Regulation of mammalian gastrin/CCK receptor (CCK2R) expression in vitro and in vivo

The gastrin/CCK receptor (CCK2R) mediates the physiological functions of gastrin in the stomach, including stimulation of acid secretion and cellular proliferation and migration, but little is known about the factors that regulate its expression. We identified endogenous CCK2R expression in several cell lines and used luciferase promoter–reporter constructs to define the minimal promoter required for transcription in human gastric adenocarcinoma, AGS, and rat gastric mucosa, RGM1, cells. Consensus binding sites for SP1, C/EBP and GATA were essential for activity. Following serum withdrawal from RGM1 and AR42J cells, endogenous CCK2R mRNA abundance and the activity of a CCK2R promoter–reporter construct were significantly elevated. Transcription of CCK2R was also increased in AGS-GR and RGM1 cells by gastrin through mechanisms partly dependent upon protein kinase C (PKC) and mitogen/extracellular signal-regulated kinase (MEK). Gastrin significantly increased endogenous CCK2R expression in RGM1 cells, and CCK2R protein expression was elevated in the stomach of hypergastrinaemic animals. In mice with cryoulcers in the acid-secreting mucosa, CCK2R expression increased progressively in the regenerating mucosa adjacent to the ulcer repair margin, evident at 6 days postinjury and maximal at 13 days. De novo expression of CCK2R was observed in the submucosa beneath the repairing ulcer crater 6–9 days postinjury. Many of the cells in mucosa and submucosa that expressed CCK2R in response to cryoinjury were identified as myofibroblasts, since they coexpressed vimentin and smooth muscle α-actin but not desmin. The data suggest that increased CCK2R expression might influence the outcome of epithelial inflammation or injury and that the response may be mediated in part by myofibroblasts

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

, CCK2R immunoreactivity and DAPI staining in full-thickness corpus 2, 6, 9 and 13 days following cryoulcer generation

<p><b>Copyright information:</b></p><p>Taken from "Regulation of mammalian gastrin/CCK receptor (CCK2R) expression and "</p><p></p><p>Experimental Physiology 2007;93(2):223-236.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2253704.</p><p>© The Authors. Journal compilation © 2007 The Physiological Society</p> Note intense epithelial staining at ulcer repair margins at 13 days, and staining in the submucosa beneath the ulcer crater (indicated by white line) at 9 days. Scale bar represents 500 μ. Sections are representative from four individual animals at each time point. , CCK2R and smooth muscle α-actin immunoreactivity in gastric corpus epithelium at the repair margin of a 9 day ulcer. Upper panel, CCK2R (FITC); centre panel, smooth muscle α-actin (Texas Red); and lower panel, overlay. , CCK2R and vimentin immunoreactivity in gastric corpus submucosa beneath the repairing crater of a 9 day ulcer. Upper panel, CCK2R (FITC); centre panel, vimentin (Texas Red); and lower panel, overlay. , deconvolved images of gastric corpus epithelial cells at the repair margin of a 9 day ulcer. Upper left panel, CCK2R (FITC); upper right panel, smooth muscle α-actin (Texas Red); and lower panel, overlay. , deconvolved images of gastric corpus submucosal cells beneath the repairing crater of a 9 day ulcer. Upper panel, CCK2R (FITC); centre panel, vimentin (Texas Red); and lower panel, overlay. For and , scale bar represents 50 μm; for and , scale bar respresents 10 μm

, CCK2R expression in mucosa and submucosa 9 days following cryoulcer generation

<p><b>Copyright information:</b></p><p>Taken from "Regulation of mammalian gastrin/CCK receptor (CCK2R) expression and "</p><p></p><p>Experimental Physiology 2007;93(2):223-236.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2253704.</p><p>© The Authors. Journal compilation © 2007 The Physiological Society</p> Bracket indicates residual ulcer crater. , CCK2R expression in mucosa and submucosa 9 days following cryoulceration, 0.5 cm away from the ulcer site. C, regenerative mucosa, adjacent to cryoulcer. , submucosa below residual ulcer crater. , normal mucosa, 0.5 cm away from site of ulcer. , normal submucosa, 0.5 cm away from site of ulcer. For and , scale bar respresents 300 μm; for , scale bar represents 50 μm. Hybridizations were performed using a mixture (1:1:1) of the three oligonucleotides described in the Methods

, sequence of the human CCK2R promoter −130 to −190 region, showing consensus -regulatory elements

<p><b>Copyright information:</b></p><p>Taken from "Regulation of mammalian gastrin/CCK receptor (CCK2R) expression and "</p><p></p><p>Experimental Physiology 2007;93(2):223-236.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2253704.</p><p>© The Authors. Journal compilation © 2007 The Physiological Society</p> Numbering is relative to the start of transcription (+1), which is taken as 193 bp upstream of the start of translation (see main text). , basal activity of CCK2R promoter–reporter constructs (size as indicated) 24 h after transfection in AGS cells. * Significantly reduced relative to 1700 bp construct ( < 0.001, = 3, ANOVA). , basal activity of CCK2R promoter–reporter constructs (size as indicated) 24 h after transfection in RGM1 cells. * Significantly reduced relative to 928 bp construct ( < 0.001, = 3, ANOVA). , basal activity of mutated 196 bp constructs 24 h after transfection into AGS cells. Mutations as indicated were made corresponding to the -regulatory elements underlined in . Values are percentage of wild-type (WT) sequence activity (= 100%). * Significantly reduced compared with WT activity ( < 0.001, = 3). , basal activity of mutated 196 bp constructs 24 h after transfection into RGM1 cells. Mutations as described in . Values are percentage of WT sequence activity (= 100%). * Significantly reduced compared with WT activity ( < 0.001, = 3, ANOVA)

, endogenous CCK2R mRNA abundance in AR42J cells in response to serum withdrawal for times indicated

<p><b>Copyright information:</b></p><p>Taken from "Regulation of mammalian gastrin/CCK receptor (CCK2R) expression and "</p><p></p><p>Experimental Physiology 2007;93(2):223-236.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2253704.</p><p>© The Authors. Journal compilation © 2007 The Physiological Society</p> Results are normalized to 18S rRNA and are expressed as percentage of full serum value (= 100%). = 4. * = 0.016, ** = 0.013, *** = 0.005 time 0, ANOVA. , CCK2R mRNA abundance in RGM1 cells in response to serum withdrawal for times indicated. Results are normalized to 18S rRNA and are expressed as percentage of full serum value (= 100%). = 3. * = 0.0016 time 0, ANOVA

The description of cough sounds by healthcare professionals

BACKGROUND: Little is known of the language healthcare professionals use to describe cough sounds. We aimed to examine how they describe cough sounds and to assess whether these descriptions suggested they appreciate the basic sound qualities (as assessed by acoustic analysis) and the underlying diagnosis of the patient coughing. METHODS: 53 health professionals from two large respiratory tertiary referral centres were recruited; 22 doctors and 31 staff from professions allied to medicine. Participants listened to 9 sequences of spontaneous cough sounds from common respiratory diseases. For each cough they selected patient gender, the most appropriate descriptors and a diagnosis. Cluster analysis was performed to assess which cough sounds attracted similar descriptions. RESULTS: Gender was correctly identified in 93% of cases. The presence or absence of mucus was correct in 76.1% and wheeze in 39.3% of cases. However, identifying clinical diagnosis from cough was poor at 34.0%. Cluster analysis showed coughs with the same acoustics properties rather than the same diagnoses attracted the same descriptions. CONCLUSION: These results suggest that healthcare professionals can recognise some of the qualities of cough sounds but are poor at making diagnoses from them. It remains to be seen whether in the future cough sound acoustics will provide useful clinical information and whether their study will lead to the development of useful new outcome measures in cough monitoring

Caspase-8-mediated apoptosis induced by oxidative stress is independent of the intrinsic pathway and dependent on cathepsins

Cell-death programs executed in the pancreas under pathological conditions remain largely undetermined, although the severity of experimental pancreatitis has been found to depend on the ratio of apoptosis to necrosis. We have defined mechanisms by which apoptosis is induced in pancreatic acinar cells by the oxidant stressor menadione. Real-time monitoring of initiator caspase activity showed that caspase-9 (66% of cells) and caspase-8 (15% of cells) were activated within 30 min of menadione administration, but no activation of caspase-2, -10, or -12 was detected. Interestingly, when caspase-9 activation was inhibited, activation of caspase-8 was increased. Half-maximum activation (t(0.5)) of caspase-9 occurred within approximately 2 min and was identified at or in close proximity to mitochondria, whereas t(0.5) for caspase-8 occurred within approximately 26 min of menadione application and was distributed homogeneously throughout cells. Caspase-9 but not caspase-8 activation was blocked completely by the calcium chelator BAPTA or bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore. In contrast, caspase-8 but not caspase-9 activation was blocked by the destruction of lysosomes (preincubation with Gly-Phe beta-naphthylamide, a cathepsin C substrate), loss of lysosomal acidity (bafilomycin A1), or inhibition of cathepsin L or D. Using pepstatin A-BODIPY FL conjugate, we confirmed translocation of cathepsin D out of lysosomes in response to menadione. We conclude that the oxidative stressor menadione induces two independent apoptotic pathways within pancreatic acinar cells: the classical mitochondrial calcium-dependent pathway that is initiated rapidly in the majority of cells, and a slower, caspase-8-mediated pathway that depends on the lysosomal activities of cathepsins and is used when the caspase-9 pathway is disabled