RESUMO
Full-length cDNAs encoding crustacean cardioactive peptide (CCAP) were isolated from several decapod (brachyuran and astacuran) crustaceans: the blue crab Callinectes sapidus, green shore crab Carcinus maenas, European lobster Homarus gamarus and calico crayfish Orconectes immunis. The cDNAs encode open reading frames of 143 (brachyurans) and 139-140 (astacurans) amino acids. Apart from the predicted signal peptides (30-32 amino acids), the conceptually translated precursor codes for a single copy of CCAP and four other peptides that are extremely similar in terms of amino acid sequence within these species, but which clearly show divergence into brachyuran and astacuran groups. Expression patterns of CCAP mRNA and peptide were determined during embryonic development in Carcinus using quantitative RT-PCR and immunohistochemistry with whole-mount confocal microscopy, and showed that significant mRNA expression (at 50% embryonic development) preceded detectable levels of CCAP in the developing central nervous system (CNS; at 70% development). Subsequent CCAP gene expression dramatically increased during the late stages of embryogenesis (80-100%), coincident with developing immunopositive structures. In adult crabs, CCAP gene expression was detected exclusively in the eyestalk, brain and in particular the thoracic ganglia, in accord with the predominance of CCAP-containing cells in this tissue. Measurement of expression patterns of CCAP mRNA in Carcinus and Callinectes thoracic ganglia throughout the moult cycle revealed only modest changes, indicating that previously observed increases in CCAP peptide levels during premoult were not transcriptionally coupled. Severe hypoxic conditions resulted in rapid downregulation of CCAP transcription in the eyestalk, but not the thoracic ganglia in Callinectes, and thermal challenge did not change CCAP mRNA levels. These results offer the first tantalising glimpses of involvement of CCAP in environmental adaptation to extreme, yet biologically relevant stressors, and perhaps suggest that the CCAP-containing neurones in the eyestalk might be involved in adaptation to environmental stressors.
Assuntos
Decápodes/metabolismo , Perfilação da Expressão Gênica , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , DNA Complementar/genética , Imuno-Histoquímica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Activated microglia, the resident macrophages of the brain, are a feature of Alzheimer's disease. Animal models suggest that when activated microglia are further activated by a subsequent systemic infection this results in significantly raised levels of interleukin 1beta within the CNS, which may in turn potentiate neurodegeneration. This prospective pilot study in Alzheimer's disease subjects showed that cognitive function can be impaired for at least two months after the resolution of a systemic infection and that cognitive impairment is preceded by raised serum levels of interleukin 1beta. These relations were not confounded by the presence of any subsequent systemic infection or by baseline cognitive scores. Further research is needed to determine whether recurrent systemic infections drive cognitive decline in Alzheimer's disease subjects through a cytokine mediated pathway.
Assuntos
Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Infecções Bacterianas do Sistema Nervoso Central/complicações , Infecções Bacterianas do Sistema Nervoso Central/metabolismo , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/etiologia , Interleucina-1/metabolismo , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Interleucina-1/sangue , Masculino , Testes NeuropsicológicosRESUMO
Ligation of the common bile-pancreatic duct induces hyperamylasemia and acute pancreatitis in rats. Pancreatic morphologic changes include edema, acinar cell damage, and mild inflammation. The pathogenesis of acute pancreatitis in this model is not understood, but may involve altered secretion and intrapancreatic activation of acinar proteases. We hypothesized that trypsinogen activation, measured by the production of plasma and pancreatic trypsinogen activation peptides (TAP), occurs early in this model. We performed the following experiments: rats were prepared with (1) bile-pancreatic ducts ligated and (2) ducts dissected but not ligated (sham). Rats were killed after 6, 24, and 48 hr. Serum amylase was measured and histologic sections were analyzed for morphologic changes. TAP was measured in both serum and pancreatic tissue homogenates using a specific polyclonal. anti-TAP antibody in an enzyme-linked immunosorbant assay. After 6, 24, and 48 hr of bile-pancreatic duct ligation, hyperamylasemia and acute morphologic changes of acute pancreatitis were observed. Evidence of acinar cell destruction was not evident until 48 hr after ligation. Levels of serum and pancreatic tissue TAP were significantly elevated at both 24 and 48 hr after ligation compared to those of sham. We conclude that increased intrapancreatic trypsinogen activation occurs early in this form of experimental acute pancreatitis and that it occurs prior to evidence of acinar cell destruction. These data and observations support the possibility that intrapancreatic protease activation contributes to the pathogenesis of ligation-induced acute pancreatitis.
Assuntos
Oligopeptídeos/biossíntese , Pâncreas/metabolismo , Pancreatite/metabolismo , Tripsinogênio/metabolismo , Doença Aguda , Animais , Ligadura , Masculino , Pâncreas/patologia , Pancreatite/etiologia , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: In healthy rats, combined bile and pancreatic juice diversion from gut has a synergistic rather than additive effect on stimulation of exocrine pancreatic protein secretion. We hypothesized that exclusion of combined bile and pancreatic juice from gut exacerbates bile and pancreatic-duct ligation-induced acute pancreatitis in rats to a greater extent than exclusion of either bile or pancreatic juice alone. METHODS: Bile and pancreatic juice (obtained fresh from donor rats) were replaced, separately or together, via a duodenal fistula beginning immediately before 6 hours of duct ligation. Pancreatic morphologic changes were evaluated with an acute pancreatitis histology score and morphometric quantitation of acinar-cell necrosis. Plasma amylase and cholecystokinin concentrations and pancreatic subcellular distribution of cathepsin B activity were determined. Characteristics of bile and pancreatic juice obtained from donor rats were also studied. RESULTS: Combined bile and pancreatic juice replacement limited the increase in acute pancreatitis histology score by 77%, acinar cell necrosis by 95%, hyperamylasemia by 77%, and hypercholecystokininemia by 99%, while preventing subcellular redistribution of cathepsin B. Amelioration of pancreatic morphologic changes was significantly greater with combined bile and pancreatic juice replacement than with replacement of either bile or pancreatic juice alone. CONCLUSION: In this experimental corollary of early gallstone-induced acute pancreatitis, combined bile and pancreatic juice exclusion from gut contributes to disease pathogenesis to a greater extent than exclusion of either bile or pancreatic juice alone.
Assuntos
Bile/fisiologia , Suco Pancreático/fisiologia , Pancreatite/prevenção & controle , Doença Aguda , Amilases/sangue , Animais , Catepsina B/metabolismo , Colecistocinina/sangue , Ducto Colédoco/fisiologia , Duodenostomia , Bombas de Infusão , Ligadura , Masculino , Necrose , Pâncreas/enzimologia , Pâncreas/patologia , Ductos Pancreáticos/fisiologia , Pancreatite/enzimologia , Pancreatite/patologia , Pancreatite/fisiopatologia , Ratos , Ratos Sprague-DawleyRESUMO
Acute necrotizing pancreatitis in opossums after bile and pancreatic duct ligation (BPDL) is a useful experimental corollary of gallstone-induced acute pancreatitis in humans. In experimental and human acute pancreatitis, a loss of segregation of the lysosomal enzyme cathepsin B and the zymogen proenzyme trypsinogen (colocalization) is implicated as the triggering event of disease pathogenesis, as cathepsin B can activate trypsinogen. The object of this study was to quantitate acinar cell necrosis and to study subcellular distribution of cathepsin B in BPDL-induced acute necrotizing pancreatitis in opossums. Bile and pancreatic ducts were ligated separately (no bile reflux) in four opossums while ducts were dissected in four sham controls. Opossums were killed 24 hr after operation. Three equidistant cross-sectional portions of each opossum pancreas were submitted to histologic examination. In blinded fashion, each focus of acinar cell necrosis was photographed and quantitated with digitizing morphometry. Numerical density (foci/cm2) and areal density (x10(3) micron 2/cm2) of focal acinar cell necrosis were determined. Differentially centrifuged pancreatic homogenates were assayed for cathepsin B, the lysosomal marker enzyme N-acetylglucosaminidase, and amylase. Morphometric quantitation of acinar cell necrosis confirmed development of acute necrotizing pancreatitis after 24 hr of BPDL in opossums. However, colocalization was not observed after BPDL, as evidenced by an absence of subcellular shift of cathepsin B activity (and N-acetyl-glucosaminidase activity) from the lysosome-enriched to the zymogen-enriched subcellular fraction. Amylase activity was increased in subcellular fractions after BPDL.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Catepsina B/metabolismo , Pancreatite/metabolismo , Pancreatite/patologia , Acetilglucosaminidase/metabolismo , Doença Aguda , Amilases/metabolismo , Animais , Ductos Biliares , Feminino , Ligadura , Masculino , Necrose , Gambás , Ductos Pancreáticos , Pancreatite/etiologia , Frações Subcelulares/metabolismo , Distribuição TecidualRESUMO
Bile and pancreatic duct ligation (BPDL) in rats does not induce severe acute pancreatitis but only mild inflammation, which is self-limiting and eventually leads to pancreatic atrophy. However, BPDL in opossums induces severe acute necrotizing pancreatitis which uniformly leads to death within 14 days. We compared pancreatic morphologic changes after 24 hr of BPDL in rats and opossums. Pancreatitis histology score and acinar cell ultrastructural changes were evaluated. In both species, BPDL was associated with significant increases in histology score compared to sham controls (5.0 +/- 0.3 vs 1.5 +/- 0.3 in rats, 5.3 +/- 0.4 vs 1.1 +/- 0.1 in opossums; mean +/- SEM, ANOVA, P < 0.05). However, there was no significant difference in histology score between rats and opossums following BPDL; histologic changes, such as white blood cell infiltration, acinar cell vacuolation, and focal acinar cell necrosis, were similar. Acinar cell ultrastructural changes after BPDL in both species included dilated endoplasmic reticulum and autophagic vacuole formation. These findings indicate that the early morphologic changes after BPDL in rats are quite similar to those seen early in the course of BPDL-induced acute necrotizing pancreatitis in opossums. As the rat is a more economical and convenient model to study than the opossum, this study supports the use of the rat model to conduct pilot studies of early events in the development of BPDL-induced acute pancreatitis. This study also suggests the potential for investigating mechanisms that may be present in the rat which protect against progressive and fatal acute necrotizing pancreatitis as observed in opossums after longer periods of BPDL.
Assuntos
Colestase Extra-Hepática/complicações , Modelos Animais de Doenças , Gambás/anatomia & histologia , Pancreatite/patologia , Doença Aguda , Análise de Variância , Animais , Ducto Colédoco/anatomia & histologia , Custos e Análise de Custo , Duodeno/anatomia & histologia , Feminino , Ligadura , Masculino , Necrose , Ductos Pancreáticos/anatomia & histologia , Pancreatite/etiologia , Ratos , Ratos Sprague-Dawley , Pesquisa/economiaRESUMO
Diversion and recirculation of bile and pancreatic juice in rats are done in studies of pancreatic exocrine secretion. Previously, the modified Bollman cage was used to restrain rats with bile and pancreatic fistulas. To mimic physiologic conditions as closely as possible and to develop a more humane model, we designed a partial-restraint tethering system to study pancreatic exocrine secretion. Eight rats were prepared with biliary pancreatic, and duodenal fistulas, of which five were given enteral supplements via a gastric fistula and three were given saline supplements via a jugular venous line. Catheters exited at the nape of the neck and passed through the hollow of a cable coil that tethered the rat. On the fourth postoperative day pancreatic juice flow and protein output were studied. The tethering system allowed grooming, feeding, and ample mobility. This model of the tethered pancreatic fistula rat is a more human model for studies of pancreatic exocrine secretion in conscious rats, compared with the Bollman cage system of near total restraint.
Assuntos
Modelos Biológicos , Pâncreas/metabolismo , Animais , Cinética , Masculino , Fístula Pancreática , Suco Pancreático/fisiologia , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Restrição FísicaRESUMO
Obstruction-induced acute pancreatitis in rats is associated with increased plasma cholecystokinin (CCK) levels. Duodenal replacement of bile reduces severity of pancreatitis and limits CCK increase. We investigated the role of CCK in the pathogenesis of obstruction-induced acute pancreatitis by pretreating rats with the somatostatin analog octreotide and the CCK antagonist L-364,718. Octreotide inhibits duodenal CCK release, and L-364,718 competitively blocks CCK receptors. We studied 31 rats after (1) sham operation (n = 7), (2) bile and pancreatic duct obstruction (BPDO) (n = 12), (3) BPDO plus octreotide (20 micrograms/kg IP and then 5 micrograms/kg/hr IV) (n = 6), and (4) BPDO plus L-364,718 (1 mg/kg IP and then 0.25 mg/kg/hr IV) (n = 6). Rats were killed after 18 hours. Pancreas weight, acute pancreatitis histology score, and plasma amylase and CCK levels were determined. Octreotide and L-364,718 limited the increase in pancreas weight. Octreotide also limited the rise in plasma CCK levels. These findings suggest that CCK may play a role in the pathogenesis of obstruction-induced acute pancreatitis.
Assuntos
Benzodiazepinonas/farmacologia , Colecistocinina/antagonistas & inibidores , Octreotida/farmacologia , Pâncreas/patologia , Pancreatite/patologia , Doença Aguda , Análise de Variância , Animais , Benzodiazepinonas/uso terapêutico , Ductos Biliares , Devazepida , Edema , Masculino , Octreotida/uso terapêutico , Pâncreas/efeitos dos fármacos , Ductos Pancreáticos , Pancreatite/tratamento farmacológico , Ratos , Ratos Sprague-DawleyRESUMO
Pancreatic exocrine stimulation by cholecystokinin (CCK) has been implicated in the pathogenesis of experimental acute pancreatitis. Bile exclusion from the gut stimulates duodenal CCK release and exacerbates obstruction-induced acute pancreatitis. Pancreatic and bile duct obstruction increases circulating CCK concentration. We hypothesized that acute pancreatitis induced by pancreatic and bile duct obstruction would be ameliorated when bile was returned to the duodenum. As many small pancreatic ducts drain into the bile duct in rats, preservation of bile flow required the use of a bile shunt. We studied acute pancreatitis and the time course of circulating CCK increase in three groups of rats after: (1) sham operation (dissection, no obstruction), (2) bile and pancreatic duct obstruction, and (3) bile and pancreatic duct obstruction with bile shunt. The rats were killed at 3-, 6-, and 18-hr intervals after operation. Their blood was collected for measurement of CCK, amylase, and bilirubin concentrations. The pancreata were excised, weighed, and processed for histological examination. The shunting of bile back to the duodenum ameliorated the acute pancreatitis along with a simultaneous limitation of the rise in CCK concentration. This suggests that bile duct obstruction, another form of bile exclusion, exacerbates pancreatic duct obstruction-induced acute pancreatitis. The elevation in CCK concentration showed an early peak indicating that the potential role of CCK in the pathogenesis of obstruction-induced acute pancreatitis is predominantly in the early phase of its development.
Assuntos
Colecistocinina/sangue , Colestase/complicações , Ductos Pancreáticos , Pancreatite/sangue , Doença Aguda , Anastomose Cirúrgica , Animais , Ductos Biliares Intra-Hepáticos/cirurgia , Bilirrubina/sangue , Constrição Patológica , Duodeno/cirurgia , Pancreatopatias/complicações , Pancreatite/etiologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
We assessed several modifications of thin-layer chromatography for evaluation of amniotic fluid lecithin/sphingomyelin ratios. For a procedure which is reliable, economical, and easy to perform, we preferred laboratory-prepared plates using SI-LICAR TLC-7GF. For spot detection, we preferred the iodine vapor method.