Pancreatic polypeptide inhibits somatostatin secretion

Pancreatic polypeptide (PP) is a major agonist for neuropeptide Y4 receptors (NPY4R). While NPY4R has been identified in various tissues, the cells on which it is expressed and its function in those cells has not been clearly delineated. Here we report that NPY4R is present in all somatostatin-containing cells of tissues that we tested, including pancreatic islets, duodenum, hippocampus, and hypothalamus. Its agonism by PP decreases somatostatin secretion from human islets. Mouse embryonic hippocampal (mHippo E18) cells expressed NPY4Rs and their activation by PP consistently decreased somatostatin secretion. Furthermore, central injection of PP in mice induced c-Fos immunoreactivity in somatostatin-containing cells in the hippocampus compared with PBS-injected mice. In sum, our results identify PP as a pivotal modulator of somatostatin secretion.     

Synthesis of N₄'-[¹⁸F]fluoroalkylated ciprofloxacin as a potential bacterial infection imaging agent for PET study

Syntheses and evaluation of fluoroalkylated ciprofloxacin analogues are described. Among these analogues, N?'-3-fluoropropylciprofloxacin (16) showed the most efficient antibacterial activity against E. coli strains (DH5α and TOP10) and a high binding affinity for DNA gyrase of bacteria. To develop bacteria-specific infection imaging agents for positron emission tomography (PET), no-carrier-added N?-3-[1?F]fluoropropylciprofloxacin ([1?F]16) was prepared in two steps from N?-3-methanesufonyloxypropylciprofloxacin, resulting in a 40% radiochemical yield (decay corrected for 100 min) via the tert-alcohol media radiofluorination protocol with high radiochemical purity (> 99%) as well as high specific activity (149 ± 75 GBq/μmol). The agent was stable (> 90%), as shown by an in vitro human serum stability assay. A bacterial uptake and blocking study of [1?F]16 using authentic compound 16 in TOP10 cells demonstrated its high specific bacterial uptake. The results suggest that this radiotracer holds promise as a useful bacterial infection radiopharmaceutical for PET imaging.     

Nucleolar protein GLTSCR2 stabilizes p53 in response to ribosomal stresses

p53 is a key regulator of cell growth and death by controlling cell cycle progression and apoptosis under conditions of stress such as DNA damage or oncogenic stimulation. As these processes are critical for cell function and inhibition of tumor development, p53 regulatory pathways are strictly monitored in cells. Recently, it was recognized that nucleolar proteins, including nucleophosmin/B23, ribosomal protein L11, and alternate reading frame (ARF), form the nucleolus-ARF-murine double minute 2 (MDM2) axis in p53 regulatory pathways, which increases p53 stability by suppressing the activity of MDM2. In this work, we show that nucleolar protein glioma tumor-suppressor candidate region gene 2 (GLTSCR2) translocates to the nucleoplasm under ribosomal stress, where it interacts with and stabilizes p53 and inhibits cell cycle progression without the involvement of the major upstream p53 regulator, ARF. Furthermore, ectopic expression of GLTSCR2 significantly suppressed growth of cancer cells in a xenograft animal model via p53-dependent pathway. Our data identify GLTSCR2 as a new member of the nucleolus-nucleoplasmic axis for p53 regulation. ARF-independent direct regulation of p53 by GLTSCR2 may be a key mechanism and therapeutic target for cell death or growth inhibition when nucleolus-ARF-p53 pathways are inactivated by genetic or epigenetic modifications of ARF, which are the second most common types of genetic change observed in human cancers.     

Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4

A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure–function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes.     

A catalog of neutral and deleterious polymorphism in yeast

The abundance and identity of functional variation segregating in natural populations is paramount to dissecting the molecular basis of quantitative traits as well as human genetic diseases. Genome sequencing of multiple organisms of the same species provides an efficient means of cataloging rearrangements, insertion, or deletion polymorphisms (InDels) and single-nucleotide polymorphisms (SNPs). While inbreeding depression and heterosis imply that a substantial amount of polymorphism is deleterious, distinguishing deleterious from neutral polymorphism remains a significant challenge. To identify deleterious and neutral DNA sequence variation within Saccharomyces cerevisiae, we sequenced the genome of a vineyard and oak tree strain and compared them to a reference genome. Among these three strains, 6% of the genome is variable, mostly attributable to variation in genome content that results from large InDels. Out of the 88,000 polymorphisms identified, 93% are SNPs and a small but significant fraction can be attributed to recent interspecific introgression and ectopic gene conversion. In comparison to the reference genome, there is substantial evidence for functional variation in gene content and structure that results from large InDels, frame-shifts, and polymorphic start and stop codons. Comparison of polymorphism to divergence reveals scant evidence for positive selection but an abundance of evidence for deleterious SNPs. We estimate that 12% of coding and 7% of noncoding SNPs are deleterious. Based on divergence among 11 yeast species, we identified 1,666 nonsynonymous SNPs that disrupt conserved amino acids and 1,863 noncoding SNPs that disrupt conserved noncoding motifs. The deleterious coding SNPs include those known to affect quantitative traits, and a subset of the deleterious noncoding SNPs occurs in the promoters of genes that show allele-specific expression, implying that some cis-regulatory SNPs are deleterious. Our results show that the genome sequences of both closely and distantly related species provide a means of identifying deleterious polymorphisms that disrupt functionally conserved coding and noncoding sequences.     

Characterization of an Escherichia coli O157:H7 plasmid O157 deletion mutant and its survival and persistence in cattle

Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic DeltapO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the DeltapO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the DeltapO157 mutant. The DeltapO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the DeltapO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the DeltapO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed approximately 50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.     

Phagocytosis of degenerating myelin in transected feline optic nerve: an immunohistochemical study

The phagocytic activity of neuroglial cells in adult feline degenerating optic nerve was investigated by immunocytochemistry at both light and electron microscopy levels. Degeneration was initiated by unilateral eye enucleation and the segment distal to the transection showing true Wallerian degeneration was examined. Following enucleation, twelve adult domestic cats were examined over a period of seven to 215 days. All cases showed slow clearance of myelin debris and absence of proliferating monocytes throughout the post-enucleation period. All phagocytic cells present were neuroglial cells, and many of these cells expressed oligodendroglial antigens. These findings demonstrate the persistence of an active population of oligodendrocytes that might play an additional functional role during Wallerian degeneration of feline optic nerve.     

Phylogenetic significance of stylar features in genus Vicia (Leguminosae): an analysis with molecular phylogeny

Tribe Fabeae consists of five genera, Lathyrus (160 spp.), Lens (4–6 spp.), Pisum (2–3 spp.), Vavilovia (monotypic), and Vicia (160 spp.), and shows a diversity in stylar features. At least six different stylar types are known in the tribe. In order to reclassify the tribe at the rank of genus, we tried to discover apomorphies in stylar features using a molecular phylogenetic study. We surveyed internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA of representative species, selected from each group having different types of styles in the tribe. We paid particular attention in sampling to members of Vicia section Vicilla, as stylar features are heterogeneous within this section. Consequently, our sample set included 15 species of section Vicilla, 23 species of other Fabeae, and two species of Trifolieae, which were used as a sister group of Fabeae. Based on our analysis, we found that a laterally compressed style and an abaxially tufted hairy style would be advanced against a dorsiventrally compressed style and an evenly hairy style, respectively, in genus Vicia. The species group, which shares the latter apomorphy, is composed of 56 species and was dispersed into 11 sections of two subgenera in the recent system of genus Vicia. We consider future revision of Fabeae should treat this species group as a single higher taxon.