[COâ and 16S rDNA Sequence Identification of Common Necrophagous Flies in Fujian Province].

ABSTRACT: Objective To identify the species of common necrophagous flies in Fujian Province by gene fragment sequences of mitochondrial cytochrome c oxidase subunit Ⅰ （COⅠ） and 16S ribosomal deoxyribonucleic acid （16S rDNA）, and to explore the identification efficacy of these two molecular markers. Methods In total 22 common necrophagous flies were collected from the death scenes in 9 different regions in Fujian Province and DNA was extracted from the flies after morphological identification. The gene fragments of COⅠ and 16S rDNA were amplified and sequenced. All the sequences were uploaded to GeneBank and BLAST and MEGA 10.0 software were used to perform sequence alignment, homology analysis and intraspecific and interspecific genetic distance analysis. The phylogenetic trees of DNA fragment sequences of COⅠ and 16S rDNA of common necrophagous flies in Fujian Province were established by unweighted pair-group method with arithmetic means （UPGMA）, respectively. Results The flies were classified into 6 species, 5 genera and 3 families by morphological identification. The results of gene sequence analysis showed that the average number of interspecific and intraspecific genetic distance of 16S rDNA ranged from 1.8% to 8.9% and 0.0% to 2.4%, respectively. The average number of interspecific and intraspecific genetic distance of COⅠ ranged from 7.2% to 13.6% and 0.0% to 6.3%, respectively. Conclusion The gene sequences of COⅠ and 16S rDNA can accurately identify the species of different necrophagous flies, and 16S rDNA showed higher value in species identification of common calliphoridae necrophagous flies in Fujian Province.

Molecular genetic analysis of heterosis in interspecific hybrids of Argopecten purpuratus x A. irradians irradians.

Argopecten purpuratus and Argopecten irradians irradians hybridization was successfully performed and the hybrid offspring displayed apparent heterosis in growth traits. To better understand the genetic basis of heterosis, the genomic composition and genetic variation of the hybrids were analyzed with amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Seven of eight universal SSR primers displayed polymorphism in the hybrids and their parental groups, and hybrids inherited both parental geno-types at each locus. Using five primer combinations in AFLP analysis, 433 loci were amplified in the hybrids and their parental groups. The frequency of polymorphisms was 88.22%. F1 hybrids inherited 88.11 and 92.88% of AFLP bands from their parents. Some loci did not follow Mendelian Law, including 48 loci in parents that were lost, and 11 new loci that were amplified in the hybrids. The parameters of Nei's gene diversity, Shannon's Information index, genetic distance, and molecular variance between groups were calculated. The genetic differentiation between two hybrid groups (0.253) was smaller than that between hybrids and their parents (0.554 to 0.645), and was especially smaller than that between two parental groups (0.769). The high genetic similarity (0.9347) and low genetic differentiation (0.2531) between two hybrid groups suggests that these hybrid groups were genetically very close. Heterozygosities of hybrid groups were higher than those of parental groups, indicating that the hybrids had increased genetic diversity.

Interleukin-6 signal transduction in human intestinal epithelial cells.

The gut is an important source of inflammatory cytokines, but there is scant information on the mechanisms of cytokine action in gut epithelium. We hypothesized that in human Caco-2 cells, IL-6 acts directly through stimulation of Stat phosphorylation and that bacterial lipopolysaccharide (LPS) causes Stat activation indirectly because of its ability to cause the autocrine secretion and action of interleukin (IL)-6. Stat1, Stat5a, and Stat5b, but not Stat3, were detected in Caco-2 cells. DNA-binding activity corresponding to activated Stat5 was stimulated in a biphasic manner by IL-6, with a transient early phase, followed by sustained activation between 8 and 48 h. LPS also stimulated Stat5-like binding, but there was no early phase of activation. Functional tests of Stat5 activation showed that IL-6 stimulated Stat5-dependent reporter gene transcription but had no effect on Stat1-dependent transcription. LPS did not stimulate Stat-dependent transcription, nor did it alter the transcriptional response to IL-6. Tyrosine phosphorylation of both Stat5a and Stat5b was induced by IL-6. We infer from these data that IL-6 acts on intestinal epithelia through a Stat5-mediated transcriptional mechanism, whereas LPS does not induce gene expression through autocrine activation of enterocyte Stat signaling. These data provide a basis for testing the in vivo regulation of gut signaling and the interaction of gut reticuloendothelial cells with epithelial signal transduction.

Differential expression of intestinal and splenic cytokines after parenteral nutrition.

OBJECTIVE: To determine the effect of parenteral nutrition (PN) on the expression of message for inflammatory cytokines in the spleen and different segments of the intestine. DESIGN: Randomized controlled trial. PARTICIPANTS: Eleven adult male Sprague-Dawley rats weighing 250 to 300 g. INTERVENTIONS: All rats underwent central venous cannulation and were randomized to two groups. Group 1 (n = 6) received saline solution infusion and chow ad libitum; group 2 (n = 5) received lipid-free PN with no oral feeding. After 7 days, the animals were killed and the spleens and segments of small and large intestine were removed. MAIN OUTCOME MEASURES: The expression of message for tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1 in the spleen and intestine was determined using a semiquantitative reverse transcription polymerase reaction. Splenic macrophages were isolated and cultured for 24 hours with and without lipopolysaccharide. Production of TNF-alpha and IL-6 was determined by bioassay followed by enzyme-linked immunosorbent assay. RESULTS: After 7 days of infusion, messenger RNA (mRNA) expression for TNF-alpha, IL-1, and IL-6 was increased in the jejunum (P < .05), and TNF-alpha mRNA and IL-6 mRNA expression was decreased in the spleen (P < .01) of PN-fed animals when compared with saline/chow controls. In addition, TNF-alpha mRNA expression was increased in the cecum (P < .05), IL-1 mRNA expression was increased in the ileum (P < .05), and IL-6 mRNA expression was increased in the cecum (P < .05) and Peyer's patches (P < .007) in the PN-fed animals. Production of TNF-alpha and IL-6 by splenic macrophages was decreased following PN infusion in both lipopolysaccharide-treated and untreated cultures (P < .05). CONCLUSIONS: Infusion of lipid-free PN induces a differential mRNA expression for inflammatory cytokines in the spleen and intestine with an overall up-regulation of the expression of inflammatory cytokines in the intestine and a down-regulation in the spleen. These data provide evidence that the regulatory mechanisms for cytokine production are different in the intestine and the spleen. Further study is needed to elaborate the mechanism of this differential expression following lipid-free PN infusion.

Effect of glutamine on phagocytosis and bacterial killing by normal and pediatric burn patient neutrophils.

Glutamine is essential for the function of lymphocytes and macrophages, where it serves, among other things, as a source of energy. Little information is available concerning the fuel that polymorphonuclear cells use for their metabolic and bactericidal functions. It was the purpose of this study to determine whether glutamine would enhance the in vitro bactericidal function of normal neutrophils and whether the amino acid would restore the observed impaired function in burn patients to or above the normal level. Twelve burn patients with total body surface area burns ranging from 32% to 87% were studied. At various postburn times, neutrophils were isolated and their ability to kill Staphylococcus aureus in the presence and absence of glutamine was determined and compared with that in normal subjects. Glutamine enhanced the bactericidal function of normal neutrophils. In every patient, at all but two postburn times, glutamine caused an improvement in the observed abnormal neutrophil bactericidal function and often restored it to or slightly above the normal level. Glutamine had no effect on the expression of C3b receptors (CR1 or CD35) or on phagocytosis by the cells. This study confirms the beneficial effects of glutamine in at least one arm of the immune system and adds evidence for the possible advantage of including this amino acid in the diets of burn and other trauma patients.

The increased potential for the production of inflammatory cytokines by Kupffer cells and splenic macrophages eight days after thermal injury.

Burn patients often experience a devastating inflammatory response to infection within the first two weeks after thermal injury. The inflammatory cytokines IL-6, TNF and IL-1 have been implicated in this condition but most studies have focused on the abnormal levels of cytokines in the plasma. In this study the production of cytokines was compared for Kupffer cells versus splenic macrophages; endotoxin (LPS) stimulation versus no stimulation; and burn (post burn days 1, 3 and 8) versus no burn (control). Corresponding serum levels of IL-6 were also determined. Kupffer cells from normal or burned animals were shown to produce much higher amounts of the inflammatory cytokines than that produced by splenic macrophages. An exception to this was the equal production of TNF by LPS-stimulated hepatic and splenic cells. Both LPS-stimulated Kupffer cells and splenic macrophages produced larger amounts of the cytokines than that produced by the unstimulated cells. There was a significant effect of thermal injury on cytokine production by LPS-stimulated Kupffer cells at post burn day 8 and on TNF production by stimulated splenic macrophages also at post burn day eight. Although there was a statistically significant effect of thermal injury at post burn day 8 on IL-1 production by unstimulated splenic macrophages, the absolute amount of cytokine produced was very small. The results suggest that by post burn day 8 the cells may have become primed to respond to a stimulus such as endotoxin (LPS), a condition that could arise in a burn patient from sepsis. Strangely, the large spike in serum IL-6 level occurred at post burn day one and the level of the cytokine returned nearly to the control value on post burn days 3 and 8.

The 1994 Lindberg Award. The production of tumor necrosis factor, interleukin-1, interleukin-6, and prostaglandin E2 by isolated enterocytes and gut macrophages: effect of lipopolysaccharide and thermal injury.

Increasing evidence shows that cells other than immune cells have the potential for producing cytokines and arachidonate metabolites. It was the purpose of this study to determine whether isolated enterocytes could produce tumor necrosis factor, interleukin-1, interleukin-6, and prostaglandin E2, to compare the production with that of isolated gut macrophages, and to determine whether a difference existed in the production of these mediators after thermal injury. Guinea pigs received a 30% total body surface area burn and were killed 24 hours after injury. Isolated enterocytes and related intestinal macrophages (5 x 10(5) cells/ml) were cultured for 24 hours in the presence and absence of endotoxin, and the supernatants were assayed for the mediators. An increase was seen in production of interleukin-6 by enterocytes and by macrophages after thermal injury. In general enterocytes and gut macrophages produced about the same amounts of the different mediators. In contrast to macrophages from other tissues, enterocytes did not produce more prostaglandin E2 after stimulation with lipopolysaccharide, and with one exception gut macrophages did not produce larger amounts of mediators after stimulation with lipopolysaccharide. Enterocytes may be a significant source of immunomediator production and could contribute to the inflammatory response.

Avoiding serious complications in laparoscopic cholecystectomy--lessons learned from an experience of 2428 cases.

Over a three-and-a-half year period, we performed 2428 cases of laparoscopic cholecystectomy (LC) and encountered 11 cases of serious procedure-related complications, including bile duct injuries in 4 patients, postoperative bleeding requiring laparotomy and haemostasis in 3 patients, bile leakage from the cystic duct stump, jejunal injury related to puncture, intraoperative injury to the duodenum and subdiaphragmatic abscess in 1 patient each respectively. Six patients required re-hospitalisation including 2 patients with pancreatitis, 1 patient with Ascaris cholangitis, 1 patient with residual stone of the common bile duct (CBD) after laparoscopic CBD exploration, 1 patient with a stone in the CBD after LC, and 1 patient with bile leakage from the cystic duct stump and peritonitis. Of the 2428 patients treated, there was only 1 operative mortality. This patient developed frequent episodes of supraventricular tachycardia. She was found to have pnuemonia on the 21st postoperative day and she died. Apart from this, 1 other patient was found to have primary cancer of the liver 1 month after LC. Based on our experience, we think that LC is safe for patients with benign disease of the gallbladder.

Effect of pentoxifylline on survival and intestinal cytokine messenger RNA transcription in a rat model of ongoing peritoneal sepsis.

OBJECTIVE: Septic animals receiving high-protein liquid diets have increased mortality and increased production of cytokines by the gut compared with animals receiving low-protein diets. The purpose of this study was to evaluate the ability of pentoxifylline to alter gut cytokine production in a rat model of prolonged acute peritonitis, to determine its effect on survival in such animals, and to determine whether alteration of gut cytokine production was associated with survival. DESIGN: Prospective, randomized animal study. SETTING: Research laboratory. SUBJECTS: Male Lewis rats weighing between 250 and 300 g. INTERVENTIONS: Anesthetized rats had placement of a gastrostomy, followed 1 wk later by implantation of a bacteria-filled osmotic minipump into the peritoneal cavity. Rats were fed a high-protein (20% total energy) enteral diet. Saline or pentoxifylline (5 or 20 mg/kg im) was administered daily beginning at the time of pump implantation. MEASUREMENTS AND MAIN RESULTS: Septic rats fed the high-protein liquid diet and given pentoxifylline in a dose of 5 mg/kg/day demonstrated improved survival compared with saline-treated animals or animals given the high dose (20 mg/kg/day) of pentoxifylline (p< .05). Administration of pentoxifylline at 5 mg/kg/day also down regulated the production of IL-6 messenger RNA (mRNA) in liver and lipopolysaccharide binding protein mRNA in the liver and intestine of septic animals given the high-protein liquid diet. CONCLUSION: Low-dose (but not high-dose) pentoxifylline administration reduced production of some, but not all, cytokines studied in the gut and liver in a rat model of acute peritonitis and this reduced production was associated with an improved survival in such animals.

Molecular cloning, characterization, and expression in Escherichia coli of full-length cDNAs of three human glutathione S-transferase Pi gene variants. Evidence for differential catalytic activity of the encoded proteins.

We report the isolation of three full-length cDNAs corresponding to the mRNAs of closely related glutathione S-transferase (GST) Pi genes, designated hGSTP1*A, hGSTP1*B, and hGSTP1*C, expressed in normal cells and malignant gliomas. The variant cDNAs result from A --> G and C --> T transitions at nucleotides +313 and +341, respectively. The transitions changed codon 104 from ATC (Ile) in hGSTP1*A to GTC (Val) in hGSTP1*B and hGSTP1*C and changed codon 113 from GCG (Ala) to GTG (Val) in hGSTP1*C. Both amino changes are in the electrophile-binding active site of the GST Pi peptide. Computer modeling of the deduced crystal structures of the encoded peptides showed significant deviations in the interatomic distances of critical electrophile-binding active site amino acids as a consequence of the amino acid changes. The encoded proteins expressed in Escherichia coli and purified by GSH affinity chromatography showed a 3-fold lower Km (CDNB) and a 3-4-fold higher Kcat/Km for the hGSTP1*A encoded protein than the proteins encoded by hGSTP1*B and hGSTP1*C. Analysis of 75 cases showed the relative frequency of hGSTP1*C to be 4-fold higher in malignant gliomas than in normal tissues. These data provide conclusive molecular evidence of allelopolymorphism of the human GST Pi gene locus, resulting in active, functionally different GST Pi proteins, and should facilitate studies of the role of this gene in xenobiotic metabolism, cancer, and other human diseases.

Production of cytokines and prostaglandin E2 by subpopulations of guinea pig enterocytes: effect of endotoxin and thermal injury.

BACKGROUND: There is increasing evidence that cells other than immune cells have the potential for producing immunomediators. This study determined whether distinct populations of enterocytes from unburned and burned animals responded differently to endotoxin regarding production of tumor necrosis factor, interleukin-1 and -6 and prostaglandin E2. METHODS: Three subpopulations of enterocytes, progressing from the villus tip towards the crypt, were obtained from washes of the small intestine. The cells were cultured in the presence of endotoxin, and the supernatants were assayed for the mediators. RESULTS: Thermal injury primed all three populations of enterocytes to produce larger amounts of tumor necrosis factor and interleukin-6 compared to cells from unburned animals. Enterocytes that were nearer the crypt produced the largest amounts of the cytokines. CONCLUSION: These observations may be important because, as gut integrity is compromised after thermal injury, enterocytes that may have previously been unexposed or less exposed to endotoxin can become a significant source of inflammatory cytokines.

Time course of production of cytokines and prostaglandin E2 by macrophages isolated after thermal injury and bacterial translocation.

The relationship of translocation of bacteria from the gut of burned guinea pigs and the in vitro production of tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6, and prostaglandin E2 (PGE2) by lipopolysaccharide (LPS)-stimulated mesenteric lymph node and splenic macrophages was investigated at two early times after thermal injury. Two hr postburn, there was a large number of translocated bacteria in the mesenteric lymph nodes and a large proportion was killed; at 24 hr postburn, there were fewer translocated bacteria, but a large proportion was viable. In some cases, there were very large differences compared to controls in the amounts of TNF, IL-6, and PGE2, but not of IL-1, produced by the macrophages at different times postburn and at different in vitro incubation times. The results suggest that the macrophages were primed by the burn or the translocated bacteria to produce in vitro different and sometimes large amounts of cytokines or PGE2 after further stimulation with LPS. Although there was no direct correlation between production of cytokines or PGE2 and time postburn, the early increased production of PGE2 by splenic macrophages could have depressed the animal's ability to kill translocated bacteria by 24 hr postburn, and could be one of the mechanisms of the cause of systemic infection after burn injury.