Hibernation reduces pancreatic amylase levels in ground squirrels

Pancreatic enzyme levels in mammals are influenced by food intake and dietary composition. In this study, we examined the activity and expression of pancreatic amylase in a hibernating mammal, a natural model for long-term fasting. Pancreatic tissues were obtained from summer-active 13-lined ground squirrels and hibernating squirrels that had not eaten for at least 6 weeks. Amylase specific activity was reduced by approximately 50% in the torpid hibernators compared with summer squirrels, and immunoblot analysis revealed that amylase protein expression was reduced by approximately 40% in the hibernators. Similar reductions in amylase specific activity were observed in interbout euthermic hibernators. These results support a strong influence of food intake on pancreatic enzyme expression in hibernating mammals. The maintenance of basal levels of this key digestive enzyme at approximately 50% of summer values despite the extended winter fast likely facilitates the rapid resumption of digestive function after terminal arousal in the spring.     

Cyclooxygenase enzymes in allergic inflammation and asthma

The cyclooxygenase enzyme system produces eicosanoids which mediate many important physiological and pathological functions. Experimental and clinical data suggest a role for this enzyme system in the pathogenesis of allergic inflammation and asthma. This article focuses on the function of this pathway in the lung, reviews evidence implicating the involvement of this pathway in asthma and allergic airway inflammation, and discusses implications for the treatment of asthmatics with cyclooxygenase inhibitors.     

Micro-PET/CT monitoring of herpes thymidine kinase suicide gene therapy in a prostate cancer xenograft: the advantage of a cell-specific transcriptional targeting approach

Cancer gene therapy based on tissue-restricted expression of cytotoxic gene should achieve superior therapeutic index over an unrestricted method. This study compared the therapeutic effects of a highly augmented, prostate-specific gene expression method to a strong constitutive promoter-driven approach. Molecular imaging was coupled to gene therapy to ascertain real-time therapeutic activity. The imaging reporter gene (luciferase) and the cytotoxic gene (herpes simplex thymidine kinase) were delivered by adenoviral vectors injected directly into human prostate tumors grafted in SCID mice. Serial bioluminescence imaging, positron emission tomography, and computed tomography revealed restriction of gene expression to the tumors when prostate-specific vector was employed. In contrast, administration of constitutive active vector resulted in strong signals in the liver. Liver serology, tissue histology, and frail condition of animals confirmed liver toxicity suffered by the constitutive active cohorts, whereas the prostate-targeted group was unaffected. The extent of tumor killing was analyzed by apoptotic staining and human prostate marker (prostate-specific antigen). Overall, the augmented prostate-specific expression system was superior to the constitutive approach in safeguarding against systemic toxicity, while achieving effective tumor killing. Integrating noninvasive imaging into cytotoxic gene therapy will provide a useful strategy to monitor gene expression and therapeutic efficacy in future clinical protocols.     

High-density single-nucleotide polymorphism maps of the human genome

Here we report a large, extensively characterized set of single-nucleotide polymorphisms (SNPs) covering the human genome. We determined the allele frequencies of 55,018 SNPs in African Americans, Asians (Japanese-Chinese), and European Americans as part of The SNP Consortium's Allele Frequency Project. A subset of 8333 SNPs was also characterized in Koreans. Because these SNPs were ascertained in the same way, the data set is particularly useful for modeling. Our results document that much genetic variation is shared among populations. For autosomes, some 44% of these SNPs have a minor allele frequency > or =10% in each population, and the average allele frequency differences between populations with different continental origins are less than 19%. However, the several percentage point allele frequency differences among the closely related Korean, Japanese, and Chinese populations suggest caution in using mixtures of well-established populations for case-control genetic studies of complex traits. We estimate that approximately 7% of these SNPs are private SNPs with minor allele frequencies <1%. A useful set of characterized SNPs with large allele frequency differences between populations (>60%) can be used for admixture studies. High-density maps of high-quality, characterized SNPs produced by this project are freely available.     

Single and interacting QTLs for cholesterol gallstones revealed in an intercross between mouse strains NZB and SM

Quantitative trait locus (QTL) mapping was employed to investigate the genetic determinants of cholesterol gallstone formation in a large intercross between mouse strains SM/J (resistant) and NZB/B1NJ (susceptible). Animals consumed a gallstonepromoting diet for 18 weeks. QTL analyses were performed using gallstone weight and gallstone absence/presence as phenotypes; various models were explored for genome scans. We detected seven single QTLs: three new, significant QTLs were named Lith17 [chromosome (Chr) 5, peak=60 cM, LOD=5.4], Lith18 (Chr 5, 76 cM, LOD=4.3), and Lith19 (Chr 8, 0 cM, LOD=5.3); two confirmed QTLs identified previously and were named Lith20 (Chr 9, 44 cM, LOD=2.7) and Lith21 (Chr 10, 24 cM, LOD=2.9); one new, suggestive QTL (Chr 17) remains unnamed. Upon searching for epistatic interactions that contributed to gallstone susceptibility, the final suggestive QTL on Chr 7 was determined to interact significantly with Lith18 and, therefore, was named Lith22 (65 cM). A second interaction was identified between Lith19 and a locus on Chr 11; this QTL was named Lith23 (13 cM). mRNA expression analyses and amino acid haplotype analyses likely eliminated Slc10a2 as a candidate gene for Lith19. The QTLs identified herein largely contributed to gallstone formation rather than gallstone severity. Cloning the genes underlying these murine QTLs should facilitate prediction and cloning of the orthologous human genes.Abbreviations: CI, confidence interval; F₁,first filial generation; F₂, second filial (intercross) generation; LOD, logarithm of the odds ratio; NZB, NZB/B1NJ; QTL, quantitative trait locus; SM, SM/J. The nucleotide sequence data for Slc10a2 were submitted to GenBank and were assigned the accession numbers AY529655 (strain SM) and AY529656 (strain NZB).     

Network structures and algorithms in Bioconductor

In this paper, we review the central concepts and implementations of tools for working with network structures in Bioconductor. Interfaces to open source resources for visualization (AT&T Graphviz) and network algorithms (Boost) have been developed to support analysis of graphical structures in genomics and computational biology. AVAILABILITY: Packages graph, Rgraphviz, RBGL of Bioconductor (www.bioconductor.org).     

Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport

When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by the loss of the extensive interphase Mt array and the formation of a bipolar mitotic spindle. Microtubules in cells stably expressing GFP-alpha-tubulin were directly observed from prophase to just after nuclear envelope breakdown (NEBD) in early prometaphase. Our results demonstrate a transient stimulation of individual Mt dynamic turnover and the formation and inward motion of microtubule bundles in these cells. Motion of microtubule bundles was inhibited after antibody-mediated inhibition of cytoplasmic dynein/dynactin, but was not inhibited after inhibition of the kinesin-related motor Eg5 or myosin II. In metaphase cells, assembly of small foci of Mts was detected at sites distant from the spindle; these Mts were also moved inward. We propose that cytoplasmic dynein-dependent inward motion of Mts functions to remove Mts from the cytoplasm at prophase and from the peripheral cytoplasm through metaphase. The data demonstrate that dynamic astral Mts search the cytoplasm for other Mts, as well as chromosomes, in mitotic cells.     

Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry

The Aequorea victoria green fluorescent protein (GFP) undergoes a remarkable post-translational modification to create a chromophore out of its component amino acids S65, Y66, and G67. Here, we describe mutational experiments in GFP designed to convert this chromophore into a 4-methylidene-imidazole-5-one (MIO) moiety similar to the post-translational active-site electrophile of histidine ammonia lyase (HAL). Crystallographic structures of GFP variant S65A Y66S (GFPhal) and of four additional related site-directed mutants reveal an aromatic MIO moiety and mechanistic details of GFP chromophore formation and MIO biosynthesis. Specifically, the GFP scaffold promotes backbone cyclization by (1) favoring nucleophilic attack by close proximity alignment of the G67 amide lone pair with the pi orbital of the residue 65 carbonyl and (2) removing enthalpic barriers by eliminating inhibitory main-chain hydrogen bonds in the precursor state. GFP R96 appears to induce structural rearrangements important in aligning the molecular orbitals for ring cyclization, favor G67 nitrogen deprotonation through electrostatic interactions with the Y66 carbonyl, and stabilize the reduced enolate intermediate. Our structures and analysis also highlight negative design features of the wild-type GFP architecture, which favor chromophore formation by destabilizing alternative conformations of the chromophore tripeptide. By providing a molecular basis for understanding and controlling the driving force and protein chemistry of chromophore creation, this research has implications for expansion of the genetic code through engineering of modified amino acids.