Independent and joint effect of type 2 diabetes and gastric and hepatobiliary diseases on risk of pancreatic cancer risk: 10-year follow-up of population-based cohort.

BACKGROUND: Type 2 diabetes mellitus, gastric and hepatobiliary comorbidities, and cancer share common risk factors: for example, tobacco, obesity, physical inactivity, high calorie intake, and metabolic disorders. Prior studies find type 2 diabetes and gastric and hepatobiliary comorbidities heightening risk of pancreatic cancer. Yet joint association of type 2 diabetes mellitus and gastric and hepatobiliary comorbidities on pancreatic cancer risk has not been assessed. METHODS: This study rates independent/joint effects of type 2 diabetes as well as gastric and hepatobiliary comorbidity on pancreatic cancer risk for a retrospective population-based cohort of 166,850 type 2 diabetics identified in 1997-1998 and followed for 10-11 years, comparing their cancer incidence with that of 166,850 non-diabetics matched for age, gender, and locale. Time-dependent Cox's proportional hazards model evaluted joint association of type 2 diabetes and chronic conditions on pancreatic cancer risk. RESULTS: A total of 1178 subjects were newly diagnosed with pancreatic cancer during follow-up, with incidence rates of 0.49 per 1000 person-years in type 2 diabetes and 0.26 per 1000 person-years in the non-diabetics. We observed greater magnitude of hazard ratios (HRs) of pancreatic cancer for patients with type 2 diabetes along with acute alcoholic hepatitis, acute pancreatitis, cholecystitis, and gastric ulcer compared with patients without type 2 diabetes or counterpart comorbidity (HR: 1.36, 95% confidence interval (CI): 1.19-1.56; 1.74, 1.23-2.45; 9.18, 7.44-11.33; and 2.31, 1.98-2.70, respectively). Main effects of type 2 diabetes were all statistically with narrow 95% CI and remained similar across risk stratification with various comorbidities: range 1.59-1.80. CONCLUSIONS: Our study demonstrates that pre-existing type 2 diabetes, acute alcoholic hepatitis, acute pancreatitis, cholecystitis, and gastric ulcer independently or jointly predict subsequent pancreatic cancer risk. Clinicians must recognise burden of these gastric and hepatobiliary comorbidities and keep clinically vigilant for their diagnosis.

First Report of Pink Seed of Lentil and Chickpea Caused by Erwinia rhapontici in Canada.

A new disease of lentil (Lens culinaris Medik.) and chickpea (Cicer arietinum L.) caused by Erwinia rhapontici (Millard) Burkh. was found in seed samples from commercial fields in Saskatchewan, Canada in 2002. Infected seeds had a pink or pinkish-brown discoloration of the seed coat. Isolation from surface-sterilized pink seeds resulted in bacterial cultures that produced a water-soluble pink pigment on potato dextrose agar (PDA). Four isolates from different lentil crops, LRC 8265, LRC 8310, LRC 8309, and LRC 8313 and one isolate from a chickpea crop, LRC 8266, were tested as previously described (2). Results of the tests were identical to those for pink bean isolates of E. rhapontici (2) with the following minor exceptions: all were negative for Voges-Proskauer; LRC 8266 was positive for tagatose; LRC 8266, LRC 8309, and LRC 8313 were negative for lactose; and LRC 8266 and LRC 8309 were positive for 5-keto gluconate. For pathogenicity tests, each isolate was inoculated into 30 pods from 6 lentil plants (cv. Laird), 30 pods from 6 desi chickpea plants (cv. Myles), and 30 pods from 6 kabuli chickpea plants (cv. Sanford) by the method described for pink seed of pea (1) and bean (2). Each pod was inoculated with 0.1 ml (0.2 ml for kabuli chickpeas) of bacterial suspension, approximately 108 CFU/ml, by injection through the mid-rib at the basal end. The same number of uninoculated and water-inoculated pods served as controls. Plants were kept in the greenhouse (20 ± 5°C) for 4 weeks, after which isolations of the pathogen were performed as described above. In duplicate experiments, all the isolates caused pink lesions on pods and seeds of lentil, desi chickpea, and kabuli chickpea. The frequency of infected seeds among the five isolates (four lentil and one chickpea) ranged from 50 to 100% on lentil, 73 to 100% on desi chickpea, and 43 to 100% on kabuli chickpea. E. rhapontici was reisolated from seeds with lesions but not from asymptomatic seeds. The study demonstrates that in addition to pea (1) and common bean (2), E. rhapontici is also the causal agent of pink seed of lentil and chickpea. The observation that lentil isolates can infect chickpea and vice versa suggests that host specificity may be lacking in E. rhapontici. To our knowledge, this is the first record of E. rhapontici on lentil and chickpea. References: (1) H. C. Huang et al. Can. J. Plant Pathol. 12:445, 1990. (2) H. C. Huang et al. Plant Dis. 86:921, 2002.

First Report of Bacterial Wilt of Common Bean Caused by Curtobacterium flaccumfaciens in Western Canada.

Bacterial wilt of common bean (Phaseolus vulgaris L.) caused by Curtobacterium flaccumfaciens pv. flaccumfaciens (Hedges) Collins & Jones (4) was found in 1947 in Ontario, Canada (3), but not in western Canada. Infected seeds exhibit yellow, orange, or purple discoloration (4). Examination of 36.7 kg of cull beans of crops grown in southern Alberta in 2001 obtained from a processing plant revealed 5.9% yellow and 0.014% orange seeds, each with wrinkled seed coats. Bacteria were isolated on potato dextrose agar. Three strains were identified using conventional tests (2), carbohydrate oxidation (GP Microplates, Biolog Inc., Hayward, CA), and cellular fatty acids (CFA) (MIDI, Inc., Newark, DE). Strains were gram-positive, motile, aerobic rods with yellow (YSB-1, YSB-2) or orange (OSB-3) colonies. Growth occurred at 27 and 37°C. The strains were positive for citrate utilization, catalase, hydrolysis of hippurate, and indoxyl acetate, and negative for urease, gelatin liquification, and oxidase. CFA profiles were ≈48% 15:0 anteiso, 37% 17:0 anteiso, 8% 16:0 iso, 3% 15:0 iso, and 3% 16:0; with17:1 anteiso A sometimes present at <2%. Acid production was weak from carbohydrates, but all oxidized many carbohydrates in the microplates. These results match C. flaccumfaciens pv. flaccumfaciens (2) in MIDI and Biolog databases. Strains were tested for pathogenicity using seed and pod inoculations. Seeds of great northern ('US1140') and navy ('AC Skipper') beans were soaked in bacterial suspension (1 to 3 × 108 CFU/ml) for 1 h, sown in Cornell Peatlite Mix (1) in Root Trainers (Spencer-Lemaire Industries, Edmonton, AB, Canada), incubated at 28°C (16-h day) and 22°C (8-h night), and examined for seedling wilt after 10 days. Seeds soaked in sterile distilled water served as controls. Testing was repeated once with 3 replicates per treatment and 10 seeds per replicate. Experiments were conducted using a complete randomization design. For pod inoculation, a suspension (0.1 ml) of each strain was injected into the midrib at the basal end of each young pod of 'AC Skipper'. Pods inoculated with sterile distilled water, 0.1 ml per pod, were used as controls. After 21 days, pods were harvested and examined. Testing was repeated once with three plants per treatment and five pods per plant. Bacteria were reisolated from hypocotyls of wilted seedlings and diseased pods. Results of seed inoculations showed all strains were pathogenic to both cultivars. Wilt incidence was 38, 35, and 57% for strains YSB-1, YSB-2, and OSB-3, respectively, on 'US1140' and 44, 40, and 63% respectively, on 'AC Skipper'. Results of pod inoculations showed 63% (YSB-1) and 55% (YSB-2) of seeds had wrinkled, yellow seed coats, and 72% (OSB-3) of seeds had wrinkled, orange seed coats. Control seedlings and seeds remained healthy. C. flaccumfaciens pv. flaccumfaciens was reisolated from wilted seedlings and seeds showing yellow or orange discoloration, but not from the controls. To our knowledge, this is the first report of bacterial wilt of bean caused by yellow and orange strains of C. flaccumfaciens pv. flaccumfaciens in western Canada. References: (1) J. W. Boodley and R. Sheldrake Jr. N.Y. State Coll. Agric. Life Sci. Inform. Bull. 43, 1977. (2) K. Komagata et al. Page 1313 in: Bergey's Manual of Systematic Bacteriology, Vol. 2, Williams and Wilkens, Baltimore, MD, 1986. (3) Z. A. Patrick, Can. J. Bot. 32:705, 1954. (4) A. W. Saettler. Bacterial wilt. Page 31 in: Compendium of Bean Diseases. R. Hall, ed. American Phytopathology Society, St. Paul, MN, 1994.

First Report of Pink Seed of Common Bean Caused by Erwinia rhapontici.

In 2001, a new disease of common bean (Phaseolus vulgaris L.) caused by Erwinia rhapontici (Millard) Burkh. was detected in seed samples from southern Alberta, Canada. Infected seeds had pink or pinkish-brown lesions on the seed coat. The disease was found in great northern (cv. US1140), pink (cv. Viva), and pinto (cv. Othello) beans at low (<0.1%) frequencies. Isolation from surface-sterilized pink seeds resulted in bacterial cultures, which produced a water-soluble pink pigment on potato dextrose agar (PDA). Seven isolates were tested for physiological characteristics using conventional tests (1) and API 50CHE test strips (bioMérieux Canada, St. Laurent, Quebec), and tested for cellular fatty acids using the MIDI system (Newark, DE). All isolates were gram-negative, motile, facultative anaerobic rods with mucoid colonies and produced a pink pigment on PDA. They were positive for citrate utilization, catalase, methyl red, and Voges-Proskauer, and negative for arginine dihydrolase, lysine and ornithine decarboxylases, urease, gelatin liquification, indole production, oxidase, and gas production. Fatty acid profiles matched with E. rhapontici (approximately 30% each 16:0 and 16:1 ω7c/15:0 iso 2OH; 12% 18:1 ω7c: 8% each 17:0 cyclo and 14:0 3OH/16:1 iso; 4 to 5% each 12:0 and 14:0). Isolates were positive for acid production from: N-acetyl glucosamine, l-arabinose, amygdalin, arbutin, cellobiose, esculin (hydrolysis), d-fructose, d-fucose, d-galactose, ß-gentiobiose, d-glucose, glycerol, i-myo-inositol, lactose, maltose, d-mannitol, d-mannose, melibiose, d-raffinose, l-rhamnose, ribose, salicin, d-sorbitol, sucrose, trehalose, and d-xylose. These results match published results for E. rhapontici (4). For pathogenicity tests, each isolate was inoculated in 30 pods from six bean plants (cv. US1140) as described for pink seed of peas (2). Each pod was inoculated with 0.1 ml of bacterial suspension, approximately 109 CFU/ml, by injection through the mid-rib at the basal end. The same number of uninoculated and water-inoculated pods served as controls. Plants were kept in the greenhouse (20 ± 5°C) for 4 weeks, after which isolations were done as described above. In duplicate experiments, all isolates caused lesions on pods extending up to 5 cm from the inoculation point with corresponding discoloration of seeds. The frequency of infected seeds varied among isolates, ranging from 20 to 50%. E. rhapontici was reisolated from seeds with lesions, but not asymptomatic seeds. The study concludes that pink seed of common bean is due to E. rhapontici, a pathogen previously reported on peas in Alberta, Canada (2), and Montana (3). References: (1) D. J. Brenner. Bergey's Manual of Systematic Bacteriology, vol.1, Williams and Wilkens, Baltimore, MD, 1984. (2) H. C. Huang et al. Can. J. Plant Pathol. 12:445, 1990. (3) B. K. Schroeder et al. Plant Dis. 86:188, 2002. (4) L. Verdonck et al. Int. J. Syst. Bacteriol. 37:4, 1987.

Gene expression in the developing mouse retina by EST sequencing and microarray analysis.

Retinal development occurs in mice between embryonic day E11.5 and post-natal day P8 as uncommitted neuroblasts assume retinal cell fates. The genetic pathways regulating retinal development are being identified but little is understood about the global networks that link these pathways together or the complexity of the expressed gene set required to form the retina. At E14.5, the retina contains mostly uncommitted neuroblasts and newly differentiated neurons. Here we report a sequence analysis of an E14.5 retinal cDNA library. To date, we have archived 15 268 ESTs and have annotated 9035, which represent 5288 genes. The fraction of singly occurring ESTs as a function of total EST accrual suggests that the total number of expressed genes in the library could approach 27 000. The 9035 ESTs were categorized by their known or putative functions. Representation of the genes involved in eye development was significantly higher in the retinal clone set compared with the NIA mouse 15K cDNA clone set. Screening with a microarray containing 864 cDNA clones using wild-type and brn-3b (-/-) retinal cDNA probes revealed a potential regulatory linkage between the transcription factor Brn-3b and expression of GAP-43, a protein associated with axon growth. The retinal EST database will be a valuable platform for gene expression profiling and a new source for gene discovery.

Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco.

4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

Peripheral nerve insult induces NMDA receptor-mediated, delayed degeneration in spinal neurons.

Injury of a peripheral nerve gives rise to adaptive functional and structural alterations in spinal neurons. We report that the rearrangement of the spinal circuitry in response to sciatic nerve transection in adult rats involves a delayed mode of degeneration of lumbar spinal cord neurons. Nuclear fragmentation was detected by the TUNEL technique 7 days after sciatic neurectomy but not after 3 or 14 days. Dying cells were preferentially located in the ipsilateral superficial dorsal horn and expressed the neuronal cytoskeletal marker SMI-31. Degeneration was prevented by continuous systemic treatment with the NMDA receptor-antagonist MK-801. These data are supportive that apoptosis is induced in spinal neurons in a transsynaptic manner by an early signal from injured afferent fibres via activation of spinal NMDA receptors.

BDNF restores the expression of Jun and Fos inducible transcription factors in the rat brain following repetitive electroconvulsive seizures.

The expression of inducible transcription factors was studied following repetitive electroconvulsive seizures (ECS), c-Fos, c-Jun, JunB, and JunD immunoreactivities were investigated following a single (1 x ECS) or repetitive ECS evoked once per day for 4, 5, or 10 days (4 x ECS, 5 x ECS, or 10 x ECS). Animals were killed 3 or 12 h following the last ECS. Three hours after 1 x ECS, c-Fos was expressed throughout the cortex and hippocampus. After 5 x ECS and 10 x ECS, c-Fos was reexpressed in the CA4 area, but was completely absent in the other hippocampal areas and cortex. In these areas, c-Fos became only reinducible when the time lag between two ECS stimuli was 5 days. In contrast to c-Fos, intense JunB expression was inducible in the cortex and hippocampus, but not CA4 subfield, after 1 x ECS, 5 x ECS, and 10 x ECS. Repetitive ECS did not effect c-Jun and JunD expression. In a second model of systemic excitation of the brain, repetitive daily injection of kainic acid for 4 days completely failed to express c-Fos, c-Jun, and JunB after the last application whereas injection of kainic acid once per week did not alter the strong expressions compared to a single application of kainic acid. In order to study the maintenance of c-Fos expression during repetitive seizures, brain-derived neurotrophic factor (BDNF) was applied in parallel for 5 or 10 days via miniosmotic pumps and permanent cannula targeted at the hippocampus or the parietal cortex. Infusion of BDNF completely reinduced c-Fos expression during 5 x ECS or 10 x ECS in the cortex ipsilaterally to the cannula and, to a less extent, also increased the expression of c-Jun and JunB when compared to saline-treated controls. BDNF had no effect on the expression patterns in the hippocampus. ECS with or without BDNF infusion did not change the expression patterns of the constitutive transcription factors ATF-2, CREB, and SRF. These data demonstrate that various transcription factors substantially differ in their response to acute and chronic neural stimulation. Repetitive pathophysiological excitation decreases the transcriptional actions of neurons over days in the adult brain, and this decrement can be prevented by BDNF restoring the neuroplasticity at the level of gene transcription.

Design of a high-flux and high-resolution VUV bending-magnet beamline.

A high-flux and high-resolution VUV beamline (4-40 eV) has been designed and is under construction at SRRC. This beamline, which collects 50 mrad of horizontal radiation, uses a 6 m cylindrical-grating monochromator with an incident angle of 70 degrees instead of the conventional normal-incidence-monochromator (NIM) design. Special features, such as movable entrance slit, bendable vertical focusing mirror and movable curved exit slit, are employed to enhance greatly the beamline performance. With both slit openings set at 10 micro m, the energy-resolving power can reach as high as 70000. Photon fluxes of 1 x 10(13) and 1 x 10(10) photons s(-1) are calculated for energy-resolving powers of 1000 and 40000, respectively. The best image size at the sample position is smaller than 0.45 x 0.2 mm.

Expression of activating transcription factor-2, serum response factor and cAMP/Ca response element binding protein in the adult rat brain following generalized seizures, nerve fibre lesion and ultraviolet irradiation.

The expression of the constitutive transcription factors activating transcription factor-2 (ATF-2), serum response factor (SRF) and cAMP/Ca response element binding factor (CREB), and the phosphorylation of SRF and CREB were studied in the untreated adult rat nervous system and following seizure activities and neurodegenerative stimuli. In the untreated rat, intense nuclear SRF immunoreactivity was present in the vast majority of neurons in the forebrain, cortex, striatum, amygdala and hippocampus, and in some scattered neurons in the medulla and spinal cord. In contrast, SRF immunoreactivity was absent in the midline areas of the forebrain, e.g., the globus pallidum and septum, and in the hypothalamus, thalamus, mesencephalon and motoneurons. Nuclear ATF-2 was expressed at high levels in apparently all neurons, but not glial cells, throughout the neuraxis except for those neuronal populations which exhibit a high basal level of c-Jun, i.e. dentate gyrus and the motoneurons of cranial and somatosensory neurons. CREB immunoreactivity was present at a rather uniform intensity in all neuronal and glial cells throughout the neuraxis. Two hours, but not 5 h or 24 h, following systemic application of kainic acid, an increase in SRF was detectable by western blot analysis in hippocampal and cortical homogenates whereas the expression of ATF-2 and CREB did not change. Phosphorylation of CREB at serine 133 and of SRF at serine 103 were studied with specific antisera. In untreated rats, intense phosphoCREB and phosphoSRF immunoreactivities labelled many glial cells and/or neurons with the highest levels in the dentate gyrus, the entorhinal cortex and the retrosplenial cortex. Following kainate-induced seizures, phosphoSRF-IR but not phosphoCREB-IR transiently increased between 0.5 h and 2 h. Following transection of peripheral or central nerve fibres such as optic nerve, medial forebrain bundle, vagal and facial nerve fibres, ATF-2 rapidly decreased in the axotomized neurons during that period when c-Jun was rapidly expressed. SRF remained unchanged and CREB disappeared in some axotomized subpopulations. Similar to axotomy, c-Jun increased and ATF-2 decreased in cultured adult dorsal root ganglion neurons following ultraviolet irradiation. The distribution of SRF and ATF-2 suggests that their putative target genes c-fos, junB, krox-24 and c-jun can be independently regulated from SRF and ATF-2. The suppression of ATF-2 and the expression of c-Jun following axotomy and ultraviolet irradiation might be part of a novel neuronal stress response in the brain that strongly resembles the stress response characterized in non-neuronal cells.

Molecular approaches to identify novel genes expressed in Arabidopsis thaliana.

Our laboratory is engaged in an effort to identify genes expressed primarily during plant embryogenesis. Genes which exhibit unique expression profiles in the plant are also being sought. To this end, several methods to identify and clone novel genes based on specific expression patterns have been developed. These methods include virtual subtraction, differential display and other PCR based technologies. In addition to this, a yeast one-hybrid approach has been established to identify transcription factors which regulate these genes. To date, this work has identified several novel genes.

Identification of novel mRNAs in immature seeds of Arabidopsis thaliana.

Although most of the major discernible morphogenetic events in plants occur after germination, the overall architectural pattern of the mature plant is established during early events of embryogenesis. So far, few genes that are expressed specifically during embryogenesis have been identified. This is due primarily to technical difficulties associated with the mass ratios of the embryo and the surrounding maternal tissue and to the lack of molecular and cellular markers to direct screening efforts. We have developed a series of molecular approaches to study the early events of embryogenesis. These include 'virtual subtraction' of a cDNA library with high specific-activity cDNA probes generated from both seed and non-seed tissue, PCR amplification of gene family members from an immature seed cDNA library using primers specific to conserved domains, differential display analysis of mRNA populations and high throughput expressed sequence tag (EST) analysis. These techniques have led to the identification and isolation of several novel seed-specific cDNAs.

Extracellular single-unit studies of suprachiasmatic neurons in brain slices. Effects of serotonin, dopamine, carbachol and LHRH.

Suprachiasmatic nucleus (SCN) is the major rhythm generating center in the central nervous system. Extracellular single-unit activities of the SCN neurons were recorded in brain tissue slices either from ovariectomized (OVX) rats or from OVX rats treated with estrogen (polyestradiol phosphate, PEP, 0.1 mg/rat, sc). Neuronal responses to luteinizing hormone-releasing hormone (LHRH) and various neurotransmitters, i.e., serotonin (5-HT), dopamine (DA) and carbachol (Carb, an acetylcholine agonist) were tested. A total of 137 units were recorded from OVX and 81 from OVX + PEP rats. The steady state firing rates of SCN neurons were between 0-9 spikes/sec in OVX and 0-23 in OVX + PEP group. Among them, most fired irregularly (86.9% in OVX and 71.6% in OVX + PEP rats), and a few fired regularly (10.2% and 25.9%, respectively). Few silent units were detected (2.9% and 2.5%, respectively). It seems that estrogen treatment somewhat increased the percentage of regular firing units. As for the agents tested in this study, LHRH by itself had no significant effect on the SCN neuronal activity; 5-HT affected more than 40% of the units, with about equal number of excitation (16.8%) and inhibition (24.4%), while Carb inhibited nearly half of the units tested (49.5%), excited only a few (10.3%). All the actions of these three agents were not significantly affected by in vivo estrogen pretreatment. Dopamine, however, excited more units (50%) in OVX than in OVX + PEP group (27.1%). Although LHRH had no direct effect by itself, it exerted a significant modulatory role on the actions of the other three agents. It either potentiated (42.9%) or inhibited (53.6%) the action of 5-HT, and it significantly potentiated the action of Carb (57.1%). For the action of DA, LHRH had a more significant potentiation effect on OVX + PEP than in OVX rats. (40.9% vs. 12.5%), while the inhibitory effect was the same. In conclusion, 5-HT, DA and Carb all exert significant effects on SCN neuronal activities. LHRH can have a modulatory role upon the action of 5-HT, DA and Carb, and estrogen pretreatment specifically affects that of DA in the SCN.