6803. Using a modified high-throughput yeast two-hybrid assay, we screened 1825 genes selected primarily from (i) genes of two-component signal transducers of Synechocystis, (ii) Synechocystis genes whose homologues are conserved in the genome of Arabidopsis thaliana, and (iii) genes of unknown function on the Synechocystis chromosome. A total of 3236 independent two-hybrid interactions involving 1920 proteins (52% of the total protein coding genes) were identified and each interaction was evaluated using an interaction generality (IG) measure, as well as the general features of interacting partners. The interaction data obtained in this study should provide new insights and novel strategies for functional analyses of genes in Synechocystis, and, additionally, genes in other cyanobacteria and plant genes of cyanobacterial origin.     

Juvenile hormone regulates extreme mandible growth in male stag beetles

The morphological diversity of insects is one of the most striking phenomena in biology. Evolutionary modifications to the relative sizes of body parts, including the evolution of traits with exaggerated proportions, are responsible for a vast range of body forms. Remarkable examples of an insect trait with exaggerated proportions are the mandibular weapons of stag beetles. Male stag beetles possess extremely enlarged mandibles which they use in combat with rival males over females. As with other sexually selected traits, stag beetle mandibles vary widely in size among males, and this variable growth results from differential larval nutrition. However, the mechanisms responsible for coupling nutrition with growth of stag beetle mandibles (or indeed any insect structure) remain largely unknown. Here, we demonstrate that during the development of male stag beetles (Cyclommatus metallifer), juvenile hormone (JH) titers are correlated with the extreme growth of an exaggerated weapon of sexual selection. We then investigate the putative role of JH in the development of the nutritionally-dependent, phenotypically plastic mandibles, by increasing hemolymph titers of JH with application of the JH analog fenoxycarb during larval and prepupal developmental periods. Increased JH signaling during the early prepupal period increased the proportional size of body parts, and this was especially pronounced in male mandibles, enhancing the exaggerated size of this trait. The direction of this response is consistent with the measured JH titers during this same period. Combined, our results support a role for JH in the nutrition-dependent regulation of extreme mandible growth in this species. In addition, they illuminate mechanisms underlying the evolution of trait proportion, the most salient feature of the evolutionary diversification of the insects.     

Signaling Cascade of Diacylglycerol Kinase �� in the Pituitary Intermediate Lobe: Dopamine D2 Receptor/Phospholipase C��4/Diacylglycerol Kinase ��/Protein Kinase C��

The pituitary gland dynamically changes its hormone output under various pathophysiological conditions. One of the pathways implicated in the regulatory mechanism of this gland is a dopaminergic system that operates the phosphoinositide (PI) cycle to transmit downstream signal through second messengers. We have previously shown that diacylglycerol kinase β (DGKβ) is coexpressed with dopamine D1 and D2 receptors in medium spiny neurons of the striatum, suggesting a plausible implication of DGKβ in dopaminergic transmission. However, it remains elusive whether DGKβ is involved in the dopaminergic system in the pituitary gland. The aim of this study is to investigate the expression and localization of DGK in the pituitary gland, together with the molecular components involved in the PI signaling cascade, including dopamine receptors, phospholipase C (PLC), and a major downstream molecule, protein kinase C (PKC). Here we show that DGKβ and the dopamine D2 receptor are coexpressed in the intermediate lobe and localize to the plasma membrane side by side. In addition, we reveal that PLCβ4 and PKCα are the subtypes expressed in the intermediate lobe among those families. These findings will substantiate and further extend our understanding of the molecular-anatomical pathway of PI signaling and the functional roles of DGK in the pituitary intermediate lobe. (J Histochem Cytochem 58:119–129, 2010)     

Preparation of 13C-labeled ceramide by acetic acid bacteria and its incorporation in mice

We prepared 2-hydroxypalmitoyl-sphinganine (dihydroceramide) labeled with a stable isotope by culturing acetic acid bacteria with ¹³C-labeled acetic acid. The GC/MS spectrum of the trimethylsilyl derivative of ¹³C-labeled dihydroceramide gave molecular ions with an increased mass of 12–17 Da over that of nonlabeled dihydroceramide. The fragment ions derived from both sphinganine base and 2-hydroxypalmitate were confirmed to be labeled with the stable isotope in the spectrum. Therefore, ¹³C-labeled dihydroceramide can be an extremely useful tool for analyzing sphingolipid metabolism. The purified [¹³C]dihydroceramide was administered orally to mice for 12 days, and the total sphingoid base fractions in various tissues were analyzed by GC/MS. The spectrum patterns specific to ¹³C-labeled sphingoids were detected in the tissues tested. Sphinganine pools in skin epidermis, liver, skeletal muscle, and synapse membrane in brain were replaced by [¹³C]sphinganine at about 4.5, 4.0, 1.0, and 0.3%, respectively. Moreover, about 1.0% of the sphingosine pool in the liver was replaced by [¹³C]sphingosine, implying that exogenous dihydroceramide can be converted to sphingosine. These results clearly indicate that ingested dihydroceramide can be incorporated into various tissues, including brain, and metabolized to other sphingolipids.     

Patient-specific modelling of pulmonary airflow using GPU cluster for the application in medical practice

In this paper, we propose a novel patient-specific method of modelling pulmonary airflow using graphics processing unit (GPU) computation that can be applied in medical practice. To overcome the barriers imposed by computation speed, installation price and footprint to the application of computational fluid dynamics, we focused on GPU computation and the lattice Boltzmann method (LBM). The GPU computation and LBM are compatible due to the characteristics of the GPU. As the optimisation of data access is essential for the performance of the GPU computation, we developed an adaptive meshing method, in which an airway model is covered by isotropic subdomains consisting of a uniform Cartesian mesh. We found that 4(3) size subdomains gave the best performance. The code was also tested on a small GPU cluster to confirm its performance and applicability, as the price and footprint are reasonable for medical applications.     

Preparation and Isolation of Oligogalacturonic Acids and Their Biological Effects in Cockscomb (Celosia argentea L.) Seedlings

A mixture of oligogalacturonic acids, the partial degradation substances of polygalacturonic acid, promoted shoot growth in cockscomb (Celosia argentea L.) seedlings, which generally had a high sensitivity for growth-promoting substances. The effect of the mixture of oligogalacturonic acids on shoot growth of cockscomb was higher than that of the polygalacturonic acid at concentrations above 30 ppm. These oligomers were loaded onto an anion exchange column, DEAE Sephadex A-25, and separated into individual oligomer sizes using the NH4HCO3 eluent system. This separation method has the advantage of using NH4HCO3 as the eluent solution; NH4HCO3 in the sample solution is effectively removed by lyophilization. Each of the isolated oligogalacturonic acids gave a single band on a fluorophore-assisted carbohydrate electrophoresis (FACE), and they showed the m/z value which corresponded to their molecular ion peaks [M-H]- on a fast atom bombardment mass spectrometry (FAB-MS) analysis. These results showed that the successive chromatography method used in this study is well suited for the preparation of oligogalacturonic acid for the plant growth test. Furthermore, we showed that the effective degree of polymerization (DP) of oligogalacturonic acid was around 8 on shoot growth of cockscomb seedlings, and the effects of both smaller and larger oligogalacturonic acids were slightly lower than that of octa-galacturonide. Octa-galacturonide promoted shoot growth of cockscomb at concentrations above 10 mM, and showed a 66% promotion at the most effective concentration of 300 mM. Root growth was slightly inhibited at concentrations above 300 mM. These results suggest that DP around 8 of oligogalacturonic acids has the function to control shoot growth in cockscomb as a growth-promoting substance.