Further characterisation of particulate neuronal nitric oxide synthase in rat small intestine

Neuronal NO-synthase (nNOS) was investigated in rat longitudinal muscle/myenteric plexus (LM/MP) tissue at the cellular and subcellular level. Using preparations and double immune staining and light and electron microscopy, we concluded that, in these preparations, nNOS is only present in neuronal cells. However, in spite of numerous attempts to morphologically identify the NOS-containing subcellular structure, no firm conclusions were possible. Consequently, the problem was approached by biochemical methods including gradient centrifugation followed by analysis of the fractions. Using a protocol involving gentle homogenisation of the tissue, we found that about 10% of the nNOS immune reactivity was particle-bound confirming previous results (Biochem. Pharmacol. 60 (2000) 145). However, applying a different protocol including strong homogenisation, we now demonstrated that about 50% of the immune reactive nNOS was sedimentable. The results suggested that particulate nNOS is associated with one single subcellular structure, which is different from the plasma membrane, rough and smooth endoplasmic reticulum, mitochondria and lysosomes. The equilibrium sedimentation characteristics of the nNOS containing particles corresponded partly to those containing vasoactive intestinal polypeptide (VIP) or synaptobrevin. Application of non-equilibrium centrifugation conditions, however, demonstrated that almost no co-localisation occurred. We conclude that, in the LM/MP tissue, nNOS is about 50% particle-bound in a subcellular structure, which is different from the VIP-containing particle and from synaptobrevin-containing exocytotic particles.     

Multiple signal pathways coupling VIP and PACAP receptors to calcium channels in hamster submandibular ganglion neurons

The Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two novel neuropeptides which produce particular biological effects caused by interaction with G-protein-coupled receptors. We have shown in a previous study where VIP and PACAP 38 inhibit voltage-dependent calcium channel (VDCC) currents (ICa) via G-proteins in hamster submandibular ganglion (SMG) neurons. In this study, we attempt to further characterize the signal transduction pathways of VIP-and PACAP 38-induced modulation of ICa. Application of 1 microM VIP and PACAP 38 inhibited ICa by 33.0 +/- 3.1% and 36.8 +/- 2.6%, respectively (mean +/- S.E.M., n = 8). Application of strong voltage prepulse attenuated PACAP 38-induced inhibition of ICa. Pretreatment of cAMP dependent protein kinase (PKA) activator attenuated VIP-induced inhibition, but not the PACAP 38-induced inhibition. Intracellular dialysis of the PKA inhibitor attenuated the VIP-induced inhibition, but not the PACAP 38-induced inhibition. Pretreatment of protein kinase C (PKC) activator and inhibitor attenuated VIP-induced inhibition, but not the PACAP 38-induced inhibition. Pretreatment of cholera toxin (CTX) attenuated PACAP 38-induced inhibition of ICa. These findings indicate that there are multiple signaling pathways in VIP and PACAP 38-induced inhibitions of ICa: one pathway would be the VPAC1/VPAC2 receptors-induced inhibition involving both the PKA and PKC, and another one concerns the PAC1 receptor-induced inhibition via Gs-protein betagamma subunits. The VIP-and PACAP 38-induced facilitation of ICa can be observed in the SMG neurons in addition to inhibiting of ICa.     

Cloning and characterization of a thermostable catfish alphaB-crystallin with chaperone-like activity at high temperatures

We have cloned, expressed and characterized catfish alphaB-crystallin (FalphaB). Genomic sequence comparison has revealed conservation of intron splicing sites and coding regions, however, the two intron sequences, 5'- and 3'-untranslated regions of FalphaB gene are shorter than those reported for other vertebrates. In contrast to mammalian homologues with a subunit association ratio (alphaA-crystallin/alphaB-crystallin) of 3:1, alpha-crystallin from catfish lens showed a ratio of 19:1. The biophysical properties and chaperone-like activity of recombinant FalphaB and porcine alphaB-crystallin (PalphaB) were studied and compared by heat denaturation, circular dichroism, intrinsic and dye-binding fluorescence, gel-filtration, and analytical ultracentrifugation. FalphaB shows 50% precipitation occurring at 72 degrees C that is higher than PalphaB at 66 degrees C. Even though FalphaB also possesses more surface hydrophilic regions than PalphaB, FalphaB still possesses higher chaperone activity to prevent aggregation of alcohol dehydrogenase at 60 degrees C. The molecular mass of FalphaB showed a smaller size (450 kDa) than PalphaB (550 kDa), which is also confirmed by analytical ultracentrifugation. In addition, FalphaB possesses better refolding potential after preheating treatment than PalphaB. FalphaB also exhibits higher chaperone-like activity than PalphaB to prevent insulin aggregation induced by dithiothreitol. In contrast to the prevalent notion that fish crystallins generally denature easily, FalphaB with chaperone-like activity appears to be more stable than mammalian homologues towards thermal and chemical denaturation.     

The native oligomeric organization of alpha-crystallin, is it necessary for its chaperone function?

Citraconylation of all the lysine residues of alpha B and alpha A disrupts the native oligomeric state of these proteins. For alpha B, the oligomerization is concentration dependent with monomers and dimers formed at low protein concentration (approximately 0.01 mg ml(-1)). For concentration higher than 0.5 mg ml(-1) tetramers are the major species. Citraconylated alpha A crystallin is mostly tetrameric at any concentration. Citraconylation had a major effect on the secondary structure of alpha B which was reflected by a significant loss of beta-sheet structure. On the other hand, the secondary structure of alpha A crystallin was not significantly effected by this chemical modification. The chaperone properties of both modified proteins were the same as the native proteins when apo alpha-lactalbumin and malate dehydrogenase were used as target proteins. The data suggest that the native oligomeric state of alpha-crystallin may not be essential for its ability to suppress non-specific aggregation.     

Fetal meconium peritonitis in single and twin pregnancy. Two cases report

We present two cases of fetal meconium peritonitis in a single and twin pregnancy, respectively. The first case diagnosis was made at 30 weeks and was confirmed after delivery of the twins by cesarean section at 37 weeks. The second case diagnosis was made at 31 week and was confirmed at 37 weeks. Meconium peritonitis is a rare prenatal complication that results from intrauterine perforation of small bowel with spillage of sterile meconium into peritoneal cavity. We now report two cases of meconium peritonitis diagnosed at 30 and 31 weeks gestation. Received: 20 March 2001 / Accepted: 13 June 2001     

Necrotizing fasciitis following liver and small intestine transplantation

Necrotizing fasciitis is a rare, subcutaneous infection. It can occur in patients after solid-organ transplantation. We herein report two patients who developed necrotizing fasciitis following combined liver and small intestine transplantation. The first patient experienced this infection 4 yr after transplantation and 1 yr after the closure of the ileostomy. The second patient suffered from necrotizing fasciitis 2 days after the transplant. Both cases were diagnosed on the physical findings, culture of subcutaneous lavage, and the computed tomography findings. The site of entrance of the organism was not clear in either case. Both patients had a fulminant course and died within 1 week from the onset, despite aggressive surgical intervention. Therefore, necrotizing fasciitis has to be recognized as a potential complication of intestinal transplantation.     

Biologic therapy of inflammatory bowel disease

Advancing knowledge regarding the biology of chronic inflammation has led to the development of specific biologic therapies that mechanistically target individual inflammatory pathways. Many biologic therapies are being evaluated for the treatment of the chronic inflammatory bowel diseases, Crohn's disease and ulcerative colitis. Biologic compounds proven to be effective for Crohn's disease include monoclonal antibodies to tumor necrosis factor (infliximab and CDP571) and to the leukocyte adhesion molecule alpha4 integrin (natalizumab). Other biologic compounds for which there is insufficient evidence to judge efficacy for inflammatory bowel disease include: p55 tumor necrosis factor binding protein (onercept); interferon alpha; interferon beta-1a; anti-interferon gamma antibody; anti-interleukin 12 antibody; p65 anti-sense oligonucleotide (blocks NF-kappaB); granulocyte colony stimulating factor, and granulocyte macrophage colony stimulating factor; anti-interleukin 2 receptor antibody; epidermal growth factor; keratinocyte growth factor 2 (repifermin); human growth hormone; anti-CD4 antibody; and anti-alpha4beta7 antibody. Biologic therapies that have been proven ineffective for inflammatory bowel disease include: interleukin 10; interleukin 11; anti-sense intercellular adhesion molecule-1; and the tumor necrosis factor receptor fusion protein etanercept. Based on the early successes of infliximab, CDP571 and natalizumab, it seems certain that biologic therapy will play an important role in the future treatment of inflammatory bowel disease.     

Establishment of a prostatic small-cell carcinoma cell line (SO-MI)

BACKGROUND: Prostatic small-cell carcinoma is an extremely rare, highly aggressive disease. We established a cell line from this tumor. MATERIALS AND METHODS: Tumor tissue obtained from a 24-year-old Japanese man was used to establish the cell line. Cultured cells and tumors transplanted into nude mice were characterized by histologic, immunohistologic, immunocytologic, and molecular biologic methods. RESULTS: An immortal culture cell line (SO-MI) was successfully established. SO-MI cells adhered weakly to plastic surfaces in vitro, showing a 52- to 72-hr doubling time. SO-MI cells were heterotopically and orthotopically transplantable in nude mice. The cells were immunoreactive for NSE, chromogranin A, and NCAM, but not for ACTH, calcitonin, serotonin, gastrin, insulin, glucagons, LCA, EMA, PAP, PSA, androgen receptor, and p53. SO-MI cells secreted NSE in vitro and in vivo. SO-MI cells at passage 30 contained 50-59 chromosomes with a modal number of 55. PCR suggested that the p53 gene was deleted in SO-MI cells. RT-PCR detected no mRNA encoding androgen receptor in these cells. CONCLUSIONS: SO-MI cells retain the neuroendocrine nature of the original tumor, and should be useful in studying possible etiologies and new treatments.     

Small intestinal bacterial overgrowth: a possible risk factor for metabolic bone disease

Small intestinal bacterial overgrowth (SIBO) is one of the causes of malabsorption syndromes. The prevalence of metabolic bone disease in patients with SIBO is unknown, but a recent prospective case-control study indicated significant contribution of SIBO to the development of metabolic bone disease. We review this and other reports in the literature and discuss the possible mechanisms causing metabolic bone disease in patients with SIBO.     

Effect of route and type of nutrition on intestine-derived inflammatory responses

BACKGROUND: Immunological links between the gastrointestinal (GI) tract and respiratory tract has been postulated in the development and maintenance of mucosal immunity. Route and type of nutrition affects mucosal immunity by reducing cell populations within the Peyer's patches of the small intestine and lamina propria as well as altering cytokine profiles within these sites. In addition to the mucosal affects, these alternations in cytokines (decreases in interleukin-4 and interleukin-10) also appear to influence the vascular endothelium of the GI tract. DATA SOURCES: This review examines the laboratory data regarding cytokine profile within the gut, endothelial adhesion molecule expression within the intestinal and extraintestinal organs, and the effect of these alterations on neutrophil accumulation and organ responses to gut ischemia/reperfusion. It also describes the effect of a specific nutrient, glutamine, on the starved gut. CONCLUSIONS: Changes induced by failure to feed the GI tract affects GI vascularity increasing expression of proinflammatory adhesion molecules. These adhesion molecules attract neutrophils and prime them for subsequent ischemic events. Lack of feeding the gastrointestinal tract acts as a "first hit" and increases the inflammatory response to a secondary insult in the lungs, liver, and GI tract. The addition of the specific nutrient, glutamine, reverses many of these defects and favorably influences the proinflammatory effects of gut starvation.