Investigation of the Interaction between the Large and Small Subunits of Potato ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA) method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies) of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield

Left atrial extension of metastatic lung tumor via pulmonary vein: report on the first case of Ewing sarcoma

Extension of metastatic lung tumors into the left atrium via pulmonary veins is rare. Here, we report the first case of Ewing sarcoma exhibiting such extension. A 31-year-old man with pulmonary metastasis from Ewing sarcoma presented with a mass in the left lung, extending to the left atrium through the left inferior pulmonary vein. As the patient was considered to be at risk of tumor embolism, the mass was excised surgically

Generation of Induced Pluripotent Stem Cells from the Prairie Vole

The vast majority of animals mate more or less promiscuously. A few mammals, including humans, utilize more restrained mating strategies that entail a longer term affiliation with a single mating partner. Such pair bonding mating strategies have been resistant to genetic analysis because of a lack of suitable model organisms. Prairie voles are small mouse-like rodents that form enduring pair bonds in the wild as well as in the laboratory, and consequently they have been used widely to study social bonding behavior. The lack of targeted genetic approaches in this species however has restricted the study of the molecular and neural circuit basis of pair bonds. As a first step in rendering the prairie vole amenable to reverse genetics, we have generated induced pluripotent stem cell (IPSC) lines from prairie vole fibroblasts using retroviral transduction of reprogramming factors. These IPSC lines display the cellular and molecular hallmarks of IPSC cells from other organisms, including mice and humans. Moreover, the prairie vole IPSC lines have pluripotent differentiation potential since they can give rise to all three germ layers in tissue culture and in vivo. These IPSC lines can now be used to develop conditions that facilitate homologous recombination and eventually the generation of prairie voles bearing targeted genetic modifications to study the molecular and neural basis of pair bond formation

Genetic regulation of glycogen biosynthesis in Escherichia coli : In vivo effects of the catabolite repression and stringent response systems in glg gene expression

The synthesis of two of the Escherichia coli glycogen biosynthetic enzymes, ADPglucose pyrophosphorylase ( glg C) and glycogen synthase ( glg A) was activated by the addition of 5 m M cyclic AMP (cAMP) to maxicells; synthesis of glycogen branching enzyme ( glg B) was unaffected. β -Galactosidase activity expressed from a gene fusion, φ(glg C- lac Z), was approximately five-fold higher in a cya + versus an isogenic cya − strain of E. coli . Addition of cAMP restored β -galactosidase in the cya − strain. The expression of φ(glg C‘−’ lac Z) encoded β -galactosidase activity in a series of spo T mutants exhibited an apparent exponential relationship to intracellular guanosine 5′-diphosphate 3′-diphosphate (ppGpp) levels. These results provide evidence for the control of glycogen biosynthesis in vivo by cAMP and ppGpp at the level of gene expression, and identify a region of DNA required for the control. The φ(glg C‘−’ lac Z) encoded β -galactosidase activity was also elevated three-to five-fold in strain AC70R1, which contains a transacting mutation ( glg Q) that affects the levels of the glycogen biosynthetic enzymes and glg C transcripts.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41337/1/284_2005_Article_BF02091831.pd

Sequences, Annotation and Single Nucleotide Polymorphism of the Major Histocompatibility Complex in the Domestic Cat

Two sequences of major histocompatibility complex (MHC) regions in the domestic cat, 2.976 and 0.362 Mbps, which were separated by an ancient chromosome break (55–80 MYA) and followed by a chromosomal inversion were annotated in detail. Gene annotation of this MHC was completed and identified 183 possible coding regions, 147 human homologues, possible functional genes and 36 pseudo/unidentified genes) by GENSCAN and BLASTN, BLASTP RepeatMasker programs. The first region spans 2.976 Mbp sequence, which encodes six classical class II antigens (three DRA and three DRB antigens) lacking the functional DP, DQ regions, nine antigen processing molecules (DOA/DOB, DMA/DMB, TAPASIN, and LMP2/LMP7,TAP1/TAP2), 52 class III genes, nineteen class I genes/gene fragments (FLAI-A to FLAI-S). Three class I genes (FLAI-H, I-K, I-E) may encode functional classical class I antigens based on deduced amino acid sequence and promoter structure. The second region spans 0.362 Mbp sequence encoding no class I genes and 18 cross-species conserved genes, excluding class I, II and their functionally related/associated genes, namely framework genes, including three olfactory receptor genes. One previously identified feline endogenous retrovirus, a baboon retrovirus derived sequence (ECE1) and two new endogenous retrovirus sequences, similar to brown bat endogenous retrovirus (FERVmlu1, FERVmlu2) were found within a 140 Kbp interval in the middle of class I region. MHC SNPs were examined based on comparisons of this BAC sequence and MHC homozygous 1.9× WGS sequences and found that 11,654 SNPs in 2.84 Mbp (0.00411 SNP per bp), which is 2.4 times higher rate than average heterozygous region in the WGS (0.0017 SNP per bp genome), and slightly higher than the SNP rate observed in human MHC (0.00337 SNP per bp)

A Universal Approach to Eliminate Antigenic Properties of Alpha-Gliadin Peptides in Celiac Disease

Celiac disease is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins, including the α-gliadins. It has been shown that α-gliadins harbor several major epitopes involved in the disease pathogenesis. A major step towards elimination of gluten toxicity for celiac disease patients would thus be the elimination of such epitopes from α-gliadins. We have analyzed over 3,000 expressed α-gliadin sequences from 11 bread wheat cultivars to determine whether they encode for peptides potentially involved in celiac disease. All identified epitope variants were synthesized as peptides and tested for binding to the disease-associated HLA-DQ2 and HLA-DQ8 molecules and for recognition by patient-derived α-gliadin specific T cell clones. Several specific naturally occurring amino acid substitutions were identified for each of the α-gliadin derived peptides involved in celiac disease that eliminate the antigenic properties of the epitope variants. Finally, we provide proof of principle at the peptide level that through the systematic introduction of such naturally occurring variations α-gliadins genes can be generated that no longer encode antigenic peptides. This forms a crucial step in the development of strategies to modify gluten genes in wheat so that it becomes safe for celiac disease patients. It also provides the information to design and introduce safe gluten genes in other cereals, which would exhibit improved quality while remaining safe for consumption by celiac disease patients

Identifying and prioritizing strategies for comprehensive liver cancer control in Asia

<p>Abstract</p> <p>Background</p> <p>Liver cancer is both common and burdensome in Asia. Effective liver cancer control, however, is hindered by a complex etiology and a lack of coordination across clinical disciplines. We sought to identify strategies for inclusion in a comprehensive liver cancer control for Asia and to compare qualitative and quantitative methods for prioritization.</p> <p>Methods</p> <p>Qualitative interviews (N = 20) with international liver cancer experts were used to identify strategies using Interpretative Phenomenological Analysis and to formulate an initial prioritization through frequency analysis. Conjoint analysis, a quantitative stated-preference method, was then applied among Asian liver cancer experts (N = 20) who completed 12 choice tasks that divided these strategies into two mutually exclusive and exhaustive subsets. Respondents' preferred plan was the primary outcome in a choice model, estimated using ordinary least squares (OLS) and logistic regression. Priorities were then compared using Spearman's Rho.</p> <p>Results</p> <p>Eleven strategies were identified: <it>Access to treatments; Centers of excellence; Clinical education; Measuring social burden; Monitoring of at-risk populations; Multidisciplinary management; National guidelines; Public awareness; Research infrastructure; Risk-assessment and referral</it>; and <it>Transplantation infrastructure</it>. Qualitative frequency analysis indicated that <it>Risk-assessment and referral </it>(85%), <it>National guidelines </it>(80%) and <it>Monitoring of at-risk populations </it>(80%) received the highest priority, while conjoint analysis pointed to <it>Monitoring of at-risk populations </it>(p < 0.001), <it>Centers of excellence </it>(p = 0.002), and <it>Access to treatments </it>(p = 0.004) as priorities, while <it>Risk-assessment and referral </it>was the lowest priority (p = 0.645). We find moderate concordance between the qualitative and quantitative methods (rho = 0.20), albeit insignificant (p = 0.554), and a strong concordance between the OLS and logistic regressions (rho = 0.979; p < 0.0001).</p> <p>Conclusions</p> <p>Identified strategies can be conceptualized as the ABCs of comprehensive liver cancer control as they focus on <it>Antecedents</it>, <it>Better care </it>and <it>Connections </it>within a national strategy. Some concordance was found between the qualitative and quantitative methods (e.g. <it>Monitoring of at-risk populations</it>), but substantial differences were also identified (e.g. qualitative methods gave highest priority to risk-assessment and referral, but it was the lowest for the quantitative methods), which may be attributed to differences between the methods and study populations, and potential framing effects in choice tasks. Continued research will provide more generalizable estimates of priorities and account for variation across stakeholders and countries.</p

Present state and future perspectives of using pluripotent stem cells in toxicology research

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed